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CURRENTS IN
BIOCHEMICAL RESEARCH

1956

6-

CURRENTS IN
BIOCHEMICAL RESEARCH

1956

Edited by DAVID E. GREEN

Twenty-seven essays charting the present

course of biochemical research and considering

the intimate relationship of biochemistry to

medicine, physiology^ and biology

INTERSCIENCE PUBLISHERS, INC., NEW YORK

Interscience Publishers, Ltd., London 1956

1956 by Interscience Publishers, Inc.

Library of Congress Catalog Card Number 56-9503

Interscience Publishers, Inc.
250 Fifth Ave., New York 1, N. Y.

For Great Britain and Northern Ireland:

Interscience Publishers Ltd.

88/90 Chancery Lane, London, W.C. 2, England

PRINTED IN THE UNITED STATES OF AMERICA
BY MACK PRINTING COMPANY, EASTON, PENNSYLVANIA

PREFACE

Ten years ago the first volume of Currents in Biochemical
Research made its debut. The cordial reception which was
given this volume by biologists, chemists, biochemists, and
clinicians indicated clearly that at appropriate intervals there
exists a real need to pause and reflect on what has been accom-
plished, to distinguish between tree and forest, to learn the
lessons of the years which have passed, and to prepare intellec-
tually for the years to come. Once again a distinguished group
of twenty-seven contributors has been invited to make a de-
cennial survey of progress in most, although by no means all,
important fields of biochemical research. They have been
asked to write as simply and as lucidly as the requirements of
scholarship permit. The objectives of these essays have been
to communicate to nonspecialists an overall impression of the
present status of the significant problems in each field, to point
up the broad strategy of current research, and finally, to specu-
late on the likely paths of future research. Above all, the essays
were intended to bring light without overwhelming the reader
with tedious detail. It would be too much to expect that each
of the twenty-seven essays has met all these exacting stipulations,
but at least I have high hopes that enough of them come sufl^-
ciently close to justify the aims of this second volume.

The past decade has witnessed a rate of progress vastly
greater than in any comparable period since the early beginnings
of biochemistry as a science more than 100 years ago. There is
little doubt that this phenomenal rate of development has been
sparked by a revolution in methodology. The emergence of
filter paper chromatography as a tool for separating minute

PREFACE

amounts of material rapidly, selectively, and even quantita-
tively, gave wings to biochemistry. In turn, this led to the
intensive exploitation and development of a large variety of
adsorption and partition procedures which almost overnight
made it not only possible, but even relatively simple to separate
and isolate constituents of any mixture, regardless of how similar
the constituents may happen to be. Isolation ceased to be a
roadblock or a major difficulty in biochemical investigation.

The spectrophotometer occupies a preeminent position
among the instruments which have facilitated the dramatic
advances in methodology. This was of course not a new
physical tool, but until some ten years ago it was a highly ex-
pensive, custom-built apparatus available only to relatively few
laboratories with considerable resources. With the advent of
the mass-produced spectrophotometer, the universal application
of spectrophotometric techniques to biochemical problems
became possible. It is no exaggeration to say that spectro-
photometry has increased manyfold the forward rate of progress
of biochemistry.

Other tools and techniques have played an equally im-
portant role in the revolution of biochemical methodology
during the past decade. The isotope technique with its count-
less ramifications and variations and the mass-produced ultra-
centrifuge and electrophoresis apparatus were also very domi-
nant factors in the over-all picture.

With such a plethora of powerful tools available, structural
problems that had long resisted solution were dusted off the shelf
and liquidated in record time. Nucleotides, nucleic acid,
polysaccharides, lipides, polypeptides, and even some proteins of
low molecular weight were no match for the many and incisive
methods which were brought to bear in the elucidation of
stucture. Even efforts at synthesis took a new lease of life now
that effective methods were available for separation of similar
reaction products, or isomers, as in the case of nucleotides. It
was a triumph of methodology to ferret out the exact structural
formula of insulin and to accomplish the synthesis of flavin

vi

PREFACE

adenine dinucleotide and vasopressin to mention but two out-
standing examples.

Methodology was not the principal limiting factor in all
structural problems. The solution of the problem of the
molecular architecture and stereochemistry underlying protein
structure required new ideas and concepts as well as more
powerful tools for analysis. The helix principle which has
been shown to apply successfully to a variety of proteins and to
nucleic acid has provided one of the keys for deciphering the
hitherto unintelligible x-ray diagrams of a variety of macro-
molecules.

A direct consequence of the advances in methodology was
the growth of enzyme chemistry from a toddler to a giant
dominating the whole of biochemistry. During the past ten
years practically all the enzymatic and chemical details of the
citric, pentose, fatty acid, and urea cycles have been elucidated.
The enzymatic synthesis of nucleic acid, lecithin, cephalin,
fatty acids, sucrose, lactose, purines, pyrimidines, and dinucleo-
tides has been accomplished by means of isolated enzymes or cell-
free enzyme systems. The chemical details of the reactions
which intervene in the enzymatic synthesis of hemin and cho-
lesterol are now almost fully documented. Hundreds of enzymes
which implement not only the above sequences but many
others too numerous to be listed have been isolated, character-
ized, and studied in detail. Indeed few of the classical problems
of metabolism remain unsolved or are not already close to
solution.

The list of coenzymes has more than doubled during the
past ten years. Lipoic acid, coenzyme A, uridine diphospho-
glucose, uridine diphosphogalactose, uridine diphosphoglucu-
ronic acid, glucose- 1,6-diphosphate, 1,3-diphosphoglyceric acid,
guanosine diphosphate, molybdenum, and cytosine diphospho-
choline are some of the more prominent recent additions to the
roster of known cofactors.

The era of each investigator preparing his own cofactors,
special chemicals, and even enzymes is now all but over. A

Vll

^^\CjAi

jf

PREFACE

whole new industry has sprung up which provides practically
every important biochemical compound relatively inexpensively
and of high purity. The importance of this development to the
rapid progress of biochemical research cannot be underesti-
mated.

Enzymology has indeed prospered by application of new
methodological tools but the traffic has gone both ways. In
turn, the enzymes have proved to be tools of such breath-taking
elegance and simplicity as to revolutionize the tactics of deter-
mining structure or of analyzing for minute amounts of material.
The elucidation of the structure of nucleic acid, of nucleotides,
and of various nucleotide cofactors has been accomplished
principally, if not exclusively, by enzymatic procedures and
with relatively small amounts of compound.

The current procedures for isolating enzymes are still
largely classical, but signs are multiplying that big changes in
methodology are imminent. Chromatography on columns of
cellulose derivatives or of calcium phosphate, electroconvection,
starch and filter paper electrophoresis, ion exchange resin
chromatography, and partition methods are portents of the
things to come.

In recent years there has been a growing recognition of the
role of integrated enzyme systems in cellular processes. The
concept of the mitochondrion as an organized mosaic of several
hundreds of enzymes linked together structurally as well as
functionally and with unique operational principles is now well
established in biochemical thinking. It is only in terms of large
molecular aggregates and of enzymes bonded together in a
precise pattern that it is at all possible to approach problems such
as oxidative phosphorylation, electron transfer, and muscular
contraction. The organized enzyme systems have provided the
bridge between classical enzyme chemistry and cellular proc-
esses. In addition to the mitochondrion, other functional
particles have been described such as the chloroplast in photo-
synthesis, and microsomes and nuclei in protein synthesis.

Progress in the field of the physical chemistry of biological

viii

PREFACE

systems and materials has been slower but still impressive.
Kinetic analysis of enzymes has graduated to a very sophisticated
and exact science which has been taken over by specialists well
grounded in physical chemical theory and practice. The con-
cepts of energy transformations and of the high-energy bond have
lost their earlier primitive character and are now couched in
the more rigorous language of quantum mechanics and valency
theory. The interpretation of the properties and behavior of
proteins and other macromolecules has now attained a high
degree of precision. Certainly the molecular picture of how
myosin contracts is constructed in terms which would have been
inconceivable even ten years ago.

It now appears that biochemistry has reached a stage in
development where fragmentation cannot be avoided. The
choice now lies between chemistry and physiology since the
meso area has all but disappeared. The classical areas of bio-
chemistry such as nutrition, vitamins, minerals, enzymology,
metabolism, and the chemical structure of biologically im-
portant compounds are becoming leaner and leaner, and the
focus is shifting inexorably either to more chemical and molecular
aspects or to the broader and as yet unsolved physiological
problems.

It is at present quite clear which chemical problems will
dominate the scene in the next few years. Certainly efforts will
be redoubled to determine the exact stereochemistry of protein
molecules after the spectacular success of the a helix concept.
Such efforts will be paralleled by companion efforts to elucidate
the structure of the active groups of enzymes. Once an under-
standing of the architecture of the active groups of enzymes has
been attained then the old problem of the mechanism of enzyme
action will be in line for renewed assault. The nature of the
forces and bonds which lead to enzyme-substrate interactions is
an area of investigation which badly needs sparking by new
leads.

The real frontier areas of biochemistry now reach to the
borderlines of physiology, genetics, cytology, medicine, and

ix

PREFACE

theoretical chemistry. The biochemical description of muscular
contraction, nerve conduction, glomerular filtration, and secre-
tion and membrane phenomena is still relatively virgin territory.
The structural analysis and interpretation of the way in which
mitochondria, nuclei, membranes, myelin sheath, and other
complex cellular structures are constructed in a chemical sense
have yet to reach even the blueprint stage. A fabulous area of
exploitation awaits the investigators who can hurdle the con-
ceptual barriers in the way of the biochemical study of hormone
action at the molecular level. If current progress on the re-
construction of in vitro systems for synthesis of protein is to
serve as a guide, then we must surely anticipate the polygamous
marriage, in the not too distant future, of biochemistry with
genetics, immunology, hematology, and virology. The older
problems of biochemistry are clearly moribund but the newer
problems have the fascination and challenge of youth.

David E. Green

February 7956

X

CONTRIBUTORS

Robert A. Alberty, Ph.D. Associate Professor of Chemistry, University

of Wisconsin, Madison, Wis. Eli Lilly Award, 1956.
H. A. Barker, Ph.D. Professor of Plant Biochemistry, University of

California, Berkeley, Calif.
James A. Bassham, Ph.D. Assistant Director, Photosynthesis Laboratory,

Bio-Organic Chemistry Group, Radiation Laboratory, University of

California, Berkeley, Calif.
KoNRAD E. Bloch, Ph.D. Higgins Professor of Biochemistry, Harvard

University, Cambridge, Afass.
Jean Botts, Ph.D. Physiologist, Division of Physical Biochemistry, Naval

Medical Research Institute, National Naval Medical Center, Bethesda,

Md.
Melvin Calvin, Ph.D. Professor of Chemistry, and Director, Bio-Organic

Chemistry Group, Radiation Laboratory, University of California,

Berkeley, Calif.
All.an M. Campbell, Ph.D. Instructor in Bacteriology, University of

Michigan, Ann Arbor, Mich.
Britton Chance, Ph.D., D.Sc. Professor of Biophysics, and Director of

the Johnson Research Foundation, University of Pennsylvania Medical

School, Philadelphia, Pa. President's Certificate of Merit, 1950;

Paul Lewis Award, 1950.
Waldo E. Cohn, Ph.D. Principal Biochemist, Oak Ridge National

Laboratory, Oak Ridge, Tenn.
Carl F. Cori, M.D. Professor of Biological Chemistry, Washington

University School of Medicine, St. Louis, Mo. Nobel Prize, 1947.
John T. Edsall, M.D. Professor of Biological Chemistry, Harvard

University, Cambridge, Mass.
Philip George, Ph.D. Research Professor of Biophysical Chemistry, The

University of Pennsylvania, Philadelphia, Pa.
G. Robert Greenberg, Ph.D. Associate Professor of Biochemistry,

School of Medicine, Western Reserve University, Cleveland, Ohio.

xi

CONTRIBUTORS

A. D. Hershey, Ph.D. Staff Member, Department of Genetics, Carnegie
Institution of Washington, Cold Spring Harbor, N. Y.

Frank M. Huennekens, Ph.D. Associate Professor of Biochemistry,
School of Medicine, University of Washington, Seattle, Wash.

Luis F. Leloir, M.D. Director of the Instituto de Investigaciones Bio-
quimicos, Buenos Aires, Argentina.

Fritz Lipmann, M.D., Ph.D. Head, Biochemical Research Laboratory,
Massachusetts General Hospital, and Professor of Biological Chemistry,
Harvard Medical School, Boston, Mass. Carl Neuberg Medal, 1 948 ;
Mead Johnson & Co. Award, 1948; Nobel Prize, 1953.

B.-VRBARA Lo\v, Ph.D. Assistant Professor of Physical Chemistry, Depart-
ment of Biological Chemistry, Harvard Medical School, Boston, Alass.

Henry R. M.ahler, Ph.D. Associate Professor of Chemistry, Indiana
University, Bloomington, Ind.

Manuel F. Morales, Ph.D. Chief of the Division of Physical Bio-
chemistry, Xaval Medical Research Institute, National Naval Medical
Center^ Bethesda, Md. American Physiological Society Travel
Award, 1950.

David Nachmansohn, M.D. Professor of Biochemistry, College of
Physicians and Surgeons, Columbia University, Nevu York, N. Y.
Pasteur Medal, 1952; Carl Neuberg Medal, 1954.

Gregory Pincus, D.Sc. Director of Laboratories, The Worcester Founda-
tion for Experimental Biology, Shrewsbury, and Research Professor of
Biology, Boston University, Boston, Mass.

Efr.mm Racker, M.D. Chitf, Division of Nutrition and Physiology, The
Public Health Research Institute of the City of New York, Inc., and
Adjunct Professor, Department of Microbiology, New York University
College of Medicine, New York, N. Y.

Harold P. Rusch, M.D. Professor of Oncology, and Director of the
McArdle Memorial Laboratory for Cancer Research, University of
Wisconsin, Madison, Wis.

Frederick Sanger, Ph.D., F.R.S. Aiember of the External Staff of the
Medical Research Council, Department of Biochemistry, University of
Cambridge, Cambridge, England.

David Shevhn, Ph.D. Professor of Biochemistry, College of Physicians and
Surgeons, Columbia University, Neiv York, A\ Y.

Xll

CONTRIBUTORS

Esmond E. Snell, Ph.D. Professor of Chemistry, and Associate Director of
the Biochemical Institute, University of Texas, Austin, Texas. Eli Lilly-
Award, 1945; Mead Johnson & Co. Award, 1946; Osborne-
Mendel Award, 1951.

S. Spiegelman, Ph.D. Professor of Bacteriology, University of Illinois,
Urbana, III.

DeWitt Stetten, Jr., M.D., Ph.D. Associate Director in Charge of
Research, National Institute of Arthritis and Metabolic Diseases,
National Institute of Health, Bethesda, and Lecturer in Medicine and
Physiological Chemistry, The Johns Hopkins University, Baltimore, Md.

D. M. Surgenor, Ph.D. Assistant Professor of Biological Chemistry,
Harvard Medical School, Boston, Mass.

Hugo Theorell, M.D., Professor. Head of the Medical Nobel Institute,
Biochemical Department, Stockholm, Sweden. Nobel Prize, 1955.

Irwin B. Wilson, Ph.D. Assistant Professor of Biochemistry, College of
Physicians and Surgeons, Columbia University, New York, N . Y .

Xlll

CONTENTS

Chemistry and Viral Growth

by A. D. Hershey 1

Photosynthesis

by J. A. Bassham and M. Calvin 29

Bacterial Fermentations

by H. A. Barker 70

Some Aspects of Vitamin and Growth Factor Research

by Esmond E. Snell 87

The Significance of Induced Enzyme Formation

by S. Spiegelman and A. M. Campbell 115

Certain Problems in the Biochemical Study of Disease

by DeWitt Stetten, Jr 162

The Hormones, Their Present Significance, Their Future

by Gregory Pincus 176

Problems of Cellular Biochemistry

by Carl F. Cori 198

Enzymes as Reagents

by Efraim Backer 215

Attempts at the Formulation of Some Basic Biochemical Questions

by Fritz Lipmann 241

Enzyme Complexes and Complex Enzymes

by Henry B. Mahler 251

Relations between Prosthetic Groups, Coenzymes and Enzymes

by Hugo Theorell 275

Enzyme-Substrate Compounds and Electron Transfer

by Britton Chance 308

On the Nature of Hemoprotein Reactions

by Philip George 338

Aspects of Protein Structure

by Barbara W. Low and John T. Edsall 378

The Structure of Insulin

by F. Sanger 434

A New Concept of Ribonucleic Acids

by Waldo E. Cohn 460

XV

71253

CONTENTS

Chemical Structure as a Guide to the Study of Biochemical Syn-
theses
by Konrad Block 474

The Role of Nucleotides and Coenzymes in Enzymatic Processes

by Frank M. Huennekens 493

The Biosynthesis of Porphyrins; The Succinate-Glycine Cycle

by David Shemin 518

Problems in the Study of Multiple Enzyme Systems

by G. Robert Greenberg 537

Enzyme Kinetics

by Robert A. Alberty 560

The Interconversion of Sugars in Nature

by Luis F. Leloir 585

A Theory of the Primary Event in Muscle Action

by Manuel F. Morales and Jean Bolts 609

Trends in the Biochemistry of Nerve Activity

by David Nachmansohn and Irwin B. Wilson 628

Blood : Some Functional Considerations

by Douglas M. Surgenor 653

An Integrated Concept of Carcinogenesis

by Harold P. Rusch 675

XVI

CHEMISTRY AND VIRAL GROWTH

A. D. HERSHEY, Department of Genetics, Carnegie Institution of
Washington, Cold Spring Harbor, New York

Introduction

The points of contact between biochemistry and the central
mysteries of biology — meaning here the reproduction and func-
tioning of hypothetical determinants of heredity — are necessar-
ily few (3). In recent years the study of viruses has emerged as
one of them, important if only because of its relative isolation.

Work by many investigators with many different viruses has
contributed to this development, not always in tangible ways.
It would be difficult, and perhaps not at present very rewarding,
to trace this genealogy. Even the work with bacterial viruses has
recently split into two parts calling for separate review (5,45).
In this discussion I shall focus attention on what happens when
bacteriophage T2 infects the common intestinal bacterium,
Escherichia coli.

The Reproductive Cycle

Morphological, biochemical, and genetic studies have re-
vealed a developmental cycle that can be represented very
simply. Figure 1 names the recognized stages and processes.
The basic information from which this cycle has been deduced
may be summarized as follows.

A. D. HERSHEY

MATURATION
VEGETATIVE RESTING

PHAGE PHAGE

RESTING PHAGE
Figure 1.

RESTING PHAGE

Extracellular virus is called resting because it is stable and
metabolically inert. Purified preparations of T2 consist of
uniform tadpole-shaped particles with a polyhedral head meas-
uring about 0.1 micron across, and a tail about 0.1 micron long
and perhaps one-fourth as broad (2,67). The surfaces of the
head and tail contain distinct antigenic proteins (36). One
particle contains about 2 X 10~^° 7 of deoxypentose nucleic
acid (DNA) (6,28), which can be expelled by osmotic shock
leaving a proteinaceous ghost that retains the shape and some
of the biological properties of the intact particle (1,21). The
ratio of protein to DNA in the whole particle is about one to one.
The DNA is variously reported to be released in the free state
(23), or combined with protein (62).

The proteins of T2 differ antigenically from the proteins of
the host. The DNA of T2 contains 5-hydroxymethyl cytosine
but no cytosine (69), in which respects it differs from all known
examples of DNA from other organisms. Both facts suggest
that virus and bacterium have pursued separate, though not
independent, evolutionary paths for a long time. Otherwise
these facts can be regarded as analytical aids pure and simple.

INJECTION

When a particle of T2 makes its specific, irreversible at-
tachment to a bacterium (living or dead), the viral DNA passes
into the cell, leaving the empty protein shell outside (27). This
husk may now be seen adhering to the cell by the end of its tail

CHEMISTK.Y AND VIRAL GROWTH

(2), and can be stripped off in a Waring blendor without affect-
ing the subsequent course of the infection (27). A spurious in-
jection, simulating osmotic shock, can be observed when virus
particles react with specific substances of bacterial origin (32),
or even with an ion-exchange resin (52). The material released
in this way consists of fibers 20 A or more in diameter and several
microns per phage particle in aggregate length (32,67). The
visible fibers are destroyed by deoxyribonuclease (32). Their
number has not been counted.

Several of the facts mentioned seem to show that injection
occurs independently of bacterial metabolism. Benzer (4)
finds, however, that an external food supply is required. The
ability of the virus to inject resists multiple lethal doses of ultra-
violet light, but is nearly as sensitive as the infective property to
formaldehyde and ionizing radiations (27,29). At present a
satisfactory m.echanism of injection cannot even be imagined.

Whatever the details of injection, it seems permissible to con-
clude that a resting particle of T2 consists of a protein syringe
that functions chiefly to get the viral DNA into the cell, and that
only the injected materials, and possibly the nonstrippable stub
of the tail (amounting to 20 per cent of the total protein of the
virus particle), participate directly in viral growth.

VEGETATIVE PHAGE

The events that follow injection are strictly dependent on a
source of nutrients external to the cell, except that starved,
infected cells tend to lose their potentiality to yield virus. Given
adequate food and a favorable temperature (usually 30° to
37° C), the normal course of events leads to cellular lysis with
the liberation of 20 to 2000 new viral particles per bacterium,
the yield depending chiefly on nutrient conditions and the length
of time (20 minutes to about 2 hours) during which cellular lysis
can be delayed.

Beginning with the injection, marked changes occur in the
biosynthetic pattern of the cell. These changes will be described
presently, but do not yet help to define vegetative phage, which

A. D. HERSHEY

is a genetic concept. This concept emerges from the following
facts.

Doermann (10) showed that infected cells broken open dur-
ing the first 10 minutes following infection do not yield any infec-
tive virus. This result has since been accounted for by the act of
injection: what goes into the cell is not virus. Doermann's
experiments went much further than this, however. He proved
that genetic determinants, that is, enumerable structures possess-
ing viral specificity, multiplied during the period in which the
cell remained free of infective virus. This proof is summarized
in a recent review (11). The term vegetative phage refers to
these noninfective structures that multiply and produce new
genetic combinations, and must be thought of as the connecting
links between parent and offspring virus. The central problem
of phage growth, therefore, is the problem of structure and func-
tion of vegetative phage.

MATURATION

The process by which vegetative phage is converted into rest-
ing phage is called maturation. From the end result we know
that it involves the reformation of envelope and tail, but we do
not know where it starts ; to answer this it is necessary to know
what vegetative phage is like. It seems possible, as will be dis-
cussed below, that vegetative phage may consist of replicas of the
injected DNA-containing fibrils. If so, maturation calls for
the complete synthesis of envelopes.

Probable intermediate stages in maturation have been identi-
fied. These consist of empty, tailless envelopes (40), accom-
panied by detached tails, or of tailed, but probably DNA-less
envelopes (46). They are found only 2 or 3 minutes before
the phage particles of which they are the presumed precursors,
and so are probably not themselves vegetative phage particles,
which must be formed appreciably earlier. The absence of
DNA is puzzling, and it seems preferable to assume that the
observed structures lose their DNA during isolation, rather than
to imagine that they receive their charge of DNA as a final act

CHEMISTRY AND VIRAL GROWTH

of maturation. The fact that nearly all the DNA in infected
cells, excepting that contained in resting phage particles, is
sensitive to deoxyribonuclease may be significant in this con-
nection. It is perhaps not quite established that the DNA-less
structures are intermediates at all. In view of these uncertain-
ties, little more can be said about them at present.

The time course of maturation is linear, beginning about 10
minutes after infection and continuing until the cells burst.
During this time vegetative phage probably continues to multi-
ply, as indicated by progressive increase in proportion of re-
combinants (41), and continued synthesis of viral protein and
DNA (46,58).

Since maturation is irreversible (22), there is no need to dis-
tinguish between mature intracellular virus and resting extra-
cellular virus. The individual particles probably play a passive
role, if any, in the release of virus from the cell.

The Priming Substance

We have seen that the interesting period in the life of a phage
particle begins when its charge of DNA enters a sensitive bacterial
cell to form vegetative phage. It is a question of considerable
ideological importance (64) whether this charge is exclusively
DNA or contains protein as well. Agreement about it has
not yet been reached at the level of experimental fact (62) and,
what is worse, it is doubtful whether direct analytical methods
can answer it satisfactorily (23). I wish to make the point that
the main biological implications of the injection phenomenon are
largely independent of this specific question. Let us assume al-
most the worst, namely, that the charge that enters the cell con-
sists of nucleoprotein fibers and that these, furthermore, are
functionless until they have made specific attachments to compli-
cated structures in the bacterium. What remains of our repro-
ductive cycle that is biologically unique?

In my opinion three very important features. First, these
hypothetical fibers would have to carry all the intelligence that

A. D. HERSHEY

T2 has acquired during its long evolutionary history (excepting
what it has delegated to the bacterium). In a sense this is the
kind of demonstration that has been the goal of experimental
genetics since its beginning, and one that remains a will-o'-the-
wisp (16,55) if it is not provided by T2. Second, these fibers
must be linear molecules, or very nearly, in which respect they
meet the theoretical requirements of a genetic material (68),
as the visible chromosomes of cells do not. Third, they can be
observed to multiply in a cell that is phylogenetically remote
from their proper origin, as the chromosomes of cells cannot.
The experimental meaning of this feature is brought out by the
fact that both nucleic acids and proteins of virus and host are
distinguishable by qualitative analytical methods. In this last
respect, the uniqueness is not a matter of opinion.

Needless to say, the blendor experiment alone does not quite
establish these claims (23). A genetic function of the nonstrip-
pable tip of the tail has first to be excluded. In itself this may be
a trivial point ; it is not likely that even the staunchest biological
holist would insist that the fertilized egg contain a representative
molecule of every specific substance the organism is capable of
producing. The question becomes important in connection with
the thesis that viral lineage (defined somewhat abstractly in
terms of viral properties that are invariant with respect to varia-
tions in the host) is determined exclusively by a structurally
homogeneous class of linearly differentiated molecules. It is
interesting also because it can be scrutinized by both genetic
and chemical methods. Both tend to exclude the tail-tip as
the primordium from which new membrane protein comes.
I shall give an example of the genetic evidence first.

The genetic control of the specificity of attachment of virus
to bacterium, which means control of the chemical structure of
the tail protein, has been studied in a particularly instructive way
by several investigators, most thoroughly by Streisinger (59).
T2 and T4 are two closely related viruses differing, among other
ways, in their tail proteins. Genetic experiments show that
this difference is determined by a single gene. We may ask

CHEMISTRY AND VIRAL GROWTH

whetlier the determinant of this character is the organ of at-
tachment itself or a different organ, that is, whether the gene
and the agent of its expression are the same or different sub-
stances. The following circumstances show that they are
difTerent.

A single bacterium infected with both T2 and T4 yields a
mixed viral progeny among which the individual particles can be
classified in two ways. First, one can determine whether their
attachment to bacteria shows the specificity of T2 or the spec-
ificity of T4. This test shows that some particles resemble T2
and some T4. Second, one can classify the particles in terms of
the progenies they yield when they multiply individually. By
this test, too, some prove to resemble T2 and some T4. The
remarkable thing about the two tests is that they do not agree:
many particles adsorb like T2 and yield progenies like T4. The
best interpretation of this result is that, owing to the special
conditions prevailing in the mixedly infected bacterium, some of
the particles receive tail protein manufactured for a T2 particle,
together with a tail-protein-determining gene derived from the
T4 lineage, and vice versa. If so the two are different struc-
tures. The genetic results just described show that tail speci-
ficity, as such, is not conserved during reproduction. Analogous
radiochemical results show that tail substance is not conserved
during reproduction. Evidently tail protein is not the seed from
which new tail protein comes. These two results seem to
show that the injected materials direct the complete resynthesis
of tail protein.

I have cited one item of a considerable body of genetic in-
formation (24) leading to the conclusion that the genetic mate-
rial of T2 consists of linear structures. All the chemical evidence
implicates DNA as the chief constituent of these structures. If
one wishes to assume that DNA is the prime hereditary material,
justification can be borrowed from the beautiful work of the past
decade with bacterial transforming systems (31), or it can be
sought directly, as will be discussed below.

7 ..;/--•: .-

A. D. HERSHEY

Metabolic Effects of Infection

Soon after infection, bacteria stop making respiratory and
other enzymes (9,51) and fail to respond to inductors of adaptive
enzymes (49), as if the metabolic equipment of the cell were
frozen at the time of infection. Protein synthesis as a whole
continues unabated, however. By contrast, the synthesis of
bacterial RNA and DNA stops abruptly, being replaced by the
synthesis of viral DNA (6). The latter point has been con-
firmed by transferring infected cells into C^'^-glucose, when it is
found that no G^^ enters DNA-cytosine (29). The bacterial
DNA that was formed before infection disappears afterward, in
contrast to bacterial RNA and protein which remain com-
paratively inert. Cytological changes suggest that the bacterial
nuclei are quickly dispersed (44) .

These facts have led to the following generalizations. (7)
Infection causes a shift from the synthesis of characteristic
bacterial constituents to the synthesis of characteristic viral con-
stituents, without major substitutions in enzymatic equipment
(9). (2) Viral genes replace bacterial genes: infection is
parasitism at the genetic level (44). For the present these
two ideas are no more than colorful, rather clouded, state-
ments of fact but, as Luria (44) has pointed out, it may be useful
to look for connections between them.

Structure of Vegetative Phage

I have summarized reasons for believing that the sub-
stance derived from a single viral particle and directly initiating
viral growth consists of one or more linear molecules. If this is
so we want to know their number and function, and whether
they multiply before or after forming more complex struc-
tures. Structure can be got at in several ways besides the
attempt to isolate vegetative phage, and some of these are
surveyed below. About function only one question can be asked
in advance of information about structure. It is concerned with

8

CHEMISTRY AND VIRAL GROWTH

the conservation of materials during multiplication, which in
turn leads back to questions about structure and function.

THE NUMBER OF LINEAR MOLECULES

The number of structures injected into the bacterium is not
known beyond the fact that one phage particle contains about
2 X 10~^° 7 of DNA, which corresponds to about 20 molecules
in the isolated form (19). Presumably the number could be
determined either by physical or genetic methods. The follow-
ing genetic experiment illustrates a principle of wide application
to questions of this kind.

T2 contains at least three regions of genetic material that
assort independently during genetic recombination (24). If a
bacterial cell is infected with two phage particles genetically
marked in one of these regions, and if the infections are spaced
by an interval of 2 minutes or more, the second particle will
generally not contribute its genetic marker to the progeny (15).
At least some of the DNA of the second particle enters the cell,
because about half of the DNA of the superinfecting particles is
quickly split into acid-soluble materials (38). The mechanism
of exclusion is otherwise unknown, but one can ask whether the
unit of exclusion is the genetically intact phage particle, or
something smaller. To answer this, one infects bacteria in
succession with two phage particles marked in two of the inde-
pendently assorting regions of genetic material, and examines
yields from single bacteria to see whether one of the two markers
can contribute while the other is excluded. A single test of this
kind (26) showed that the two markers were always excluded
together. For what it is worth, this result suggests that the
genetic unit called vegetative phage (or its precursor) is also a
structural unit.

PHYSICAL STRUCTURE

The composition of vegetative phage is difficult to investi-
gate, first because there is no unambiguous way to identify
interesting structures if they could be isolated ; second because

A. D. HERSHEY

any method used to break open the infected cell might alter im-
portant structural relations.

Direct microscopy of infected cells (50) has not revealed any
characteristic intracellular structures that might be vegetative
phage particles. Similarly, examination of extracts of infected
bacteria (40) brought to light only normal cell components,
resting phage particles, and empty, tailless membranes that have
been interpreted as early products of the maturation cycle.
Watanabe, Stent, and Schachman (63) labeled viral precursor
DNA by infecting cells with P^^-containing phage, and then sub-
jected extracts prepared at various times to centrifugal analysis.
Citrate was used to inhibit bacterial deoxyribonculease, but
otherwise the conditions of lysis may have been favorable for
enzymatic degradation. They found that most of the viral
precursor DNA in the lysates sedimented like free nucleate.

None of these results either contradicts or justifies the as-
sumption that vegetative T2 consists of free molecules of DNA.

VIRAL PRECURSOR DNA

Early tracer experiments showed that the phosphorus used
to make viral progeny comes from three distinct sources:
materials assimilated by the bacterium before infection (8),
materials assimilated after infection (8), and parental materials
(53). Weed and Cohen (66), Stent and Maal0e (58), and
French et al. (18) found that atoms derived from any of these
three sources entered unequally into the viral progeny formed at
successive times, suggesting the existence of a precursor pool
containing a limited amount of phosphorus. These facts
prompted a search for viral precursor DNA, which was greatly
facilitated by the interim discovery that the DNA of T2 contains
the distinctive constituent 5-hydroxymethyl cytosine (69).
This search brought to light the following information.

Cytosineless DNA, that is, DNA having the composition of
the DNA of viral particles, begins to form in infected bacteria
promptly after infection, and already measures roughly 50 phage

10

CHEMISTRY AND VIRAL GROWTH

equivalents per bacterium 10 minutes after infection, at which
time infective virus particles begin to form. Also at later times,
infected cultures contain more cytosineless DNA than can be ac-
counted for by infective particles, and the amount of surplus
DNA remains approximately constant at 50 to 100 phage
equivalents per bacterium (28). Tracer experiments (22) show
that this surplus DNA is the principal immediate precursor of
the particles. First, because phosphorus incorporated early into
the surplus DNA disappears from it later on, as if it were being
transferred to infective viral particles by an efficient, irreversible
process. Second, because the kinetics of the transfer, whatever
the source of labeled phosphorus, calls for a precursor pool con-
taining 50 to 100 phage equivalents of phosphorus per bacterium,
in agreement with direct measurements of the amount of surplus
DNA. This agreement shows that the precursor pool is sampled
more or less at random when a phage particle is formed. In
this sense it must contain chiefly a single precursor, as opposed to
a sequential series of precursors. This important distinction,
based on analytical methods admittedly not very exact, is re-
called below by the term unitary pool.

IDENTITY OF PRECURSOR DNA AND VEGETATIVE PHAGE

The idea of vegetativ^e phage is based on genetic experiments
pointing to the existence of a mating pool or, more generally, a
genetic precursor pool. The chemical experiments reveal a
pool of viral precursor DNA. These two pools can be com-
pared with respect to unity, size, and time of formation, and
should resemble each other in all these respects if vegetative
phage is or contains the viral precursor DNA.

Strictly speaking, the pool of precursor DNA cannot be a
unitary pool, because at intermediate stages during maturation,
any DNA contained in the immature particles would belong
neither functionally to a randomly sampled pool, nor analyti-
cally to mature phage particles. The effect of this complication
would be to cause the pool of precursor phosphorus, assayed

11

A. D. HERSHEY

kinetically, to be smaller than the pool of precursor DNA meas-
ured directly. The fact that no such discrepancy is found
means that the time precursor DNA spends in the maturation
cycle is short compared to the washout time of the pool (about
8 minutes). However, since the methods are far from precise,
and viral growth in different cells is not synchronized, only ex-
treme alternatives can be excluded; for example, linear multi-
plication such that each offspring particle enters its maturation
cycle directly at birth. To this extent the chemical findings
suggest a geometric mechanism of synthesis of DNA. More to
the point, however, is the likelihood that the amount of precursor
DNA determined analytically should exceed appreciably the
amount contained in the genetically defined vegetative struc-
tures.

The chemical facts show that the infected cell contains 50
to 100 phage equivalents of viral precursor DNA, much of
which belongs to a unitary pool in the sense that it is sampled at
random for the maturation of infective particles. This pool is
largely filled 10 minutes after infection, at the time maturation
begins.

What are the characteristics of the genetic pool? Luria's
(43) analysis of viral mutation showed that genetic deter-
minants increase geometrically; this is probably equivalent to
saying that they share a pool from which samples are drawn at
random during maturation. This was shown more directly by
Visconti and Garen (61), who found that genetic markers con-
tributed by superinfecting phage, entering the bacterium 7 or 8
minutes after primary infection, were sampled from the same
pool with markers derived from the primary infection. More-
over, the kinetics of genetic recombination suggest that the
mating pool has already reached its maximal size, estimated at
30 vegetative particles per bacterium, at the time maturation
begins (41). Thus the genetic pool and the viral precursor DNA
pool are similar with respect to unity, size, and time of formation.
It is a reasonable though not quite necessary inference that the
precursor DNA is contained in the genetic pool.

12

CHEMISTRY AND VIRAL GROWTH
VIRAL PRECURSOR PROTEIN

Cohen (7) found that there was no interruption of protein
synthesis at the time of infection, and that protein continued to
accumulate until viral growth ceased. His findings have been
confirmed by measuring the assimilation of S^^ into acid-in-
soluble materials after infection (29). These measurements
show that bacteria synthesize protein at nearly identical rates
before, during, and for some time after infection. When does
the synthesis of viral protein start?

This question has been attacked in two ways. Luria (44)
and his collaborators studied the formation of viral antigens in
infected cells. Their results led to the conclusion that viral
antigens are formed relatively late during the process of viral
growth, as though they were products of the maturation cycle.
If this is so, the protein synthesized immediately after infection is
not virus specific.

Hershey et al. (29) analyzed viral precursor protein by
tracer methods. They fed S^^ to infected bacteria for intervals
of 5 minutes, and measured the amount of S^^ subsequently
incorporated into virus and total intrabacterial protein, re-
spectively. The amount incorporated into protein of all kinds
was virtually independent of the period of assimilation tested,
but the proportion of this incorporated into virus varied greatly.
For the period to 5 minutes after infection, only 13 per cent
went into virus; for 5 to 10 minutes, 25 per cent; for later times,
50 to 60 per cent. This means that infected bacteria synthesize
two classes of protein, one viral precursor and one not, and that
the maximal rate of precursor protein synthesis is not reached
until shortly before the beginning of maturation (15 minutes
after infection under the conditions of these experiments).
Kinetic analysis of sulfur assimilation during the period in which
virus was accumulating at a linear rate showed that the interval
between assimilation of sulfur atoms and their incorporation into
unspecified protein averaged about 2 minutes. The interval
between assimilation and incorporation into mature virus aver-
aged about 10 minutes. Hence labeled viral precursor protein

13

A. D. HERSHEY

persists in the infected cell for an average of 8 minutes. Labeled
viral precursor DNA, under the same conditions, persists about
14 minutes. This difference seems to show that the cell con-
tains more precursor DNA than precursor protein, and therefore
that the DNA destined for a given phage particle is formed some-
what earlier than the protein destined for that particle.

Both the serological and tracer methods are consistent with
the idea that protein synthesis is a terminal phase in the forma-
tion of the phage particle, but neither has yielded satisfactory
proof of this idea. A more promising approach might be to
look for conditions under which maturation is inhibited while
DNA synthesis proceeds, or conditions permitting DNA syn-
thesis but not protein synthesis. Cultures containing proflavine
furnish an example of the first class. This substance appears to
block only a late step in maturation and permits both protein
and DNA synthesis (44). One inhibitor of protein synthesis has
been tested. Cohen (7) finds that 5-methyl tryptophan blocks
both protein and DNA synthesis in infected bacteria. This
suggests either that vegetative phage contains protein or that
DNA synthesis is dependent on the synthesis of protein for other
reasons. The attempt to confirm or refute this conclusion by
studying the effects of specific metabolic blockade seems at
present to offer the most general method of defining the com-
position of vegetative phage.

RNA ECONOMY IN INFECTED BACTERIA

Cohen (8) showed that the amount of RNA in infected
bacteria remains constant from the moment of infection, and
that not more than 2 to 9 per cent of its phosphorus is replaced by
newly assimilated phosphorus during 60 minutes of viral growth.
Manson (48) confirmed the latter result by a more rigorous
method ; in his experiments replacement up to 5 per cent of the
total RNA could not be excluded because of the presence of
uninfected bacteria in the cultures. In order to see what these
results mean it is necessary to make some quantitative com-
parisons.

14

CHEMISTRY AND VIRAL GROWTH

The amount of DNA in an uninfected bacterium growing in
glucose-ammonia medium averages about 40 phage equivalents
(8 X 10~^ 7). After infection, viral precursor DNA is synthe-
sized at the rate of about three phage equivalents per bacterium
per minute and accumulates to roughly 40 equivalents per
bacterium. The amount of bacterial phosphorus (assimilated
before infection) that is used to make viral DNA measures about
40 phage equivalents. The total amount of preassimilated RNA
phosphorus per bacterium is nearly 400 phage equivalents.

If 5 per cent of the total RNA were undergoing moderately
rapid turnover, E.NA synthesis could easily keep pace with DNA
synthesis, without having been detected in the experiments cited
above. It has been suggested that this happens (22).

If 10 per cent of the total preassimilated RNA were con-
verted into viral DNA, this source would account for an ap-
preciable part of the observed use of preassimilated materials by
the virus, and would not readily be detected as a significant
decrease in RNA content of the culture. As a matter of fact,
one does observe a significant decrease in acid-insoluble RNA
when measured in terms of preassimilated isotope, and indirect
evidence suggests that some of the purine and pyrimidine carbon
enters DNA (29).

These remarks are intended to show that an active role of
RNA in the growth of T2 has not been entirely excluded. Since
the T2-infected bacterium is the only known biological system in
which synthesis of RNA during growth may not occur, more
penetrating experiments designed to test this possibility are
called for.

MATERIALS CONSERVED DURING REPRODUCTION

A special method of defining the composition of vegetative
phage — perhaps a minimum composition, perhaps an irrelevant
one — consists in tracing those structures that pass intact from
parental to offspring phage. It is now generally agreed that
only the constituents of the parental DNA are transferred (17,

15

A. D. HERSHEY

27,35). It is also agreed that the efficiency of transfer never
exceeds 40 or 50 per cent (18,22,65). The mechanism of
transfer, and therefore its significance, remains undecided. The
question of mechanism is conveniently discussed in terms of
alternative explanations for the low efficiency of transfer.

7. The low efficiency of transfer may result from losses
occurring in some but not all the infected bacteria, either be-
cause preparations of phage always contain inviable particles
or because for other reasons some of the bacteria yield few or no
viral progeny. Although losses from both causes undoubtedly
occur and cannot at present be measured satisfactorily, most
observers have considered this explanation inadequate. Losses
of this type, of course, have nothing to do with the mechanism of
transfer.

2. The loss may be an essential feature of viral growth,
but occur before multiplication starts. This would imply that
about half the parental DNA goes into vegetative phage, and the
other half serves a different, presumably nongenetic, function.
This possibility has been tested in two ways. Maal0e and Wat-
son (47) measured the efficiency of transfer of P^^ during two
successive cycles of growth, and found only the usual transfer of
40 per cent during the second cycle. This showed that the
transferred atoms from the parental DNA are incorporated into
both transferred and nontransferred parts of the DNA of the
progeny. Hershey et al. (29) found that the four bases of the
DNA of C^^-labeled T2 are transferred with equal efficiency.
These two results show that the loss is random, that is, if the
parental DNA splits into two parts that perform different func-
tions, and if only one part is conserved among the progeny, the
choice is probably made between identical parts. In this form
the idea of functional differentiation within the viral DNA is
unattractive but cannot be considered disproved.

3. All the parental DNA may enter vegetative phage
where it is subject to a succession of small losses during replica-
tion. The losses would have to amount to 5 or 10 per cent per
generation to account for an observed transfer of 50 per cent

16

CHEMISTRY AND VIRAL GROWTH

Losses of this magnitude might well be expected on general
grounds. An attempt was made to test this idea by feeding P^^
to infected cells during the first 4 minutes after infection (22).
It was found that the labeled DNA content of the infected cells
rose to a maximum during the first 25 minutes, and that 70 to
90 per cent of this was eventually incorporated into virus. This
high efficiency fails to exclude the hypothesis under consideration
because of the slow entry of P^^ into viral precursor DNA. It
does show, however, that maturation is not the step that limits
the efficiency of transfer from parents to offspring.

4. Finally, perhaps only a small part or none of the
parental DNA goes into vegetative phage directly. Half of it,
after playing some unspecified role, could then be broken down
and fed into the general pool of DNA precursors. This idea was
at first supported by Kozloff's finding that parental DNA
phosphorus is transferred more efficiently than parental DNA
nitrogen (34,35). However, subsequent and technically su-
perior experiments showed equal transfer of phosphorus and
purine carbon (65), and equal transfer of the four bases (29).
Moreover, the intermediates must be nucleotides or larger frag-
ments, because free bases or nucleosides, which compete effec-
tively with CO2 (33) or glucose (20) as a source of viral DNA
carbon, fail to compete during the transfer from C^Mabeled
virus (29). In view of the seeming contradictions, Kozloff"'s
result ought to be reinvestigated by the isolation of doubly
labeled nucleotides. In the meantime, the bulk of evidence is
opposed to, but does not disprove, the idea of indirect use of
parental materials.

At the moment, enumeration of these alternatives (the list
is not exhaustive) is of value chiefly to show that the relatively
low efficiency of transfer does not permit any conclusions about
the mechanism of transfer. In addition, it may be pointed out
that the transfer of purine and pyrimidine carbon (except cyto-
sine) from bacterial DNA to viral DNA is nearly or quite per-
fectly efficient (29). Since this may surely be taken as an ex-
ample of "indirect" transfer, the assumption that the transfer

17

A. D. HERSHEY

from parents to progeny involves breakdown and resynthesis of
DNA leaves the low efficiency of the process unexplained.

All the evidence short of isolation and identification leads
to the conclusion that vegetative phage contains large amounts
of DNA. The possibility that DNA is its principal functional
component is suggested by the analysis of resting phage particles,
by examination of the injection mechanism, and by the com-
position of the material conserved during viral growth. A more
general confirmation of this idea has not yet proved possible but
promises to emerge from considerations to which we now turn.

CORRELATION OF MATERIAL AND GENETIC CONTINUITY

A direct correlation between the transfer of genetic markers
and of labeled DNA from parental to offspring virus would not
only answer the question about biochemical pathways but also
rule out the unpleasant possibility that most of the viral DNA
resembles more closely in function the white of an egg than it
does the nuclear apparatus of an ovum. Such a demonstration
would, in fact, reduce genetic questions about T2 to purely bio-
chemical questions about structure and function of DNA. Koz-
loff (34) recognized this very early but was unable to find any
correlation between genetic and biochemical results.

Kozloff"'s basic experiment is the following. Bacteria were
simultaneously infected with isotopically labeled phage, pre-
viously exposed to ultraviolet light or x-rays, and with un-
labeled, unirradiated phage. Previous experiments of this type
with genetically marked phage had shown that the genetic
contribution from the irradiated phage to the mixed yield of
virus could be suppressed by a sufficient dose of radiation.
Kozloff reasoned that the contribution of isotope ought also to be
suppressed if the material transfer had any direct connection
with genetic function. Since he did not find any appreciable
effect of irradiation on isotope transfer, Kozloff' (34,35) concluded
that the material transfer from parents to progeny probably had
nothing to do with genetic function, and that it might very well
involve breakdown to nonspecific fragments en route. Watson

18

CHEMISTRY AND VIRAL GROWTH

and Maal0e (65) and French et al. (18) described similar results
without, however, subscribing to KozlofF's conclusion.

Subsequent experiments (29) have shown that the earlier
results can be questioned on strictly technical grounds. For
this purpose it is necessary to distinguish between the effects of
ultraviolet light and ionizing radiations.

Phage inactivated by ionizing radiations are found to be
largely incapable of injecting their DNA into bacterial cells,
although their ability to attach to the cells is not affected.
Following mixed infection with irradiated and unirradiated
virus, the viral yield after cellular lysis is heavily contaminated
with the original particles of irradiated virus. For this reason it
is not possible to measure isotopic contribution from the ir-
radiated virus to the progeny of the mixed infection (29).

The early experiments with phage inactivated by ultra-
violet light are questionable for two reasons. First, the genetic
potency of ultraviolet-killed phage in mixed infections is only
now being investigated in a quantitative fashion (12) and proves
to be much greater than previously supposed. Second, the
isotopic contribution is not independent of the radiation dosage,
as Kozloff believed, but falls slowly and continuously with in-
creasing doses (29). This effect cannot be attributed solely to
loss of the ability to inject ; the latter property is extremely re-
sistant to ultraviolet light. The careful experiments that will be
required to assess the significance of the correlation between the
behavior of genetic and isotopic tracers remain to be done, but
it is clear that a correlation exists.

A very clear qualitative correlation can be demonstrated for
the following special situation. As previously mentioned, phage
particles attaching to bacteria that have already been infected
2 minutes or so earlier fail to contribute genetic markers to the
progeny, and half the DNA of the superinfecting particles is
quickly split into acid-soluble material. At first it was thought
that the chemical breakdown might be the cause of the genetic
exclusion. However, French et al. (18) found that no chemical
breakdown occurs when the bacterial deoxyribonuclease is

19

A. D. HERSHEY

inhibited by streptomycin, and that even under these conditions
there is no transfer of isotope from superinfecting phage to the
offspring of the primary infection.

We have recently studied superinfection a Httle further, in-
hibiting bacterial deoxyribonuclease when desired by reducing
the concentration of magnesium in the cultures to 10~^ M (29).
At this low magnesium concentration, injection and growth of
the primary infecting phage, and the transfer of its phosphorus
to viral offspring, are normal. Independently of magnesium
concentration, superinfecting phage injects only half its DNA
(or half the particles inject), and both genetic and isotopic
markers derived from it are excluded from the viral yield. The
half that is injected may or may not be broken down, depending
on the magnesium concentration. When the bacterial de-
oxyribonuclease is inhibited, superinfecting phage is excluded
from genetic participation in growth and half its DNA enters
the cells and remains intact without metabolic participation in
viral growth. Unfortunately, the significance of this parallel is
doubtful because the mechanism of exclusion is unknown.

RADIOGENETIG STRUCTURE ANALYSIS

Viral radiogenetics began some years ago when Luria (42)
proposed that phage particles inactivated by ultraviolet light
could be revived by a process of genetic substitution. His idea
was appealing, first, because it promised a test of the much older
idea that radiations produce localized genetic damages to biologi-
cal materials and, second, because it led to rather special as-
sumptions about the mechanism of viral growth. His idea sub-
sequently lost favor without really being disproved, partly be-
cause Dulbecco (13) showed that the inactive particles could
also be revived by something resembling repair, partly because
the quantitative predictions of the substitution hypothesis failed
at high radiation doses (14), partly because the early radiogenetic
experiments suffered from what is best described as lack of
faith.

Stent (57) and Doermann (12) have recently revived the

20

CHEMISTRY AND VIRAL GROWTH

idea of reactivation by genetic substitution. Their experiments
are of tlie following type. Bacteria are infected with two kinds of
phage differing by several genetic markers, one kind carrying
radiation damage and one not. In Stent's experiments, the
damages result from decay of P^^ incorporated into the virus
during prior growth. In Doermann's experiments they are
produced by irradiating phage with ultraviolet light. In both
instances the damage can be ascribed plausibly to localized
effects on the viral DNA, and both types of damage show the
same behavior in radiogenetic experiments.

The following description refers specifically to Doermann's
experiments. He uses T4 particles irradiated with ultraviolet
light until they are unable individually to infect a bacterium in
the usual way. Such particles may be called dead, and the
radiochemical process producing them may be called primary
killing. The dead particles can be revived in several ways. In
what follows we are concerned with only one of them, called
cross-reactivation. It shows the following characteristics.

7. Yields from individual bacteria infected with one or
more live plus one dead phage particle frequently show some but
not others of the genetic markers introduced with the dead
particle. The response of the individual markers to varying
radiation dosage corresponds to a target volume that is 4 per cent
of the target volume for primary killing.

2. Markers known from genetic tests to be unlinked are in-
activated independently of each other.

3. For moderate radiation dosages, markers derived from
the irradiated phage particles are either absent entirely, or
appear in numerous copies in yields from individual bacteria.

4. Inactivation of genetically linked markers occurs in a
correlated manner.

Doermann interprets these facts in the following way.
Most of the radiation damage results from localized photo-
chemical reactions in genetic material; otherwise correlated
inactivation of unlinked markers would be observed. Un-
damaged pieces of genetic material are rescued by recombina-

21

A. D. HER.SHEY

tion away from damaged pieces, since both the rescue and au-
thentic recombination are subject to the same Hnkage rules.
The photosensitive target measured in these experiments is the
average size of pieces that can be rescued by recombination.
It measures one twenty-fifth of the total genetic material and is
large compared to the size of the individual markers, since
linked markers tend to be rescued together.

Stent's conclusions are consistent with these and lead further
in one direction. Since the primary lethal damage produced by
assimilated P^^ results from one out of twelve atomic disintegra-
tions (30), at least this fraction of the DNA must be associated
with genetic material. The fraction must, in fact, be larger,
because the radiochemical efficiency is dependent on tempera-
ture (56).

These methods are evidently capable of yielding rather pre-
cise descriptions of genetic material, possibly in physical terms,
certainly in radiochemical terms that can be compared with
genetic descriptions. Moreover, like the earlier results of Luria,
they call for interesting assumptions about the mechanism of
viral growth. The problem is to explain how rescue by re-
combination is achieved without greatly setting back the multi-
plication of the rescued piece relative to comparable genetic
material that does not require rescue. Such a setback does, in
fact, appear when the rescue is made more difficult by high
radiation dosages. In this situation we come face to face with
the problem of structure and function of vegetative phage.
Since both the facts and ideas bearing on the interpretation of
the radiogenetic experiments are changing rapidly, it would be
rash to predict how they are to be reconciled with each other.

Relation between Virus and Cell Nucleus

There are a number of indications from other phage-
bacterium systems that some bacteriophages engage directly
with components of the bacterial nucleus (5,29,37,45,70).
These are only beginning to be worked out and cannot be dis-

22

CHEMISTRY AND VIRAL GROWTH

cussed here. For T2 none of the biological tests of this idea are
applicable, but chemical tests are.

As already mentioned, the first cytochemical effect of the
infection is a rapid dispersal of the bacterial chromatin (44).
By chemical analysis, one observes a more or less complete dis-
appearance of cytosine-containing DNA, and the atoms of this
DNA are rapidly incorporated into viral DNA. The conver-
sion appears to be highly efficient. The direct analytical re-
sults indicate a phosphorus transfer of 50 per cent (22,54), sub-
ject to the assumption that all the transferred phosphorus comes
from bacterial DNA. This becomes nearly 100 per cent when
corrected for the fact that only half the phosphorus of the bac-
terial DNA fraction, but nearly all the phosphorus in isolated
phage, is really DNA phosphorus (28). Similar conclusions
follow when the transfer of C^ ^-labeled bacterial precursors is
measured. All the DNA purine and pyrimidine carbon (ex-
cept cytosine), and all or most of the carbon of specifically
labeled bacterial DNA-thymine, appears either in viral DNA or
in residual bacterial DNA in infected bacteria (29). The con-
version is more or less complete, and occupies the first 30 minutes
of viral growth.

One would like to interpret these facts as an illustration of
Luria's idea of parasitism at the genetic level. Before doing so,
two questions have to be answered. First, does the virus cause
the breakdown of bacterial DNA, or merely block its resyn-
thesis? Second, is bacterial DNA the only characteristic bac-
terial substance that is used to make virus?

The first question has been answered by showing that
bacterial DNA, and indeed characteristic bacterial substances in
general, do not turn over at rates comparable to rates of syn-
thesis in growing bacteria (25). Moreover, bacterial RNA does
not turn over appreciably in infected bacteria (8,22,48). State-
ments made about turnover are, of course, intelligible only in
terms of actual experiments. Two examples will suffice here.
The presence of thymidine in unlabeled culture micdium during
viral growth will specifically suppress the conversion of labeled

23

A. D. HERSHEY

bacterial DNA- thymine into viral DNA-thymine (29). The
presence of thymidine in unlabeled culture medium during
bacterial growth will not suppress the conservation of labeled
bacterial DNA-thymine (25). In fact, the amounts of radio-
carbon in all the DNA-bases remain constant during 6 hours of
growth at one generation per hour in media containing any of a
number of competitive substrates. One concludes that infection
stimulates a decomposition of bacterial DNA that is different in
rate or kind from any that may occur during bacterial growth.
It may be added that the decomposition in infected bacteria is
not prevented at concentrations of magnesium too low to permit
the action of intracellular deoxyribonuclease as tested in another
way (29), so that the effect may not be due merely to the activa-
tion of this enzyme (35).

Bacterial DNA is not the only source of preassimilated
material available for the synthesis of viral substance. There is
another source of purines and pyrimidines, probably RNA (29).
There is also a small amount of preassimilated sulfur, which may
or may not be derived from protein, that can be used to make
viral protein (29). However, these materials are used at rates
altogether negligible compared to the rate of conversion of
bacterial to viral DNA.

It is reasonable to conclude that the preferential breakdown
and re-use of bacterial DNA indicates some close relationship,
structural or spatial or both, between the two kinds of DNA in
infected bacteria.

T2 and Other Organisms

At the present time, if one wished to invent a genetic mate-
rial, one would almost certainly choose a DNA constructed
along the lines proposed by Watson and Crick (64). A few
organisms have actually chosen DNA, the evidence being very
good for several bacteria (31), and what I have related above
for T2.

These facts immediately raise two questions. The first is

24

CHEMISTRY AND VIRAL GROWTH

whether all organisms have chosen DNA for a genetic material,
which might imply that the choice had been made only once.
At the present time this seems unlikely, because a number of
plant viruses probably lack DNA. Evidently one of the tasks of
the plant virologists is to make very sure of this.

The second question is the following. In an organism that
does make use of DNA, is DNA the sole agent of genetic con-
tinuity, or is genetic specificity passed from substance to sub-
stance like the token in a relay race? The answer to this ques-
tion can only be guessed, but one can hope to get it from T2, as
I have tried to show.

More generally, one wonders what connection there can be
between the linear molecules that pass from pneumococcus to
pneumococcus, or from T2 to its host, and the relatively enor-
mous chromosomes of most cells. A similar question puzzled
geneticists, of course, long before anything was known about
inheritance in bacteria or viruses (68). I think it is doubtful
whether T2 can throw any light on this question except, perhaps,
by renewing interest in it.

More specifically, what connection can there be between
genetic recombination in T2 and reciprocal crossing-over be-
tween chromosomes? Here the answer may be none (60).
Not only because the discrepancy in size forbids analogy; also
because recombination in T2 does not seem to be reciprocal
(39).

One suspects that in T2 the mechanical and biochemical
bases of inheritance are reduced to their simplest terms; indeed,
this is what one hopes. If this proves to be true, it will be unfair
to complain that information about this organism cannot be
applied directly to more complicated ones.

Conclusion

In studying viral growth, one necessarily studies many
biochemical problems simultaneously. The main ones involve
interrelationships among deoxyribonucleic acids, ribonucleic

25

A. D. HERSHEY

acids, and proteins, during the synthesis of at least two of these
classes of substance. But to state them in this way is to ignore
the unique problems presented by T2. These depend on the
fact that the material thread connecting parental and offspring
virus seems to be an analytically recognizable deoxyribonucleic
acid and nothing else. The real biochemical problems for the
present are the relation of this substance to viral inheritance, on
the one hand, and the degree of autonomy of nucleic acid repli-
cation, on the other. No outstanding solution to these problems
has come from the work with T2. Yet many experiments that
might have yielded very depressing results have failed to do so.
On the contrary, their net effect is to increase the hope one
attaches to the many penetrating experiments that remain to
be done.

References

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2. Anderson, T. F., Cold Spring Harbor Symposia Quant. Biol., 18, 197 (1953).

3. Beadle, G. W., in D. E. Green, ed.. Currents in Biochemical Research.
Interscience, New York-London, 1946.

4. Benzer, S., personal communication.

5. Bertani, G., Cold Spring Harbor Symposia Quant. Biol., 18, 65 (1953).

6. Cohen, S. S., Cold Spring Harbor Symposia Quant. Biol., 12, 35 (1947).

7. Cohen, S. S., J. Biol. Chem., 174, 281 (1948).

8. Cohen, S. S., J. Biol. Chem., 174, 295 (1948).

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10. Doermann, A. H., Carnegie Inst. Wash. Yr. Bk., 47, 176 (1948).

11. Doermann, A. H., Cold Spring Harbor Symposia Quant. Biol., 18, 3 (1953).

12. Doermann, A. H., Symposium on Mechanisms of Genetic Recombina-
tion, Oak Ridge Natl. Lab., April, 1954, J. Cellular Comp. Physiol, 45,
Suppl. 2, 51 (1955).

13. Dulbecco, R., J. Bacterial., 59, 329 (1950).

14. Dulbecco, R., J. Bacterial., 63, 199 (1952).

15. Dulbecco, R., J. Bacterial., 63, 209 (1952).

16. Ephrussi, B., Nucleo-cytoplasmic Relations in Microorganisms. Clarendon
Press, Oxford, 1953.

17. French, R. C, J. Bacterial., 67, 45 (1954).

18. French, R. C, A. F. Graham, S. M. Lesley, and C. E. van Rooyen,
J. Bacterial, 64, 597 (1952).

26

CHEMISTRY AND VIRAL GROWTH

19. Garen, A., and E. Reichmann, personal communication.

20. Graham, A. F., and L. Siminovitch, Can. J. Microbiol., in press.

21. Herriott, R. M., J. BacterioL, 61, 752 (1951).

22. Hershey, A. D., J. Gen. Physiol., 37, 1 (1953).

23. Hershey, A. D., Cold Spring Harbor Symposia Quant. Biol., 18, 135 (1953).

24. Hershey, A. D., Advances in Genet., 5, 89 (1953).

25. Hershey, A. D., J. Gen. Physiol., 38, 145 (1954).

26. Hershey, A. D., unpublished.

27. Hershey, A. D., and M. Chase, J. Gen. Physiol., 36, 39 (1952).

28. Hershey, A. D., J. Dixon, and M. Chase, J. Gen. Physiol., 36, 111 (1953).

29. Hershey, A. D., A. Garen, D. Fraser, and J. D. Hudis, Carnegie Inst.
Wash. Yr. Bk., 53, 210 (1954).

30. Hershey, A. D., M. Kamen, J. Kennedy, and H. Gest, J. Gen. Physiol.,
34, 305 (1951).

31. Hotchkiss, R. D., in Dynamics of Virus and Rickettsial Infections. Blakiston,
New York, 1954.

32. Jesaitis, M. A., and W. F. Goebel, Cold Spring Harbor Symposia Quant.
Biol., 18, 205 (1953).

33. Koch, A. L., F. W. Putnam, and E. A. Evans, Jr., J. Biol. Chem., 797,
105 (1952).

34. Kozloff, L. M., J. Biol. Chem., 794, 95 (1952).

35. Kozloff, L. M., Cold Spring Harbor Symposia Quant. Biol., 18, 207 (1953).

36. Lanni, F., and Y. T. Lanni, Cold Spring Harbor Symposia Quant. Biol.,
78, 159 (1953).

37. Lederberg, E. M., and J. Lederberg, Genetics, 38, 51 (1953).

38. Lesley, S. M., R. C. French, A. F. Graham, and C. E. van Rooyen,
Can. J. Med. Sci., 29, 128 (1951).

39. Levinthal, C, Genetics, 39, 169 (1954).

40. Levinthal, C, and H. W. Fisher, Cold Spring Harbor Symposia Quant.
Biol., 18, 29 (1953).

41. Levinthal, C, and N. Visconti, Genetics, 38, 500 (1953).

42. Luria, S. E., Proc. Natl. Acad. Sci. U. S., 35, 253 (1947).

43. Luria, S. E., Cold Spring Harbor Symposia Quant. Biol, 76, 463 (1951).

44. Luria, S. E., in Dynamics of Virus and Rickettsial Infections. Blakiston,
New York, 1954.

45. Lwoff, A., BacterioL Revs., 77, 270 (1953).

46. Maal0e, O., and N. Symonds, J. BacterioL, 65, 177 (1953).

47. Maal0e, O., and J. D. Watson, Proc. Natl. Acad. Sci. U. S., 37, 507 (1951).

48. Manson, L. A., J. BacterioL, 66, 703 (1953).

49. Monod, J., and E. WoUman, Ann. inst. Pasteur, 73, 937 (1947).

50. Mudd, S., J. Hillier, E. H. Beutner, and P. Hartman, Biochim. et Biop/iys.
Acta, 70, 153 (1953).

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51. Pardee, A. B., and I. Williams, Ann. inst. Pasteur, 84, 117 (1953).

52. Puck, T. T., Cold Spring Harbor Symposia Quant. Biol., 18, 149 (1953).

53. Putnam, F. W., and L. M. Kozloff, J. Biol. Chem., 782, 243 (1950).

54. Siddiqi, M., L. M. Kozloff, F. W. Putnam, and E. A. Evans, Jr., J.
Biol. Chem., 799, 165 (1952).

55. Sonneborn, T. M., Proc. Intern. Congr. genet. 9th Congr. Bellagio, Italy,
1953.

56. Stent, G. S., Cold Spring Harbor Symposia Quant. Biol., 78, 255 (1953).

57. Stent, G. S., Proc. Natl. Acad. Sci. U. S., 39, 1234 (1953).

58. Stent, G. S., and O. Maal0e, Biochim. et Biophys. Acta, 70, 55 (1953).

59. Streisinger, G., Thesis, University of Illinois, Urbana, 1954.

60. Sturtevant, A. H., Symposium on Mechanisms of Genetic Recombina-
tion, Oak Ridge Natl. Lab., April, 1954, J. Cellular Comp. Physiol.,
45, Suppl. 2, 237 (1955).

61. Visconti, N., and A. Garen, Proc. Natl. Acad. Sci. U. S., 39, 620 (1953).

62. Volkin, E., Federation Proc, 7J, 315 (1954).

63. Watanabe, I., G. S. Stent, and H. K. Schachman, Biochim. et Biophys.
^fto, 75, 38(1954).

64. Watson, J., and F. H. C. Crick, Nature, 777, 111 (1953).

65. Watson, J., and O. Maaljzie, Biochim. et Biophys. Acta, 70, 432 (1953).

66. Weed, L. L., and S. S. Cohen, J. Biol. Ch.m., 792, 693 (1951).

67. Williams, R. C, Cold Spring Harbor Symposia Quant. Biol., 78, 185 (1953).

68. Wright, S., Physiol. Revs., 27, 487 (1941).

69. Wyatt, G. R., and S. S. Cohen, Biochem. J. (London), 55, llA (1953).

70. Zinder, N., Cold Spring Harbor Symposia Quant. Biol, 78, 261 (1953).

28

PHOTOSYNTHESIS*

J. A. BASSHAM and M. CALVIN, Radiation Laboratory and
Department of Chemistry, University of California, Berkeley, California

During the past decade, considerable advance in the under-
standing of the complex process of photosynthesis has been
realized. This achievement has resulted both from the use of
new methods of investigation and from the stimulation of interest,
partly as a result of these new techniques, which has led to very
widespread participation in the study of this problem throughout
the scientific world. Fortunately, compilation and discussion of
this immense amount of work (17,26,27,34,46,47,65) — together
with review articles of the most recent work (30,36,41) — have
generally kept pace with the work itself. In the present instance,
therefore, no responsibility for a complete inclusion of published
work will be assumed, but rather, an attempt will be made to
present some current opinions regarding selected aspects of
photosynthesis, together with some speculations in areas that
may be expected to prove fruitful in the near future.

From the discoveries of recent years, it has become in-
creasingly apparent that photosynthesis includes a number of
cyclic processes which are coupled to one another in such a way

* The preparation of this paper and the original work described were
sponsored by the U. S. Atomic Energy Commission.

29

J. A. BASSHAM AND M. CALVIN

that there is a continuous flow of energy from one cycle to another
resulting in the conversion of light energy into the potential
energy of new chemical bonds. These cyclic processes, although
similar to better known cyclic processes occurring during
respiration in plants and animals, do not seem to be in any
instance simple reversals of respiratory cycles but do appear to
have many points of contact with respiratory reactions, including
common intermediates and enzymes. These points of contact
may well lead to interaction between the two processes which
could alter the course of each (18). It appears that the organiza-
tion of the green plant, both structural and enzymatic, may
provide mechanisms for partially preventing such interactions
where they would prove deleterious to the efficiency of the
photosynthetic reaction, and possibly for permitting or even
inducinsT them where interaction is beneficial.

^&

Function of the Chloroplast

One such device for isolating photosynthesis from respiration
is the chloroplast itself. It appears likely that the entire reaction
of photosynthesis, from the absorption of light, carbon dioxide,
and water to the formation of various end products, may occur
in the chloroplasts. This fact was indicated long ago by the
observed formation of starch granules in chloroplasts. (However,
starch and other high molecular weight compounds are also
formed outside the chloroplasts from simple sugars, amino acids,
and other low molecular weight compounds which diffuse out
of the chloroplast.) It has long been known that isolated chloro-
plasts can, under suitable conditions, retain the ability to evolve
oxygen at rates comparable to those of photosynthesis, and at the
same time, transfer reducing power to a suitable oxidizing agent.
Efforts to demonstrate CO2 reduction with isolated chloroplasts
were generally unsuccessful in the past. However, Gerretsen
(32) found a decrease in the oxidation-reduction potential in
illuminated chloroplast suspensions when they were supplied
with carbon dioxide and concluded that there might be an uptake

30

PHOTOSYNTHESIS

of carbon dioxide, though to only about 3% of the rate of photo-
synthesis. Boychenko and Baranov (7) have demonstrated the
incorporation of carbon dioxide into reduced organic compounds
by isolated chloroplasts under illumination. This incorporation
was determined through use of carbon-fourteen labeled CO2,
providing a very sensitive method for detecting reduced carbon.
This result recently has been confirmed by Arnon et al. (2). The
rate of carbon reduction compared with the rate of carbon
reduction in a corresponding amount of photosynthesis in intact
leaves was not more than 0.5%, and many of the intermediates
of the carbon reduction cycle have not yet been found. In any
event, if it turns out that the rate is small compared with photo-
synthesis and that only some steps involved in the complete cyclic
reduction during photosynthesis are present in the isolated
chloroplasts, these facts may be taken merely as an indication
that some of the factors involved in the rather complex reduction
cycle have been lost from the chloroplasts during their isolation,
or that only limited amounts of the necessary enzymes are carried
down with them.

We could think of the chloroplast as a container of a complex
arrangement of enzymes and cofactors, some of which are lost
when the chloroplast is removed from the cytoplasmic environ-
ment of the cell. Possibly such loss could be prevented if the
chloroplasts could be preserved throughout their isolation in a
solution exactly duplicating that contained in the cell. Perhaps
it would be worth while to prepare such a solution by disrupting
cells and removing chloroplasts and cell-wall fragments by
centrifugation. This solution could then be used during the
preparation of chloroplasts from fresh cells.

The difference in susceptibility to inactivation during chloro-
plast isolation displayed by the carbon-reducing apparatus as
compared to that found with the system for the photolysis of
water can be considered as a difference in susceptibility to loss of
factors or to the disruption of the organization of various systems.
It appears that the enzymatic apparatus for the absorption of light,
the conversion of light energy to chemical energy, the decom-

31

J. A. BASSHAM AND M. CALVIN

position of water by this energy, and the formation of oxygen
and reducing power are all rather resistant to inactivation,
provided relatively simple buffer solutions are employed in the
isolation of the chloroplasts. This seems to indicate the presence
of an organized enzymatic apparatus, perhaps with microscopi-
cally large and intricate structure and with all necessary cofactors
and prosthetic groups firmly attached. Such a view is con-
sistent with the apparent structure of the grana and lamellae as
revealed by electron microscope studies (40,57).

The carbon reduction cycle, however, must be easily
inactivated or separated during preparation of isolated chloro-
plasts. This may indicate that the enzymes involved in this cycle
are not part of an organized structure, except insofar as the
chloroplast structure tends to enclose these enzymes and retain
within itself a space with high reducing potential.

It seems likely that this reducing power does not diffuse out
of the chloroplast. If it did, one might expect greater inhibition
of respiration in the light than is actually observed in cells which
contain chloroplasts (12,31). It is significant that inhibition of
respiration during photosynthesis is most pronounced in some
organisms which do not contain organized chloroplasts (11,13)
and this fact again indicates that one role of the chloroplast
structure may be to retain reducing power at a high level for
photosynthesis. Another bit of evidence relating to this possi-
bility is the observation that the conversion of photosynthetic
intermediates to respiratory intermediates is inhibited in the
light (18). This might be attribtued to the reducing condition
within the chloroplast. In this case, the inhibition is believed to
be due to the reduction of a specific cofactor rather than to a
general reduction of metabolic intermediates which might other-
wise enter the oxidative pathway.

The mechanism by which the chloroplast might retain the
reducing power is not entirely clear, since such carriers of
reducing power as TPNH might be expected to diffuse out of the
chloroplast rather freely. One possibility is that the primary
carrier of the reducing power is itself a protein complex resistant

32

PHOTOSYNTHESIS

to dialysis. Thus some type of metalloprotein might carry the
reducing power. Another possibility is that reduced thioctic
acid, a dithiol, might be a carrier of reducing power and might
be attached by its carboxyl group to form a part of a lipid. It
may be simply that the enzymes which reduce CO2 in the chloro-
plast are so active that the reducing power is largely used before
it can diffuse out of the chloroplast.

The other reagent requirement for the reduction of CO2 at
photosynthetic rates seems to be a high level of ATP, as will be
seen later in this discussion. Bradley (D. F. Bradley, private
communication) finds that the level of ATP is higher in the dark
than in the light if the plant is in an atmosphere of 4% CO2 in
air, lower in the dark than in the light if the atmosphere is
nitrogen. Strehler (54) finds that there is an increase in the level
of ATP in the cell upon turning on the light after a period of
darkness. This increase occurred during the induction period
when the rate of carbon reduction had not yet reached its
maximum value. These facts might be explained in the following
way. In the dark with O2, respiration and the production of
ATP by some of the energy available from respiration proceed
in both the chloroplast and the space outside. When the light
is turned on the rate of production outside the chloroplast is not
immediately affected, but inside the chloroplast there is a com-
bination of several effects. Normal dark respiration ceases owing
to the production of reducing power and in particular reduced
thioctic acid (18), while at the same time the production of ATP
through energetic coupling of the recombination of some photo-
chemically produced oxidizing and reducing agents begins (4).
The result of these various rate changes is an initial increase in
the level of ATP. As the rate of CO2 reduction increases, the
demand for ATP increases. This results in a decrease in the
level of ATP in the chloroplast, perhaps to a value much less
than during the dark time, and ATP will begin to flow into the
chloroplast from the cellular space outside. In an atmosphere
of N2, on the other hand, respiration will cease in the dark, lead-
ing to a lower level of ATP in both chloroplast and cytoplasm,

33

J. A. BASSHAM AND M. CALVIN

but upon turning on the light some ATP will be formed in the
chloroplast, owing to the oxidation (back reaction) of some of the
photochemically generated reducing power by O2 liberated from
photosynthesis, or by an intermediate oxidant. The formation of
ATP by the oxidation of an intermediate reductant (i.e.,
TPNH) is known from studies of oxidative phosphorylation.
That some of the ATP was formed during the transfer of the
electrons between intermediate reductant (TPNH) and inter-
mediate oxidant (i.e., metalloflavin and/or cytochromes) is also
an intergrai part of this process, although ultimately the electrons
([H]) are transferred to molecular O2. The experimental
observation of the formation of ATP by the recombination of
photo-produced intermediate reductants and oxidants without
the possibility of the intervention of molecular oxygen has been
made by Frenkel (28) using the plastids isolated from purple
bacteria which are incapable of making molecular O2. A simi-
lar observation has been made by Arnon and co-workers (2)
using specially prepared whole chloroplasts from spinach. In
the latter case it was necessary to add other redox systems to
replace molecular O2, such as the naphthoquinone related to
vitamin K, or ascorbic acid. In addition some participation
of added FMN was demonstrated.

In regard to the location of the processes of photosynthesis,
then, the chloroplast or its immediate environs seems to be the
unique location of all important parts and may provide a con-
tainer or matrix for the complex system of enzymes and cofactors
involved in the carbon reduction cycle as well as a support for
the pigment and enzyme structure which carries out energy
conversion and decomposition of water.

The Carbon- Reducing Enzymes

Two powerful tools have been brought to bear on the problem
of the path of carbon dioxide reduction during photosynthesis
during the past decade : tracer elements and paper chromatog-
raphy. It is unlikely that our present degree of knowledge of

34

PHOTOSYNTHESIS

carbon reduction would be anywhere near as far advanced had
either of these tools been missing. In any event, it is now possible
to trace the entire path of carbon reduction from the entry of
carbon dioxide into the plant cell to the formation of sugars and
other end products (4). The essential steps in this process are
( 7) the carboxylation of a sugar phosphate, ribulose diphosphate,
to give two three-carbon molecules, both of which are probably
phosphoglyceric acid (see discussion later in this section) ; (2)
the reduction (with the aid of ATP) of phosphoglyceric acid by
reducing agents formed from water by the photochemical re-
actions ; and (3) the rearrangement of most of the molecules of
reduction product, phosphoglyceraldehyde, to give (with the aid
of ATP) more ribulose diphosphate for continued carboxylation,
with a smaller part of the sugar phosphates being drained
ofT as end products. The individual steps in this process are
shown in Figure 1, together with the enzymes believed to be
involved. The carbon balance of this system is indicated in the
following scheme.

3C5 + 3CO2 J^^I^ 6PGA (phosphoglycerate)

6PGA -^^^ 6C3

6ATP

2C3 > Ce

Ce + 2C3 > C5 + C7

Cv + C3 > 2C5

Q A.TP

12 [H] + 3C0.2 — > C3 + 3H2O

A slightly different version of the sugar rearrangement might
be proposed in which different reactions replace those between
fructose-6-phosphate and glyceraldehyde phosphate by trans-
ketolase to give ribulose-5-phosphate and a four-carbon aldose
which then combines with dihydroxyacetone phosphate (aldo-
lase?) to give sedoheptulose-l,7-diphosphate. The modified
version would postulate that two molecules of fructose-6-
phosphate are split and recombined by transketolase and trans-
aldolase to give directly sedoheptulose-7-phosphate and ribulose-

35

J, A. BASSHAM AND M. CALVLNJ

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36

PHOTOSYNTHESIS

5-phosphate. Although it is not possible to choose unequivocally
between these two possibilities at present, evidence obtained
from degradation of the various radioactive sugar phosphates
obtained from soy bean leaves which were exposed to G^^02 for
a very short time, indicates that the original proposal (as shown
in Figure 1) is correct. These degradation results are shown in
Table I, which is derived from Table I of reference 4 by assuming
that the carbon atoms numbers 1, 2, and 6 have the same C^^
level as that found in carbon 7.

TABLE I

Distribution of C^^ in Sedoheptulose
Isolated from Soy Bean Leaf

Time of exposure to G'^02, sec.

H2C— OH

c=o

I
HO— C— H

I
HC— OH

I
HG— OH

HC— OH

H2G— OPO3H

In either version for sugar rearrangement carbons numbers 4
and 5 of sedoheptulose are derived from carbons 3 and 4 of fructose,
respectively. However, in the original version (Figure 1)
carbon 3 of sedoheptulose is derived from the carbon 1 of
glyceraldehyde phosphate directly, whereas in the modified
version carbon 3 of sedoheptulose is derived from carbon 3 of
fructose and therefore should have the same labeling at all times
as carbon 4 of the sedoheptulose. Since this is not the case, the
original version of the rearrangement seems more likely, espe-
cially since at very short exposures of the plant to C"02, carbon 3
is much more highly labeled than carbon 4, as would be expected

37

0.4

0.8

2

2

33

39

8

18

49

38

2

2

J. A. BASSHAM AND M. CALVIN

if it were derived from carbon 1 of the more recently formed
dihydroxyacetone phosphate.

It is to be noted that the difference in labeling between
carbons 3 and 4 of fructose (hence 4 and 5 of sedoheptulose) was
explained as arising from the formation of dihydroxyacetone
phosphate (from which carbon 3 of fructose is derived) sub-
sequent to the formation of glyceraldehyde phosphate (the
precursor of carbon 4 of fructose) ,

Of all the enzymes indicated in Figure 1 only two, the
enzyme for the carboxylation of RuDP and the enzyme required
for the phosphorylation of ribulose monophosphate, have not
been found so far in tissues other than green plants. The
preparation of the carboxylation enzyme in a cell-free extract
has been reported by Quayle and co-workers (45), and Weissbach
et al. have isolated an enzyme preparation which causes the phos-
phorylation of RuMP to RuDP (63). It appears that the
phosphorylation and the carboxylation may be unique steps in
the carbon reduction cycle. If these steps are unique to photo-
synthesis, they may be so only because they either require
enzymes not found outside green plants or because the particular
concentrations of metabolites within the chloroplast are required
if these reactions are to proceed at a finite rate. Some evidence
bearing on this point has been obtained in this laboratory. The
purple photosynthetic bacterium, Rhodopseudomonas, is not only
capable of photoreducing COo with H2 but it can also reduce
CO2 in the dark with Ho or with organic substrates provided O2
is present (53,58). This is usually spoken of as an energetic
coupling of some of the energy released in the reaction

2H2 + O2 > H2O

to help accomplish the reduction reaction

2H2 + CO2 > (CH2O), + H2O

Since the free energy change for the latter reaction as written is
negative, it appears that the presence of a stronger oxidizing
agent than CO2 is required to produce some component or

38

PHOTOSYNTHESIS

reagent necessary to permit the reaction to proceed. This
reagent may be ATP, and the oxidant required may be either
0-2 or another oxidant produced photochemically. It is the
reaction of this oxidant (or Oo) with the activated (Ho) which
can produce ATP by the mechanism almost certainly similar to
that ordinarily known as "oxidative phosphorylation." Only
under special conditions such as these, in which a high level of
H (from Ho) and ATP are present simultaneously, may we expect
the photosynthetic carbon cycle to operate (53).

The precise detailed mechanism of the carboxylation re-
action is not yet known with certainty. The first step may be
the enolization of the ribulose diphosphate to form a double
bond between carbon atoms 2 and 3. An addition of the carbon
dioxide may then take place with the shift of an electron from
tlie hydroxyl hydrogen on carbon 3 to one of the carboxyl
oxygens. This hydrogen would come off as hydrogen ion leaving
a carbonyl group at carbon 3. The resulting compound would
be a /3-keto acid.

H2CO(P) HsCOCD

f O ^^C C-OH

CO2 + HC-OH »► C-OH *■

HC-OH HC-OH

H2C-0(P) H2G-0(P)

H.C-OCP) H2C-0(P)

^^C--C-OH -O2C-C-OH

\ ^C^OH ^ C=0+H+

' HC-OH HC-OH

H2CO® H2C-0(P)

Once formed, the j8-keto acid appears to undergo "acid
splitting" into two molecules of PGA. This is analogous to the
splitting of acetoacetate to two molecules of acetate. "Ketone

39

J. A. BASSHAM AND M. CALVIN

splitting" would produce CO2 and a 3-keto pentose. Other
possibilities are hydrogenation of the carbonyl group followed by
a rearrangement which would result in one molecule of PGA
and one molecule of phosphoglyceraldehyde ; or a direct splitting
or "reverse benzoin" type reaction which would result in the
formation of one molecule of phosphoglyceraldehyde and one of
3-phosphohydroxypyruvic acid.

Since PGA is known to be one if not the only product of the
carboxylation reaction in vivo, it is necessary to consider only
the "acid splitting" and reductive splitting. There was con-
siderable evidence for the acid split leading to PGA only, even
before the enzyme was studied in vitro. Studies were made of the
change in concentrations of RDP and PGA which occur in algae
immediately after turning off the light (18 and Bradley, private
communication). It was found that the concentration of PGA
rose very rapidly under these conditions while that of RuDP
dropped very rapidly. This was explained as resulting from a
cessation in the reduction of PGA due to the sudden decrease in
photochemically formed reducing agent, but at the same time,
a continuation of the carboxylation of RuDP leading to the
formation of PGA. The latter reaction, therefore, was not
apparently affected at once by the lack of illumination. This
result indicates that the formation of PGA from RuDP and CO2
does not necessarily involve a reduction, although it is possible
to postulate for this reaction a reducing agent with a longer half
life in the dark than the reducing agent required for the reduction
of PGA.

Finally, recent work in this laboratory (J. Mayaudon,
private communication) with a somewhat more purified enzyme
preparation, and with C^^-labeled RuDP and CO2, indicates that
the only product of the carboxylation reaction in vitro is PGA.

Despite these arguments, there still remains some possibility
that the first product of the carboxylation reaction, the six-
carbon j8-keto acid, might undergo different fates in the light
and in the dark. In the dark, splitting to two molecules of PGA
would proceed as discussed above; but in the light, with

40

PHOTOSYNTHESIS

reducing agent (TPNH or DPNH) in plentiful supply, the j3-keto
acid might be reduced and then split by a rearrangement to one
molecule of PGA and one of phosphoglyceraldehyde. This
would provide a route for the formation of one molecule of
triose phosphate without the requirement of an ATP molecule.
This possibility should be kept in mind during the following
discussion of the energy requirements of the carbon reduction
cycle, which is based on the assumption that each molecule of
triose phosphate formed requires the supply of a molecule of
ATP.

Estimation of the free energy change for the reaction of
RuDP with CO2 and water to produce two molecules of PGA
showed a net free-energy change at physiological conditions
which was either zero or negative (4), indicating that the
reaction would proceed under the influence of a suitable catalyst
without energetic coupling with some other reaction. It thus
appears that energy is supplied to the cycle in the form of TPNH
or ATP. Both are involved in the reduction of PGA to phos-
phoglyceraldehyde, whereas only ATP is needed for the phos-
phorylation of RuMP. For each carboxylation, using one
molecule of CO2 and producing two molecules of PGA, two
TPNH molecules and two ATP molecules would be converted
to TPN+ and ADP -f PO4 in the subsequent reduction of PGA
to phosphoglyceraldehyde. The enzymatic formation of RuDP
from ribulose monophosphate has been shown by Weissbach
et al. (63) to require a molecule of ATP. Since the enzymatic
conversion of triose phosphate to ribulose monophosphate proba-
bly does not require the expenditure of any ATP or other sub-
stances which supply energy through energetic coupling reactions
the net supply of energy to the carbon reduction cycle for each molecule of
CO2 reduced is that required for the formation of two molecules of
reduced TPN and three molecules of A TP as follows:

2(TPN+ + H2O > TPNH + H+ + V2 O2) AF = +103 kcal.

3 (ADP + PO; > ATP) AF = +32 kcal.

which adds up to 135 kcal. (If one molecule of triose phosphate

41

J. A. BASSHAM AND M. CALVIN

is obtained without consuming ATP, then the total requirement
is 2 TPNH and 2 ATP molecules, or 124 kcal.) Since the
energy required for the reaction

CO2 + H2O > (C6Hi206)v. + O2

is about +116 kcal., about 19 kcal. apparently have to be
expended to make the cycle operate at a high rate. The 135

SUCROSE

I
I

*
HEXOSE PHOSPHATES,*^

I
I

TRIOSE PHOSPHATES

PENTOSE CYCLE

PHOSPHOGLUCONIC
ACID'-.

..-:»SEDOHEPTULOSE PHOSPHATES

2 [H] <-
ATP

PHOTOSYNTHETIC
CARBON CYCLE

■2[H]
ATP

PENTOSE PHOSPHATES*

-ATP

RiSULOSE DIPHOSPHATE
CO2

ATP<-

PEPA

PYRUVIC ACID

I
I

/'x THIOCTIC ACID

/ \^REQUIRED

ACONITIC ACID

ISOCITRIC ACID

2M

CO,

it

2iH]

ACETYL
CoA

CITRIC ACID

A
I

/

FAT

OXALOACETIC
ACID

OXALOSUCCINIC ACID
\
\

\ *C02

I

KREBS CYCLE KETOGLUTARIC ACID

SUCCINIC ACID

MALIC ACID

^ ^2 Hi

fumaric acid

Fig. 2. The relation of carbon paths in photosynthesis and respiration.

kcal. (or 124 kcal.) requirement, on the other hand, represents
the absolute minimum of energy, which must be obtained from
light, exclusive of any losses in the photochemical and other

42

PHOTOSYNTHESIS

energy transfer reactions. We shall return to a discussion of the
energy requirements for these other processes later.

The relation between photosynthesis and respiration is
shown in Figure 2, where respiration is indicated by dotted lines
and photosynthesis by solid lines. The points at which reducing
agents and ATP are utilized in photosynthesis or produced in
respiration are indicated.

It can be seen that the path of carbon reduction in photo-
synthesis is far from a simple reversal of its path in respiration.
However, the conversion of PGA to hexose in photosynthesis and
the reverse in respiration appear to follow siinilar if not identical
paths. Moreover, some of the pentose cycle path seems to have
some steps in common with photosynthesis.

THE LIGHT REACTION

In considering the most characteristic reaction of photo-
synthesis, the light reaction, it is necessary to keep in mind the
physical arrangement of the chloroplast structure as it is now
thought to exist. The fine structure of the chloroplast (or of the
grana into which some chloroplasts seem to be divided) is
believed to be laminar, with very thin, perhaps monomolecular
layers of chlorophyll alternating with thicker layers of proteins
and lipoproteins. Although the organization and thickness of
these layers seem to vary with the species, particularly in some
algae as compared with higher plants, it seems likely that the
electrochemical fields that exist at the chlorophyll-protein and
lipoprotein interfaces are similar in all cases. Thomas et al. (56)
have recently studied the Hill reaction in particles of sublaminar
size. Measurement of oxygen evolution as a function of the
particle size showed an ability of particles as small as 10^ A^ in
volume to carry out the Hill reaction with about 50% the specific
activity of intact grana, but a rapid decrease in such ability with
smaller particles, and no activity with particles with volumes of
less than 2 X 10^ A^ at a given light intensity. However, higher
light intensities resulted in Hill reaction activity with even
smaller particles, so it was concluded that if there is a physical

43

J. A. BASSHAM AND M. CALVIN

unit of about 10^ A^ volume, or 100 A diameter, it is capable of
producing oxygen in the Hill reaction even if partially frag-
mented.

Earlier, Milner et al. (42), using the photochemical reduction
of 3,6-dichlorobenzene indophenol as a measure of activity,
studied the Hill reaction with particles of subgranar size and
obtained activity with a mixture of particles, the majority of
which were about 20 A in diameter. The same workers (43),
after measuring Hill reaction activity with these particles of about
one-fourth that of intact chloroplasts, found that the particles
could be aggregated by precipitation with a variety of salts in
the presence of 15% to 20% methanol in such a way as to
produce particles with increased activity. Loss of lipid material
resulted in loss of activity.

These experiments indicate that there are physical units,
about 100 A in diameter and containing about 200 chlorophyll
molecules, which are capable of carrying out the Hill reaction
nearly as efficiently (^-^60%) as intact chloroplasts. If these units
are broken down further, Hill reactivity falls off rapidly, but is
present to some extent with considerably smaller particles,
especially with high light intensities. Moreover, much of the
original activity can be restored by reaggregation. The require-
ments for photodecomposition of water with chlorophyll seem
to be some aggregation of chlorophyll, lipids, and protein.
Rodrigo (51) has studied associations of a few molecules of
chlorophyll, finding no shift of the red absorption peak to 6800
A. However, when chlorophyll was mixed with some ground-up
leaves which contained no chlorophyll initially, some shift of the
red absorption peak toward 6800 A and some oxygen evolution
in light with quinone were observed. The implication of this
result seems to be that a degree of aggregation of the chlorophyll
molecules sufficient to shift the red peak as far as 6800 A is
required before the Hill reaction can function. It is interesting
to note in this connection that the absorption spectra of chloro-
phyll in crystals of varying size formed from ethyl chlorophyllide
in acetone has been studied by Jacobs and Holt (37) and a shift

44

PHOTOSYNTHESIS

to the longer wavelengths with increasing crystal size observed.
A maximum shift of the red peak to about 7450 A was found,
beyond which there was no further shift with larger crystals.
This shift is ascribed to resonance interaction between identical
chromophores and to migration of the resonance energy through
the array.

The phenomenon of energy migration through the pigment
aggregate brings us to a consideration of the light absorption
process. According to present views (24) it appears that most
of the energy absorbed by plant pigments for subsequent con-
version to chemical energy is either absorbed directly by chloro-
phyll a or transferred to chlorophyll a. The excited state is
presumed to differ in energy from the ground state by only about
42 kcal./mole, corresponding to 6800 A light, the longest wave-
length light that brings about photosynthesis with high efficiency.
It is postulated that the extra energy that is absorbed at shorter
wavelengths, either by chlorophyll a or other pigments, is con-
verted to vibrational energy and eventually lost as heat. Thus
the course of energy transfer from chlorophyll on would be
unaff"ected by the wavelength of the light absorbed. It has long
been known, in fact, that the yield of oxygen evolved per quan-
tum of light absorbed is as high for red light as at any other
wavelength.

On the other hand, light absorbed at wavelengths around
4800 A produces a relatively lower quantum yield, indicating
that pigments which absorb in that range may transfer their
energy to chlorophyll inefficiently or may transfer some of their
energy to other chemical reactions inefficiently. In the latter
case, the course of subsequent steps in photosynthesis should be
to some extent affected by the light energy converted to chemical
energy without passing through the excited chlorophyll a stage.
Such an effect has recently been reported by Voskrenskaya (59),
who has studied the products of carbon reduction, using G'^ as
a function of the wavelength of the incident light. She reports
an enhanced ratio of protein to carbohydrate in blue light as
compared with red light. This effect seems to be more pro-

45

J. A. BaSSHAM and M. CALVIN

nounced at longer periods of time than those required for the
early steps in the reduction of carbon described earlier. It
seems likely that such effects are due to changes in the relative
rates of transformation for various photosynthetic intermediates
into other substances. These changes in rate of specific reactions
are probably photocatalytic and the light energy is used only in
the activation or deactivation of an enzyme.

Another photoactivation has been reported by Warburg
et al. recently (61). In this case it was found that the mano-
metrically measured quatum yield with either red or green light
was greatly affected by catalytic amounts of added blue light.
Some rather special conditions for the culturing of the algae used
in the measurements appear to be necessary for tliis effect to be
seen, since in other experiments reported by Warburg, high
quantum efficiencies were obtained with red light only, so that
the role of the blue light again appears to be in the activation of
an enzyme, but in this case, one which affects the efficiency of
the energy conversion path. Another possible interpretation of
this effect will be presented in the section on the quantum
requirement.

The step in photosynthesis which is perhaps most character-
istic is the efficient conversion of energy of an excited state of
chlorophyll a to the stored energy of new chemical bonds. The
first point to consider is the quantity of energy actually available
for transformation. It has been frequently proposed that the
primary excited state of chlorophyll a has such a short half life
(10~^^ sec.) that direct conversion of the electronic energy of
this state to some chemical reaction or the transfer to some other
pigment might not take place before the energy was lost by
fluorescence. Consequently, it was believed that transition to a
metastable triplet state with concurrent loss of some of the
electronic energy must first occur, followed by conversion or
transfer of the energy of the triplet state. However, according
to Scheibe (52), transfer of electronic energy in a condensed
pigment system can occur in 10~^* sec. It seems possible,
therefore, that in the aggregated chlorophyll-lipid-protein

46

PHOTOSYNTHESIS

system, the full 42 kcal. might be transferred to a suitable proxi-
mate acceptor, at least insofar as competition with fluorescence
is concerned.

The conversion of electronic energy to chemical energy may
involve either the transfer of electronic energy to some acceptor
molecule other than chlorophyll followed by energy conversion,
or the transfer of an electron from the chlorophyll aggregate
(array) to some other molecule and a corresponding recovery of
an electron by the positively charged chlorophyll molecule group
from some other source. Either of these types of conversion
seems possible at the present time, and we will consider both.

In order to accept electronic energy from chlorophyll and
convert it to chemical energy, a compound, which could be
called the quantum converter, should possess several properties.
It would have to be closely associated with, or incorporated in,
the chlorophyll aggregate, and should possess some state differing
from its ground state by about 30 to 40 kcal., in order to accept
the energy of a quantum from the chlorophyll. It would convert
this energy by some process which occurs in a time that is short
compared to the time required for the return of the energy to
chlorophyll or its dissipation in a nonuseful way, and in so doing
would form new chemical configurations which would store most
of the energy received from the chlorophyll. By new chemical
configurations, we mean only that some nuclei would have moved
sufficiently to prevent the loss of the received energy either by
its return to the donor or conversion to heat ; that is, the energy
would now be trapped in a new configuration which could not
return easily to the former one.

When the energy had undergone this conversion, the
quantum converter, in its new form, would pass the energy on.
Since, in this picture, the most energetically difficult step, the
photolysis of water, has yet to occur, we will suppose that the
new form of the quantum converter would then react with water
to produce a reducing agent and some form of hydroxyl or per-
oxide compound which can ultimately liberate oxygen. A
requirement for this reaction is that the resulting bond energies

47

J. A. BASSHAM AND M. CALVIN

plus about 30 to 40 kcal. be about the same as the bond energies
of broken bonds, if the reaction is to provide a means of efficient
energy conversion. The products of the reaction should not be
able directly to recombine easily in such a way as to produce
water again (back reaction) but should be able to react separately
to produce ultimately oxygen and reducing power. Finally,
the quantum converter molecule should be able to return to its
orginal state, after having transferred its electrons (reducing
power) and oxygen to other molecules.

In addition to the requirements imposed by the mechanism,
there is the necessity that the quatum converter be present in
sufficiently high concentration in the chloroplast to account for
the observed rates of quantum conversion, both in steady-state
photosynthesis and in flashing-light experiments. This require-
ment of concentration will depend, of course, on the time
required for the quantum converter to undergo one cycle, from
acceptance of the quantum of electronic energy and to return to
its original state.

Thioctic acid (lipoic acid) has been proposed as a com-
pound which might satisfy all the above requirements (3,16).
It was suggested that following the absorption of a quantum
by chlorophyll, this energy is transferred to thioctic acid,
causing the S — S bond of the latter molecule to break to
give a diradical. It was postulated that this diradical then
reacts with water, forming a sulfhydryl and a sulfhydroxyl
group. Dismutation of this reaction product results in a dithiol
molecule and a disulfenic acid. The dithiol would then reduce
DPN or TPN and would itself be reoxidized to thioctic acid,
while the disulfenic acid would undergo a series of reactions
resulting in the reformation of thioctic acid and the liberation of
oxygen. All of these reactions from the dismutation onwards
would probably involve catalysis by metalloproteins.

The lipophilic properties of thioctic acid and its small
molecular size would permit close association with the chloro-
phyll aggregate and might account for the apparent lipid
requirement of the Hill reaction. The formation of the diradical

48

PHOTOSYNTHESIS

by breaking the strained ring has been suggested as the mechanism
for storing the accepted electronic energy. Estimates of the bond
energy for the S — S bond in simple open-chain molecules ranging
from 50 to 70 kcal. together with estimates for the reduction of
the dissociation energy of the S — S bond due to ring strain in
the 6,8-trimethylene disulfide ring of 10 to 25 kcal. indicate the
possibility that formation of the free radical could store 30 to 40
kcal. (3). A number of studies with model systems (6,8-trimethyl-
ene disulfide in u.v. light) were presented which indicated,
among other things, an ability of the trimethylene disulfide to
react with ROH (alcohol) or water under illumination to form
a thiol and a sulfenic acid or ester.

+ light + ROH
S — S

H OR

Two pieces of biological evidence were off'ered to support
the suggestion that thioctic acid participates in the quantum
conversion. It had already been observed that in photosynthe-
sizing plants the conversion of photosynthesis intermediates to
Krebs cycle intermediates is inhibited during illumination, but
occurs rapidly in the dark. Since this conversion involves the
formation of acetyl CoA from pyruvic acid, a reaction which
requires thioctic acid as a cofactor (14,33,44,48,49), it had been
suggested (18) that this reaction is blocked in the light because
most of the thioctic acid is maintained in the reduced dithiol
form within the chloroplast. The reduction of thioctic acid
could occur, of course, as a secondary reaction resulting from the
formation of some other primary reducing agent in the light-
energy conversion. However, the favorable characteristics for
quantum conversion which the thioctic acid possesses together
with the indication that it was reduced in the light led to the
supposition that it might be in the primary reaction.

When algae are allowed to take up added thioctic acid in the
dark for several minutes and then killed with quinone and the

49

J. A. BASSHAM AND M. CALVIN

Hill reaction studied in the light (9) it was found that the initial
rate of oxygen evolution is increased by as much as 50% over
the rate observed in the control in which no thioctic acid was
added. This result could be interpreted as evidence for the
participation of thioctic acid in the primary conversion step
or as an acceleration of a dark reaction transfer of reducing
power to quinone. Thus the biological evidence for the role
of thioctic acid as a quantum converter is suggestive but
not unequivocal. However, since the oxygen evolution in the
Hill reaction is insensitive to inhibition by parachloromercuri-
benzoate, a more limited role of thioctic acid in which it acts
only as an acceptor of an electron from chlorophyll seems the
more likely than the above role, in which it removes this electron
from oxygen after receiving energy from excited chlorophyll.

Of the various possible chemical reactions of chlorophyll
under the influence of light we shall consider only the transfer
of an electron. Since there is evidence that the light absorption
process functions with chlorophyll in an aggregated system, it is
interesting to consider, instead of the reaction of a single chloro-
phyll molecule, the possibilities that exist with some sort of
orderly array of chlorophyll molecules. This array is probably
not actually crystalline chlorophyll but may well be an orderly
arrangement of chlorophyll molecules associated with other
molecules and protein or lipoprotein. We may think of the
electronic system of such an aggregate as a single unit in which
the TT electrons of the chlorophyll molecules interact. The
absorption of an electromagnetic quantum will raise one
electron of this system from the ground state in which it is con-
fined to a single molecule into a state in which it may migrate
throughout the array, i.e., into a conduction level. If there is
built into this structure a permanent polar character such as
exists at a "p-n" junction, for example, these photoconduction
electrons will diff'use toward the positive end of the permanent
dipole, leaving a positive hole to diffuse in the opposite direction.

Thus a separation of charge will be induced by the light
which may be neutralized by a suitable electron acceptor at one

50

PHOTOSYNTHESIS

end, and a corresponding electron donor at the other end, to
drop electrons into the positive holes that have been photo-
created. In order to complete the separation of the electron and
the positive charge and make use of the electrical energy avail-
able, all that is needed is either a semiconductor which will
transmit only electrons or positive charges, or else an acceptor
molecule which can accept either an electron or a positive charge
(i.e., contribute an electron) or both, in such a way as to produce
an irreversible change.

The possibility of a semiconductor is an interesting one in
that it provddes a possible function for the lipid constituents. One
might consider most of the lipid material as an insulator with
occasional conductor molecules to pass electrons through.
Such a function might be served by a conjugated molecule like
a carotenoid which could accept an electron at one end and give
an electron to a suitable acceptor at the other end. At the same
time, the positive charges left behind would be reacting with
water, probably through the agency of some metalloprotein, to
produce oxygen. The electron acceptor, in the case of the Hill
reaction, could be the supplied oxidant such as quinone, while
in photosynthesis the acceptor could be the primary carrier of
reducing power (thioctic acid).

The conductor function for a carotenoid compound might
explain the stimulation of photosynthesis by catalytic amounts
of blue light observed by Warburg et al. (61). Warburg has
suggested the participation of carotenoid somewhere in the
transfer of electrons between the photochemical reaction and
the reduction of carbon dioxide as an explanation of the blue-
light effect. It should be noted, however, that Stanier (R.
Stanier, private communication) has studied mutants of Rhodo-
spirillum which contain no carotenoids but which nevertheless
are able to carry out reduction of carbon dioxide during photo-
synthesis.

The advantage of the semiconductor arrangement would be
the physical separation of the points at which reducing agent
and oxidizing agents are formed. Also the nonspecificity of

51

J. A. BASSHAM AND M. CALVIN

oxidants required for the Hill reaction could be explained, since
they could be reduced directly by electrons supplied by the
photoactivation of chlorophyll rather than by some specific
primary reducing agent. During actual photosynthesis, a
specific reducing agent would undoubtedly be formed and would
provide a more efficient transfer of the energy available from the
electrons obtained from the photochemical reaction. We would
expect that this compound would have special properties which
would particularly suit it to the task of accepting electrons and,
in its reduced form, transferring energy to the carbon reduction
apparatus. It might be expected that this compound would
stimulate the Hill reaction owing to its special qualifications for
receiving electrons. For example, if thioctic acid were the com-
pound receiving the electrons from the chlorophyll during
photosynthesis, it might be expected that thioctic acid would
stimulate the Hill reaction under suitable conditions even though
not required for the Hill reaction to function.

Such a stimulation has been observed by Bradley and
Calvin (9), who studied the rate of oxygen evolution in the Hill
reaction as a function of added thioctic acid. When quinone
was added as an oxidant at a concentration which produced the
highest rate of evolution of oxygen with quinone alone, it was
found that the addition of thioctic acid in a molar concentration
only 0.1 as great asthatof the quinone produced a 35% stimulation
of the initial rate. Moreover, this stimulation was observed with
6,8-dithiooctanoic acid (6-thioctic acid) only, whereas 5-thioctic
acid and 4-thioctic acid were not eff'ective. The reduced form
(dithiol) of thioctic acid and the more oxidized form (the sulf-
oxide) were also found to be ineffective. It appears that, barring
some enzymatic specificity which seems unlikely in the Hill reac-
tion, the special property of 6-thioctic acid which is responsible for
its activity is the ring strain of the five-membered ring which
facilitates breaking of the sulfur-sulfur bond.

One advantage of considering the chlorophyll as an aggre-
gated system is that it permits a more reasonable mechanism for
the transfer of one electron at a time, with each electron re-

52

PHOTOSYNTHESIS

quiring one quantum of light energy. This corresponds to the
well-known four-quantum theory in the primary step, since the
transfer of four electrons, requiring four quanta, are required to
form one molecule of O2.

Reactions between a single molecule of chlorophyll, water,
and hydrogen or electron acceptor are difficult to formulate,
since one is faced in that case with the necessity either of absorb-
ing two consecutive quanta in one chlorophyll molecule in order
to have enough energy to form oxygen and reducing agents of the
strength of TPNH (about 51 kcal.) or else of forming oxygen and
a reducing agent of insufficient strength which could be used in
subsequent reactions involving dismutations of energy to form
better reducing agents. Another alternative is that a single
molecule of chlorophyll, activated by one quantum, reacts with
some activated, hydrated compound in which the O — H bond
could be more readily broken, and thus has left over enough
energy to form a good reducing agent. It may well be that
water is in fact incorporated into some compound to weaken
the O— H bond before the O — H bond is broken regardless of
the mechanism of electron transfer, but it is doubtful if this
activation is sufficient to permit the formation of oxygen and
reducing power of the strength of TPNH at the same time by
one quantum.

Recent proposals involving reactions in which one molecule
of chlorophyll provides both electrons in a given H2O photolysis
include the one by Levitt (39), who suggests that a chlorophyll
molecule, excited by one quatum of light, gives up an electron
to thioctic acid forming oxidized chlorophyll, after which the
molecule of oxidized chlorophyll absorbs a second quantum
and transfers a second electron. The resulting dipositive chloro-
phyll then reacts with water to produce hydrogen ion and oxygen.
In the meantime, the thioctic acid has been reduced by the two
electrons to the dithiol.

On the other hand, Wessels (64) has proposed a one quan-
tum per two electrons reaction in which a much weaker reducing
agent, reduced vitamin K, is produced. This reducing agent is

53

J, A. BASSHAM AND M. CALVIN

then used in part to produce ATP and in part to produce a
reducing agent of the level of TPNH through the expenditure
of ATP in a coupled reaction. Since the formation of reduced
vitamin K and oxygen from water and vitamin K is said to
require 39 kcal., the energy of one quantum would have to be
used with nearly 100% efficiency. Such an efficient mechanism
will be especially attractive if the very low quantum require-
ments reported by Warburg prove correct. The presence of
vitamin K in chloroplasts and its concurrent formation with
chlorophyll (23) are also favorable to this suggestion. Finally,
its oxidation-reduction potential is close to that measured with
illuminated chloroplast preparations (50). The mechanism
proposed by Wessels for the formation of ATP from reduced
vitamin K is reasonable but energetically very inefficient in that
only one ATP is formed for each molecule of reducing agent
used. No specific mechanism was proposed for the formation
of TPNH from TPN+ by the oxidation of reduced vitamin K
and the conversion of ATP to ADP and inorganic phosphate, but
this reaction would be a bit uphill energetically, since the dif-
ference in redox-free energies between TPNH and reduced
vitamin K is apparently about 12.5 kcal., slightly more than the
10 or 11 kcal. now thought to be available from ATP hydrolysis.
Besides this, the proposal of Wessels requires the steps in the
liberation of O2 from whatever intermediates may be formed in
the reaction of oxidized chlorophyll with water to proceed with
virtually no change in free energy. In other words, this proposed
mechanism includes one very inefficient step and a number of
steps which are nearly 100% efficient in energy transfer. Al-
though this is entirely possible, we find it slightly more satisfying,
from a thermodyamic viewpoint, to suppose that most of the
steps involved in the energy transfer from one system to another
proceed efficiently but with a small loss of energy in each, thus
providing a smooth driving force throughout the entire process
which will not require the enzymes at any stage to cope with
infinitesimal concentrations of substrates.

54

PHOTOSYNTHESIS
INTERMEDIATE TRANSFER SYSTEMS

We have already anticipated, in the discussion of both the
carbon reduction cycle and the light reaction, some of the re-
actions involved in the transfer of reducing power and energy
from the light reaction to carbon reduction and in the evolution
of oxygen from whatever products are formed in the breaking of
the O — H bond. Let us assume, for the time being, that the
theories above, which require that tlie absorption of a quantum
for each electron taken from water (whether by quantum con-
version by thioctic acid or by transfer of an electron by the
chlorophyll aggregate from water to reducing agent) are correct.
Then there is ample energy in four quanta of 6800 A light (168
kcal./mole) to bring about the photolysis of two water molecules
and the formation of two molecules of reducing agent of strength
equal to TPNH:

2[H20 -f TPN+ = 1/2 Oo + TPNH + H+] AF - +103 kcal

The entire excess of energy, some 65 kcal. minus whatever
was lost in the primary absorption and conversion processes,
will be available for the evolution of oxygen from the inter-
mediates formed from the oxidation of water. It is possible that
some of this energy might be used in the formation of some ATP
from ADP and inorganic phosphate. Whether or not this occurs
is very important in the evaluation of possible quantum require-
ments. We have already arrived at a quantum requirement of
two molecules of TPNH and three molecules of ATP for each
molecule of carbon dioxide reduced. If one molecule of the
primary reducing agent must be oxidized in order to form two or
three molecules of the required ATP by some reaction similar
to that which couples the energy of the oxidation of DPNH to
the formation of ATP (38), tlien the total requirement for
equivalents of reducing agent will be seven or six, requiring
seven or six quanta (4). If all the ATP molecules could be
supplied from the energy left over from the evolution of oxygen
as suggested above, then the quantum requirement will obviously
be only four.

55

y. A. BASSHAM AND M. CALVIN

The decomposition of water will require by far the greater
portion of the energy available from the primary photochemical
reaction. From the half-reaction potential of the primary reduc-
ing agent, which in our scheme must be about 0.3 v., we can say
that the relative energy stored by the transfer of one electron to
the reducing agent is about 7 kcal./mole (taking the energy re-
quired to transfer an electron to 1 A^ H+ in contact with H2, g,
1 atm., as zero). If there is a loss of about 5 kcal. in the transfer
of the electron from the chlorophyll aggregate, there is left, from
a 42 kcal./mole quantum, about 30 kcal./mole to be stored in
each positive charge ("hole"). This would then be a poten-
tial of about 1.3 V. The potential required for the half reaction

2H2O = H2O2 + 2H+ + 2 ^-

is about 1.2 v. at/?H 7 and 10"^ Af H.2O2. Thus the reaction will
go as written, provided the very high activation energy required
for the removal of electrons from water-oxygen atoms, which
would result in formation of hydroxyl radicals in solution, can
be overcome. We may suppose that the hydration of a suitable
surface on the granar fragment, perhaps resulting in actual
hydrated compounds, results in an orientation of — OH groups
which permits the formation of O — O bonds concurrently with
the removal of the electrons from the water.

The formation of the positive and negative potentials dis-
cussed above requires some mechanism for obtaining just the
right distribution of energy to achieve the necessary oxidation
and reduction. This can be accomplished by extending some-
what further the proposal for the separation of charges through
the agency of semiconductors. We may think of the subgranar
unit as a photoelectric battery. The driving force for this battery
is the light energy absorbed by the chlorophyll. The absorption
of light produces in the aggregate conduction electrons and their
corresponding positive "holes." This part of the structure can
be considered a conductor after light absorption. On either side
of the chlorophyll aggregate is a layer of semiconducting material.
This material may be lipid or lipoprotein. One layer contains a

56

PHOTOSYNTHESIS

permanent structural excess of electrons (?2 volume), permitting the
conduction of positive charges. The other layer would contain a
permanent structural deficiency of electrons {p volume), permit-
ting the conduction of electrons. With the creation of mobile
charges by light absorption there would follow a flow away from
the chlorophyll aggregate and across the semiconducting layers.
If the potential between these two layers could be measured, it
would be found equivalent to ^^42 kcal./mole minus the 5
kcal./mole loss postulated above, or 1.6 v. If the circuit between
these two "electrodes" is completed by chemical reactions, as
it is in photosynthesis, the potential at each electrode, relative
to the ground state of the system, will be simply that required
by the oxidants or reductants with which the electrons and
positive charges must react. Thus, in terms of our arbitrary
"ground," which is the standard hydrogen electrode potential,
the 1.6 V. potential across the "battery" will be distributed as
1.3 V. positive and 0.3 v. negative, since these are the potentials,
relative to our "ground," with which the electrodes must react.
From this point of view it appears that the recently developed
solar battery (22) may have been preceded by a similar but much
more efficient process in photosynthetic organisms !

The evolution of oxygen from hydrogen peroxide according
to the reaction

H2O2 = V2 O2 + H2O

proceeds with a positive potential of 0.37 v. for \0~^ M peroxide,
so that another 8.5 kcal./mole of excess energy is expended per
electron. There is some question as to whether this reaction
could be catalyzed by catalase (55), owing to the apparent lack
of inhibition of the Hill reaction by cyanide. However, this
reaction can be catalyzed by a number of simple inorganic
compounds, so perhaps this problem is not too serious. More-
over, inhibition of oxygen evolution in the Hill reaction by
orthophenanthroline has been observed (29), so some iron-
containing enzyme may be involved. Finally, the recent work
of Chance (21) has demonstrated the formation of catalase-

57

J. A. BASSHAM AND M. CALVIN

H2O2 complexes under aerobic conditions. It appears that this
complex can form with concentrations of H2O2 as low as 10~^ M,

Consideration of the above mechanism of oxygen evolution
suggests that the place where energy might possibly be available
for use in forming ATP, or perhaps additional reducing agent, is
in the liberation of oxygen from peroxide. Some 17 kcal. are
available from each molecule of peroxide. However, it is
difficult, with only our present knowledge, to visualize the
possible mechanism of this energetic coupling. On the other
hand, it is attractive to suppose that the peroxide may be used
in part to oxidize some ferrocytochrome (19) in a reaction
catalyzed perhaps by peroxidase (19). Thus the requirement
for an oxidizing agent to react ultimately with the primary
reducing agent could be met without requiring oxygen. This
is rather useful because of the evidence that molecular oxygen
is not required for photosynthesis. Allen (1) has reduced the
concentration of molecular oxygen in contact with photosyn-
thesizing organisms to a value of 0.004 mm. Hg and finds, at
this level, no diminution of the rate of photosynthesis. The
absence of a back reaction involving molecular oxygen was also
shown by the studies of Brown (10) with the mass spectrometer
and iso topically labeled oxygen.

It is interesting to suppose that the ferrocytochrome
oxidized by H2O2 is reduced cytochrome /, discovered by Hill
and Scarisbrick (35). It was found that this compound was
present in considerable amounts in green chloroplast material
and that it had an oxidation-reduction potential of +0.365 v.,
about 0.1 V. better as an oxidizing agent than cytochrome c.
Since the oxidizing potential of H2O2 for the physiological
conditions as given above is +1.2 v., there is a potential dif-
ference between these two half reactions of 0.8 v., or a negative
free energy change of 37 kcal. /mole for the oxidation of two
reduced cytochrome / molecules with one H2O2 molecule.

The oxidized cytochrome could then react with other
electron carriers, perhaps other cytochromes, and eventually,
with the primary reducing agent, or with TPNH. Since the

58

PHOTOSYNTHESIS

oxidation-reduction potential of the latter is now considered to
be —0.324 v. (15), there is a potential difference between cyto-
chrome and TPN+ of 0.65 v., or a negative free energy of 30.3
kcal. for the oxidation of one mole of TPNH by oxidized cyto-
chrome /.

Very little is known about the mechanism of formation of
ATP during the oxidation of TPNH. There is enough energy
available in each of the steps suggested above to bring about
the formation of 2 moles of ATP with ease. We can suppose
that the general type of mechanism for the oxidative formation
of ATP might involve esterification of a hydroxyl group with
inorganic phosphate, dehydrogenation of an adjacent C — C or
C — N bond with a suitable oxidizing agent to form a "high-
energy phosphate group," a reaction with ADP to form ATP
and an unsaturated alcohol, and finally, reduction of the
unsaturated alcohol to the orginal substance.

H
-C-

H

-C-

H

-f HOPO3H

H
-C-

OH

H
-C-
I H

OPO3H-

+ H2O

H
-G-

H
-G

— + R

I H
OPO3H-

■^ RH2 H C— C—

I H

OPO3H-

-G^=G-

I H

OPO3H-

+ ADP

-C=

OH

=C h ATP

H

-G— C h R'H2

I H

OH

H H

-> — G G h R'

I H

OH

-> RH2 + R' + ATP

R + R 'H2 + ADP + HsPOr —

The — CH2 group of the reacting alcohol can also be
— NH — . Considering the number of steps involved in the above
mechanism, we could expect that at least 5 kcal. negative free-

59

J. A. BASSHAM AND M. CALVIN

energy change would be required for the entire process, so that
about 15 kcal./mole might be required for the formation of ATP
by such a mechanism. It is thus possible that anywhere from 1 to
4 molecules of ATP might be formed for each molecule of primary

H

I

H-0

Fe

H
I
,0-H

II _ II

+ AMP-O-P-0 + HO-P-0"
I I

OH 0"

Coenzyme
and/or protein

AMP-O-P
I

l?/6^ !?/0-

Fe**

P-OH
1
.0

AMP-O-P *PrOH;--

I I

Fe***

Coenzyme
and/or protein

Coenzyme
and/or protein

^v^.

° n °

ll/0\ II
AMP-O-P P-0"

n

II /0\ II

AMP-O-P

~P-0"

+ e

Fe***

\

Fe'

Coenzyme
and/or protein

2 HgO

Coenzyme
and/or protein

*
ATP

Fig. 3. A suggested mechanism for ATP formation via oxidation and reduction

of a metalloenzyme.

reducing agent used, provided electron carriers of suitable
intermediate potentials are available. What these might be is
difficult to say, but some cytochromes, for example, cytochrome
b, lie intermediate between cytochrome / and TPNH. It is

60

PHOTOSYNTHESIS

conceivable that there is still another type of process for the
generation of ATP which differs fundamentally from the one
outlined above, involving the intermediate formation by
oxidation of a "high-energy phosphate group" in the form of an
enol ester or anhydride. This is suggested by the increasing
knowledge of "oxidative phosphorylation" and the participation
of metalloproteins in this process, particularly metalloflavins
and/or porphyrin, together with the fact that tripositive (higher
valence state) ions form stronger and more compact complexes
than do dipositive ions (lower valence state). An example of
the manner in which such a process might operate is shown in
Figure 3. The metal (in this case Fe) in its reduced form
(Fe+2) could bind into a complex the terminal phosphate of
ADP and one orthophosphate ion. This complex, upon oxida-
tion to the higher valence state (Fe+^), would contract and
induce the displacement of an OH~ from orthophosphate by
the — 0~ atom of the terminal phosphate of ADP, thus produc-
ing the stable ATP chelate of the Fe+3, in order that the ATP
be liberated for other uses, the Fe+^ must be reduced again to
Fc+\ for which the chelation constant is much smaller. The
cycle is thus complete, the net result being the transfer of an
electron from the reducing agent to some oxidizing agent of
higher potential via the Fe atom with the trapping of some (if
not all) of the energy of this transfer in the form of ATP. It is
interesting to note that Chance (20) has observed an apparent
requirement of the reduction step for the liberation of ATP in
the course of oxidative phosphorylation.

The process postulated for the formation of primary
reductant and H2O2 has a quantum requirement of 4 for each
H2O2 formed or 2 for each RH2 formed. The over-all quantum
requirement will depend on the number of RH2 molecules
which must be used to form ATP. If all required ATP can be
supplied from respiration reactions outside the chloroplast, as
may be the case at very low light intensities, the over-all quantum
requirement will be 4. At high light intensities the over-all
quantum requirement will be 10, 7, 6, or 5, depending on

61

J. A. BASSHAM AND M. CALVIN

whether the number of ATP molecules formed per RH2 burned
is 1, 2, 3, or 4, respectively.

The controversy regarding the minimal requirement of
photosynthesis has not been settled. The recent experiments
reported by Warburg et al. (60) are very convincing, since
quantum yields of four and even three are reported with high
light intensities for long periods of time and without corrections
for respiration, thus effectively answering criticisms based on the
possibility that the reported quantum requirements are due to
contribution of respiratory energy to photosynthesis.

This leaves only criticisms based on the evaluation of the
manometric technique. No such evaluation will be attempted
here, but it may be worth while to point out one possible diffi-
culty. Calculations of oxygen evolution and carbon dioxide
uptake by the two-vessel method depend on the assumption of
constant solubilities of these gases. However, the solubility of
carbon dioxide may change significantly if the j&H of the medium
changes, and this in turn could be influenced by the secretion of
acid by the algae. For example, it has been observed in this
laboratory that in high light intensities, algae produce glycolic
acid. Tolbert (N. E. Tolbert, private communication) has
found that glycolic acid formed in strong light by algae is secreted
into the medium. One might speculate that perhaps blue light
might activate some acid-secreting enzyme system, though there
is at present no evidence for this.

In view of this and many other difficulties inherent in the
manometric determination of quantum yields, it has seemed
desirable to try other methods of measuring oxygen liberation
for quantum requirement calculations. One such study is that
of Brackett et al. (8), who used a polarographic determination of
oxygen and calculated quantum requirement as low as six. A
relatively simple and straightforward experiment has now been
carried out using an oxygen analyzer employing paramagnetic
measurement of oxygen (5).

A suspension of algae was placed in a thin plastic cell of
large area. A mixture of 4% COo was passed through this

62

PHOTOSYNTHESIS

suspension, then through an oxygen analyzer and a carbon
dioxide analyzer. The circulation of gas through this closed
system was accomplished by means of a pump. The indications
from the analyzers were continuously recorded on a multipoint
recorder. A uniform light field of 6300 A light was obtained
from a spiral neon tube with suitable filters and incident and
transmitted light intensities measured with a bolometer, which
was frequently calibrated against three standard lamps. Small
variations in the light field were mapped by a small photo-
electric cell and suitable corrections made.

The measurements of photosynthetic rate were dependent
only on the measured change in the percentage oxygen in the
system and the known volume of the system. Both of these are
directly measured quantities which can be, and were, checked
frequently with standard gas mixtures. Virtually no variation
was found from time to time. The energy measurements were
also simple and straightforward, since they involved essentially
the measurement of energy absorption in a thin layer of large
area.

The algae, Chlorella pyrenoidosa, were grown according to
previously described conditions (6) in 4% COo. The quantum
requirements of these algae were tested after a variety of pre-
conditions. The best condition found was selected and determi-
nations were made as a function of light intensity. The values
of the quantum requirement were determined both for the un-
corrected rate of photosynthesis and for the rate, which we will
call photosynthesis, obtained by subtracting the dark respiration
rate from the uncorrected rate.

This correction seems justified in view of Brown's (10) study
in which isotopic oxygen was used to demonstrate that no sig-
nificant change in rate of respiration of Chlorella pyrenoidosa took
place during alternate 1 5 or 20 minutes of light and dark. The
same paper, as well as the earlier ones by Emerson and Lewis
(25), Weigl et al. (62), and Brackett et al. (8), showed an increase
in the dark respiration rate (and the light rate as well in Brown's
paper) which is produced by conditioning the plants with photo-

63

J. A. BASSHAM AND M. CALVIN

synthesis as compared with leaving them in the dark for several
hours. This indicates a photosynthesis enhancement of the rate

17

16

15

14

13

12

•/♦

10

9-

8 -

QUANTUM REQUIREMENT OF
PHOTOSYNTHESIS BY Chlorella

• UNCORRECTED
O CORRECTED

5 P/R

10

15

I

10 15 20 30

q X 10-'^

40

Fig. 4. Relation between quantum requirement in photosynthesis and
the ratio of photosynthesis to respiration rate.

of respiration over the rate required by the plants while resting
in the dark.

64

PHOTOSYNTHESIS

c

■*-"
CS

u

Dh
CO

V
u

-a
c

C3

V

G
>^

CO

O

o

a,

o
o

t-l
+->

u

o
XI

a

CO

O

a

c
o

Sh

re!

u

C

4-»

bo

65

J. A. BASSHAM AND M. CALVIN

If each of the experimentally determined quantum require-
ments is plotted against the ratio of photosynthesis to respiration
(/?/r), an interesting result is obtained. At high ratios of
/)/r, where it would be expected that respiration could contribute
relatively little of the ATP required for photosynthesis, both
corrected and uncorrected quantum requirements approach the
same value, about 7.4. At low values oi p/r, where respiration
could contribute relatively more ATP, the corrected quantum
requirement approaches four while the uncorrected quantum
requirement becomes very great. This result, shown in Figure
4, lends credence to the theory that the two molecules of primary
reductant required for the reduction of one molecule of CO2 are
generated by four quanta but that when the ATP required for
the reduction of CO2 must be formed by reactions consuming
the reducing agent, there is a net requirement of about six or
seven quanta for each CO2 molecule reduced.

When the reduction in quantum requirement at low light
intensities is multiplied by the total number of molecules of
oxygen evolved and when the product (number of quanta
"saved" by respiration) is compared with the enhancement of
respiration due to photosynthesis, it is found that about seven
quanta are "saved" for each extra molecule of oxygen taken up
by photosynthesis enhancement of respiration over the resting
dark respiration. Since about seven molecules of ATP are
produced by each molecule of oxygen consumed in respiration,
this result is consistent with the theory that respiration con-
tributes energy to photosynthesis in the form of the reactivity of
ATP and is also consistent with the requirement discussed earlier,
of about one quantum for each molecule of ATP formed by burn-
ing photochemically produced reducing agent.

The relationships of energy transfer in respiration and in
photosynthesis are shown in Figure 5. It will be seen that in this
scheme the principal "innovations" required for photosynthesis
as compared with respiration are the photochemical "battery"
and the use of perhaps two specialized electron carriers, thioctic
acid and cytochrome /. As more is known about details of the

66

PHOTOSYNTHESIS

energy-transferring processes, we may expect that additional
special steps will be found.

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67

J. A. BASSHAM AND M. CALVIN

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69

BACTERIAL FERMENTATIONS

H. A. BARKER, Department of Plant Biochemistry, University of California,

Berkeley. California

Early interest in bacterial fermentations was stimulated
mainly by a need to determine the chemical changes occurring
in food, soil, and other materials under anaerobic conditions or
to discover and produce compounds that might have industrial
applications. Abundant information on these aspects of fer-
mentation has accumulated over the years. More recently, the
center of interest has shifted to the analysis of the chemical
mechanism of fermentations, since anaerobic bacteria frequently
provide relatively simple and convenient systems for the study
of basic metabolic processes.

The usefulness of bacteria for metabolic studies is dependent
in part upon two characteristic properties, the high rate and the
extraordinary degree of specialization of their energy-generating
mechanisms. These properties are perhaps even more highly
developed in anaerobic than in aerobic bacteria. They facilitate
the analysis of problems of intermediary metabolism by providing
a biological material that is relatively free of complicating side
reactions.

Although individual species use highly specialized catabolic
reactions, anaerobic bacteria as a group possess the ability to

70

BACTERIAL FERMENTATIONS

attack a great variety of substrates including carbohydrates,
polyalcohols, amino acids, purines, pyrimidines, and many
organic acids. Consequently they provide a wide range of
specialized catabolic processes for study.

Fermentation Patterns

Not all organic compounds are fermentable. The question
arises as to why some compounds are utilized by anaerobic
bacteria and others are not. No answer is possible in terms of
molecular structure because the fermentability of a compound
depends both on its structure and on the enzymatic make-up of
the organism to which it is offered. A partial answer can be
given only in terms of the nature of the reactions which a com-
pound can undergo in a particular enzymatic system.

An essential but not always sufficient requirement for
fermentability is that the compound or products derived from
it must be able to serve both as a reductant and as an oxidant.
Furthermore, the potential difference between the oxidant and
reductant systems must be great enough (^0.25 v.) to provide
the energy necessary to synthesize the various structural and
functional components of the cell from simple molecules. Since
most biologically important organic compounds are susceptible
to enzymatic oxidation, the more unique requirement for fer-
mentability is the formation of a suitable oxidant system.

The oxidizing and reducing systems of bacterial fermenta-
tions are provided in several more or less distinct ways. In the
simpler fermentations the substrate itself or a phosphorylated
derivative is both oxidized and reduced. Examples of this are
the dismutation of pyruvate to lactate, acetate, and carbon
dioxide by some lactic acid bacteria, and the fermentation of
glycerol by Escherichia freundii, in which the oxidation of glycero-
phosphate to dihydroxyacetonephosphate and other subsequent
products is coupled with the reduction of glycerol to trimethylene
glycol (22). In a few fermentations the substrate is converted
to two different compounds, one of which is oxidized and the

71

H. A. BARKER

Other reduced. This is true in sugar fermentations by lactic acid
bacteria which produce glycerol. The dihydroxyacetonephos-
phate initially derived from carbons 1 to 3 of glucose presumably
is reduced, and the glyceraldehyde initially derived from carbons
4 to 6 is oxidized.

A fermentation pattern more frequently encountered is that
in which part or all of the substrate, usually after some prepara-
tory reactions, is oxidized and then is transformed into an hydro-
gen acceptor. The simple lactic acid fermentation of glucose is
of this type; glyceraldehydephosphate is oxidized and the
product is converted to pyruvate, which is reduced to lactate.
A variation of this pattern is known to occur in two fermentations
in which the reduction precedes rather than follows the oxidation
in the path of substrate degradation. This situation is encoun-
tered in the fermentations of uric acid (30) and of orotic acid
(18). In the former, the first step is the reduction of uric acid
to xanthine, which is probably coupled with oxidation of glycine
and pyruvate derived from xanthine. In the orotic acid fer-
mentation, an initial reduction of orotic acid to dihydroorotic
acid is coupled with the oxidation of an as yet undetermined
compound.

Besides the true fermentations just described, several other
closely related catabolic processes sometimes referred to as
"anaerobic oxidations" are utilized by anaerobic bacteria.
These processes are similar to the true fermentations in that they
provide energy for growth, and they frequently produce acids,
gases, and odors characteristic of fermentations. They differ
from true fermentations in that two major substrates are required,
one to serve as an oxidant, the other as a reductant. The latter
is usually an organic compound, although hydrogen gas and
carbon monoxide are used as reductants by some species. The
oxidant may be either an organic or an inorganic compound
according to the nature of the organism. The best known
example of the use of organic oxidants is in the so-called Stickland
reaction (23) catalyzed by Clostridium sporogenes and several other
Clostridia. In this reaction an amino acid such as alanine or

72

BACTERIAL FERMENTATIONS

valine is oxidized, and another amino acid such as glycine or
proline is reduced.

The main inorganic oxidants used by anaerobic bacteria
are nitrate, sulfate, and carbonate. The ability to use one of
these compounds as a major oxidant is generally restricted to
specialized groups of bacteria. Nitrate is reduced to nitrous
oxide and nitrogen by denitrifying bacteria, sulfate is converted
to hydrogen sulfide by sulfate-reducing bacteria, and carbon
dioxide is reduced either to methane by the strictly anaerobic
methane-producing bacteria (32) or to acetic acid by organisms
such as C. aceticum (37) and C. thermoaceticum (38). Inorganic
oxidants are advantageous in that they make possible the utiliza-
tion of a wider variety of organic compounds, and the more
complete oxidation of individual substrates, than is possible in a
true fermentation.

Selection of Organisms

The selection of an organism capable of attacking a given
compound or performing a particular reaction sequence can
often be made by consulting a reference book on systematic
bacteriology. However, because bacteriologists have generally
shown a strong preference for the use of a limited number of
substrates, of which the sugars are most prominent, it sometimes
happens that no organism is known which possesses a highly
developed ability to attack a particular compound in which an
investigator is interested. Under these circumstances a suitable
organism frequently may still be obtained by use of the enrich-
ment culture method.

The advantages of the enrichment culture method, origi-
nally developed by Winogradsky and Beijerinck in the last
century, appear not to be widely recognized; certainly, the
method has not been applied as extensively as might be desirable.
The method depends on the principle that organisms capable of
decomposing a given compound can be obtained from a mixed
population such as that in soil by using a culture medium in

73

H. A. BARKER

which the compound in question constitutes the main energy
source. In such a medium only organisms that can muhiply at
the expense of the added substrate become numerous. Aerobic
or anaerobic species can be selected by supplying or removing
oxygen, and other conditions such as the nitrogen source, the pH,
or the temperature may be varied so as to favor specific groups of
bacteria. Bymaking several transfers in the appropriate medium,
organisms with the desired substrate specificity and environmental
requirements can be "enriched" to the extent that their isolation
in pure culture can be achieved by conventional methods.

The application of the enrichment culture method to the
study of the fermentation of nitrogenous compounds, for example,
has resulted in the isolation of several previously unknown
bacteria that are potentially useful for metabolic studies. With
uric acid as an enrichment substrate under anaerobic conditions,
two closely related purine-fermentating bacteria, Clostridium
acidi-urici and C. cylindrosporum, can be isolated from soil (3).
These bacteria show a high degree of biochemical specialization,
since they decompose uric acid, xanthine, and guanine with
great vigor but do not attack a variety of other common sub-
strates (2). Enrichment cultures with orotic acid have yielded
an organism, Zymobacterium oroticum, which has been used to
study the enzymatic synthesis and degradation of the pyrimidine
ring (18,35). Other bacteria fermenting uracil, thymine, or
allantoin have been obtained by the same method. A number
of amino acid fermenting bacteria have also been isolated from
enrichment cultures. Examples of such organisms are Clostrid-
ium tetanomorphum, which attacks histidine and glutamate (34),
C. propionicum, which ferments alanine, serine, and threonine (8),
and Diplococcus glycinophilus, which uses only glycine and certain
of its di- and tripeptides (8).

Many similarly specialized bacteria, fermenting other com-
pounds, undoubtedly can be obtained by the enrichment
method. It is a useful tool with which to isolate organisms
favorable for the study of a variety of biochemical reactions.

The remainder of this article will be devoted to a discussion

74

BACTERIAL FERMENTATIONS

of the way in which our knowledge of specific aspects of some
bacterial fermentations has been arrived at.

Initial Stages of Carbohydrate Fermentation

One of the most significant developments in the field of
fermentation biochemistry in the past few years has been the rec-
ognition of the existence of several nonglycolytic pathways of car-
bohydrate breakdown. Some indications of nonglycolytic path-
ways were obtained long ago by balance experiments, but only
since the application of tracer techniques has the evidence for such
pathways become conclusive and generally been recognized.

Two fermentative bacteria have been shown to use pre-
dominantly nonglycolytic pathways, namely Leuconostoc mesen-
teroides and Pseudomonas lindneri.

Leuconostoc mesenteroides catalyzes a so-called heterolactic
fermentation of glucose, the main products being equimolar
amounts of lactic acid, ethanol, and carbon dioxide. The
significant feature of this fermentation, from the point of view
of its mechanism, is the constancy in the yields of the three
products. In other bacterial fermentations giving the same
products, the ratio of lactate to ethanol, for example, varies
considerably with pYi and other environmental factors. In the
Leuconostoc fermentation, on the contrary, half of the glucose is
always converted to lactate and half to ethanol and carbon
dioxide. This result is inconsistent with the glycolytic mecha-
nism, in which both halves of the glucose molecule are funneled
through a common intermediate, pyruvate, which can be
converted either to lactate or to ethanol.

More definitive evidence for a nonglycolytic mechanism of
glucose fermentation in Leuconostoc was obtained by Gunsalus
and Gibbs (14). They found that the fermentation of glucose-
l_Ci4 gives labeled carbon dioxide, and glucose-3,4-C^^ gives
carboxyl-labeled lactate and ethanol-l-C^l Their data indicate
that the carbon dioxide is derived from glucose carbon 1, the
methyl and carbinol groups of ethanol from glucose carbons 2

75

H. A. BARKER

and 3, respectively, and the lactate carbons, starting with the
carboxyl group, from carbons 4, 5, and 6 of glucose. The
detailed mechanism of glucose fermentation by Leuconostoc has
not yet been worked out, but there is considerable evidence that
glucose-6-phosphate and 6-phosphogluconate are intermediates.

The alcoholic fermentation of P. lindneri differs from that of
yeast in respect to the fate of the first three carbons of glucose
(13). Whereas in yeast glucose carbons 1 and 2 are converted
to ethanol and carbon 3 to carbon dioxide, in the P. lindneri
fermentation carbon 1 goes to carbon dioxide and carbons 2
and 3 to ethanol. The carbinol carbon of ethanol is derived
from carbon 2 of glucose, in contrast to the Leuconostoc fermenta-
tion in which the methyl carbon of ethanol comes from the
carbon 2 of glucose. The carbon distribution pattern of the P.
lindneri fermentation is sufficiently similar to that observed in
glucose oxidation by Pseudomonas saccharophila to suggest that the
underlying mechanisms of the two processes may be the same.
Glucose-6-phosphate, 6-phosphogluconate, and 2-keto, 3-deoxy,
6-phosphogluconate have been shown to be intermediates in
glucose breakdown by P. saccharophila (11,20).

Two other fermentative bacteria that use nonglycolytic
mechanisms to some extent are Escherichia coli and Propioni-
bacterium pentosaceum. E. coli possesses enzymes catalyzing both
the glycolytic (10) and the nonglycolytic ribulose-phosphate
(29,31) mechanisms of glucose breakdown. The available
evidence indicates that the glycolytic path is used for anaerobic
decomposition of sugar, whereas both paths are used in the oxida-
tion of glucose. The experiments of Cohen (9) with glucose-
1-G^^ indicate that at least 14 to 37 per cent of the glucose
decomposed aerobically is metabolized via the pentose pathway.
The remainder of the glucose presumably is oxidized by the
glycolytic path, although specific and conclusive evidence for
this has not been obtained.

Propionic acid bacteria probably use both glycolytic and
nonglycolytic pathways of glucose fermentation to a significant
extent. Evidence for the glycolytic pathway includes demon-

76

BACTERIAL FERMENTATIONS

strations of the phosphorylation of glucose by ATP (4) and the
conversion of glucose or fructose- 1,6-diphosphate to phospho-
glycerate by dried or toluene-treated cell suspensions (36), and
the conversion of phosphoglycerate to the normal fermentation
products by proliferating cells. More direct evidence for the
distinctive steps of the glycolytic pathway, the formation of
fructosediphosphate and its conversion to triose phosphate by
the aldolase reaction, is required, particularly in view of the
fact that all previous studies with propionic acid bacteria have
been done with crude enzyme preparations. Alternate inter-
pretations of the experimental data are therefore possible. For
example, the observation that toluene-treated cell suspensions
converted fructose- 1,6-diphosphate to phosphoglycerate in the
presence of fluoride and an oxidant, does not definitely establish
the existence of the aldolase reaction. An alternative inter-
pretation is that fructosediphosphate is converted to glucose-6-
phosphate which is oxidized to phosphoglycerate via phospho-
gluconate, pentose phosphates, and glyceraldehyde phosphate.
The recent tracer experiments of Leaver and Wood (17)
have demonstrated rather conclusively that the glycolytic path
is not the only mechanism of glucose fermentation by propionic
acid bacteria. The fermentation of glucose-l-C^'* gave carbon
dioxide with a higher specific activity than that of any carbon of
the other products. The quantitative data indicate that at least
20 per cent and possibly considerably more of the glucose is
decomposed by oxidation of carbon 1 to carbon dioxide, as
would occur in a nonglycolytic pathway. The fermentation of
glucose-3,4-C^^ resulted in the appearance of C^^in noncarboxyl
positions of acetate, propionate, and succinate. This result,
when considered in conjunction with other information concern-
ing the propionic acid fermentation, is inconsistent both with a
glycolytic pathway and with the Leuconostoc mesenteroides and
ribose phosphate pathways of glucose breakdown. The isotope
distribution data can be interpreted in terms of glucose oxidation
by the 2-keto-3-deoxy-6-phosphogluconate pathway of Pseudo-
monas saccharophila (20). However, no specific information is

77

H. A. BARKER

available concerning the occurrence of this pathway in propionic
acid bacteria.

The glycolytic pathway is probably used as a fermentation
mechanism by several bacteria, in addition to E. coli. For
example, several reactions of this pathway have been demon-
strated with Streptococcus faecalis (24), particularly the formation
and decomposition of fructose diphosphate. Clostridium perfringens
has been shown to contain the enzyme aldolase, indicative of a
glycolytic mechanism (1). With several other bacteria, such as
C. thermoaceticum (39), Lactobacillus casei (12), and Butyribacterium
rettgeri (25), the distribution patterns of C^"* in products obtained
from the fermentation of C^^-labeled glucose indicate a pre-
dominantly glycolytic pathway, although this has not yet been
verified by critical enzymatic experiments.

The examples given above demonstrate that at least three
different pathways exist for the anaerobic decomposition of
glucose to three-carbon compounds by bacteria. There is no
reason to believe that these are the only such pathways. Of the
many anaerobic bacteria that are known, only a very few have
been studied extensively enough so that any definite conclusion
can be reached concerning their mechanisms of glucose decom-
position. Also virtually nothing is known about the energy-
utilizing mechanisms that are associated with nonglycolytic
sugar decomposition. Only a good beginning has been made in
the study of this fundamental aspect of bacterial fermentations.

The discovery of several alternate pathways of sugar
fermentation and other biochemical processes has had a con-
siderable effect on the comparative biochemical point of view.
Not long ago, many microbiologists in particular believed that
all bacteria are built on essentially the same basic metabolic
pattern. The conspicuous differences in fermentation products
which were known to exist were thought to be attributable to
differences in relative rates of various reactions or to the omission
or addition of specific enzymatic steps. The possibility that a
given process, such as the conversion of glucose to pyruvate,
might occur by more than one mechanism was not seriously

78

BACTERIAL FERMENTATIONS

considered; the existence of two such pathways appeared
superfluous and even unreasonable. Therefore the discovery of
various alternative pathways for single processes has required a
revision of the concept of a basic metabolic pattern in terms of
processes rather than in terms of specific chemical mechanisms.
It may also be noted that alternate metabolic pathways
provide a useful set of characters for the analysis of phylogenetic
relationships among microorganisms. Past attempts to deduce
phylogenetic relationships from gross inorphology or fermenta-
tion product patterns have not been particularly successful, in
part because the numbers of characters available were too
restricted. This difficulty at least can be overcome by the
comparison of complex metabolic pathways which provide many
enzymatic steps linked together in process patterns that probably
represent the culmination of long sequences of evolutionary
development.

Butyric Acid Fermentation

Clostridium kluyveri is a good example of an anaerobic
bacterium that has been useful in the study of a fundamental
biochemical process, namely, the synthesis of fatty acids (5).
This organism was discovered more or less fortuitously while
making enrichm.ent cultures for methane-producing bacteria
with ethyl alcohol as the sole organic substrate. Examination
of the fermentation products in such cultures revealed that
butyric and caproic acids were frequently formed in large yields
along with acetic acid, and were always associated with the
presence of a Clostridium later called C. kluyveri. The formation
of these acids from ethyl alcohol provided direct confirmation for
the old theory that fatty acids containing an even number of
carbon atoms are built up from a C2 compound.

Further study of the substrate requirements for butyrate
synthesis by pure cultures of C. kluyveri demonstrated that both
ethanol and acetate are essential for the process, which occurs
stoichiometrically according to the equation
CH3CH2OH + CH3COOH > CH3CH2CH2COOH + H2O

79

H. A. BARKER

Tracer experiments indicated that the ethanol is oxidized to the
acetate level before being converted to butyrate. Thus acetate
or an activated derivative was identified as a major precursor of
butyrate. Later, Lynen's acetyl-coenzyme A (19), formed from
acetaldehyde and coenzyme A by a DPN-dependent oxidation,
was shown to be the activated form of acetate.

Even before the isolation of acetyl-CoA by Lynen, the
evidence for the existence of such a compound was greatly
strengthened by Stadtman's discovery that the exchange of in-
organic phosphate with acetyl phosphate in C. kluyveri extracts
is catalyzed by an enzyme, phosphotransacetylase, which is
coenzyme A dependent (33). This exchange could be most
easily interpreted by means of the following reaction

acetyl phosphate + CoA > acetyl-CoA + phosphate

which was subsequently shown to occur. The phosphotrans-
acetylase reaction has been very useful in enzymatic studies of
acetate metabolism, since it provides a convenient method of
forming acetyl-CoA.

A central problem in the metabolism of fatty acids was the
identity of the intermediates between acetate and butyrate.
Early investigations of fatty acid metabolism in animals sug-
gested that acetoacetate and j8-hydroxybutyrate might be
involved, but conclusive evidence for or against the participation
of these compounds was not forthcoming until the development
of cell-free enzyme systems that metabolize fatty acids. With
extracts of C. kluyveri, acetoacetate, /3-hydroxybutyrate, and other
possible C4 compounds in the same oxidation states were shown
definitely not to be intermediates in the interconversion of ace-
tate and butyrate (16). This and other evidence led to the
development of the idea that the intermediates are not simple C4
molecules but are C4-coenzyme compounds. This hypothesis
has been fully confirmed by the discovery of the role of the
acetyl, butyryl, crotonyl, j8-hydroxybutyryl, and acetoacetyl
derivatives of coenzyme A in both animal and bacterial systems
(19,21,33).

80

BACTERIAL FERMENTATIONS

Although many of the steps in the enzymatic conversion of
ethanol and acetate to butyrate and caproate are now known,
our understanding of the role of this process in the energy
transformation of the bacteria is very inadequate. Presumably
the formation of the G4 and Cs fatty acids, which involves a free-
energy change of approximately 12 kcal. per mole of ethanol
consumed, constitutes the main source of energy for synthetic
activities of the organism. This implies that the catabolic
reaction should result in a net formation of high-energy phos-
phate or other suitable energy donor. But as yet there is no
evidence that this occurs. The oxidation of acetaldehyde to
acetyl-CoA yields a high-energy thioester bond which can be
converted to a high-energy phosphate bond by the phosphotrans-
acetylase reaction. However, the thioester bond is not available
for this purpose, since it is required for the formation of aceto-
acetyl-CoA from acetyl CoA.

There appear to be two ways in which useful energy might
be provided during the C. kluyveri fermentation. One is by an
oxidation of acetaldehyde which is not coupled with butyrate
synthesis but with the formation of hydrogen gas as illustrated
in the following reaction, where Pi represents phosphate:

CH3CHO + Pi + ADP > GH3COOH + ATP + H2

It should be noted that hydrogen is formed in appreciable
amounts during the C. kluyveri fermentation. The free-energy
change is unfavorable (approximately 4 kcal.) for the above
reaction to proceed from left to right as written. However, by
coupling this reaction with strongly exergonic reactions of ATP,
such as the hexokinase reaction, which are involved in the
synthesis of cellular constituents, the over-all reaction would be
slightly exergonic (approximately —2 kcal.).

A second possible mechanism for net ATP formation in the
C. kluyveri fermentation is an oxidative phosphorylation de-
pendent on electron transport from ethanol and acetaldehyde
to the unsaturated fatty acid electron acceptors such as crotonyl-
CoA. The potential difference between the ethanol-acetalde-

81

H. A. BARKER

hyde and the butyryl-CoA-crotonyl-CoA system (21) appears
to be of the order of 0.2 v., which is more than enough to permit
the formation of one high-energy phosphate bond from ortho-
phosphate. Such a coenzyme-hnked phosphorylation could
account for the importance of butyrate and caproate synthesis
in the metabolism of C. kluyveri. However, as yet there is no
evidence for such a mechanism. In fact, it has not been possible
to observe a reduction of crotonyl compounds by reduced DPN
and TPN, which are formed in the oxidation of ethanol and
acetaldehyde.

Purine Fermentations

The fermentation of purines by C. acidi-urici and C. cylindro-
sporum presents some interesting and as yet partially unsolved
biochemical problems.

The main products of the fermentation of uric acid, xan-
thine, or guanine are ammonia, carbon dioxide, and acetate,
sometimes accompanied by small amounts of formate and
glycine (2,27) . Early experiments with cell suspensions indicated
that the mechanism of the purine fermentation is quite different
from that of uric acid oxidation via allantoin in animals and
aerobic bacteria. This conclusion was based in part on the
observation that neither allantoin nor urea is decomposed under
conditions in which uric acid is rapidly fermented.

The first definite indication of the path of uric acid break-
down was the discovery that glycine is formed in appreciable
amounts and is also decomposed when uric acid is simultaneously
available to the organism (2). The dependence of glycine
decomposition on the presence of uric acid suggested some sort
of interaction between the two substances. This was further
supported by the observation that certain cell suspensions of C
acidi-urici exhibit a conspicuous lag in uric acid decomposition,
which can be eliminated completely by the addition of glycine.
Later work (30) suggests that the oxidation of glycine is coupled
with a reduction of uric acid to xanthine.

82

BACTERIAL FERMENTATIONS

The formation of glycine and formate from purines points
to a similarity between tlie mechanisms of the bacterial fermenta-
tion and the synthesis of purines in animals. This similarity has
been further defined by the use of tracer methods (15,26).
Nitrogen atoms in the 1, 3, and 9 positions of the purine have
been shown to be converted to ammonia, and the nitrogen in
position 7 appears in glycine. Carbon from positions 2 and 6
goes to carbon dioxide, from positions 4 and 5 to the carboxyl
and methylene carbons of glycine, and from position 8 to formate.
The only conspicuous difference between the fermentation and
synthetic processes with respect to the tracer data is in the fate or
source of the carbon in position 2. In the uric acid of the pigeon
this carbon atom is derived from formate, whereas in the
fermentation it is converted to carbon, dioxide by a path not
involving formate. This suggests that the purine which is actu-
ally degraded by the bacterial system, as contrasted with the
purine added as a substrate, is more oxidized in the 2 position
than is the hypoxanthine ribotide which appears to be the first
purine derivative synthesized from nonpurine precursors in
animals (7).

Several types of evidence, which will not be given here,
indicate that uric acid, guanine, and hypoxanthine are converted
to xanthine before the purine ring structure is disrupted. The
decomposition of xanthine has been studied with crude cell-free
extracts (6,27,30), and has been shown to proceed in accordance
with the following equation

1 xanthine + 6H2O >

3 ammonia + 2 carbon dioxide + 1 formate + 1 glycine

which represents a nonoxidative process. Unlike intact cells,
the cell-free extracts used in these experiments do not form
appreciable amounts of acetate.

A question of special interest in xanthine decomposition is
the point of attack on the purine molecule. Previous studies on
purine synthesis in birds indicated that the ribotide of 4-amino-
5-carboxamidoimidazole combines with "formate" to yield a

83

H. A. BARKER

hypoxanthine derivative. If a similar but reverse process occurs
in the fermentation of xanthine, an initial attack on the bonds
between the 1,2 or 2,3 positions in the purine would be required.
Actually all available evidence is against an initial splitting of
these bonds. For example, 4-amino-5-carboxamidoimidazole
is not attacked nor does it accumulate under conditions favorable

HN-CO HN-CO HgN COOH

0^ C-^^-CO -^iU °9 ^^^CH H,0 OC C-N<.

HN-C-NH^ HN-C-NH' *- HN-C-NH"

URIC ACID XANTHINE 4-URE1D0-5-CARB0XY-

IMIDAZOLE

-NH3
-CO2

H N-CH '^ COOH

2NH3+ I +HCOOH^^ CH-N^ ;^ f'^^CH

COOH H^N-C-NH' H2N-C-NH'

_^

4-AMlNO-IMIDA- 4- AMI N0-5-CARB0XY-
ZOLE IMIDAZOLE

r

CH,0H CH, CH, ^^

I 2 -NH, I 3 .2H I 3 +C0-
CHNH2 ^ CO 1- COOH 2

COOH COOH

Fig. 1. Fermentation of uric acid. The solid arrows indicate known
reactions, the dotted arrows postulated reactions.

for xanthine decomposition. Moreover, in the presence of suit-
able inhibitors the major product of xanthine decomposition by
extracts has been identified as 4-ureido-5-carboxyimidazole
(28) which is formed by a rupture of the bond between the 1 and
6 positions. The ureido group of this compound is next attacked
enzymatically with the formation of ammonia, carbon dioxide,
and 4-amino-5-carboxyimidazole (27). The latter is decarbox-
ylated and then the imidazole ring of the resulting 4-amino
derivative is cleaved to give glycine, ammonia, and formate in
the cell-free system. This is undoubtedly a multistep process,
the details of which have not yet been worked out.

84

BACTERIAL FERMENTATIONS

In the fermentation of purines by living cultures of C.
acidi-urici, glycine and formate do not accumulate in considerable
amounts, whereas acetate is a major product. Tracer experi-
ments have established that both glycine and formate are con-
verted in part to acetate. Formate carbon goes into the methyl
group of acetate. The methylene carbon of glycine enters both
carbons of acetate, whereas the carboxyl carbon of glycine is
converted mainly to carbon dioxide. The mechanism of acetate
formation from glycine and formate has not been established
definitely, but a pathway involving serine is indicated by in-
direct evidence (30). Serine is rapidly converted to pyruvate
by cell-free extracts and pyruvate is oxidized to acetate and
carbon dioxide. Also glycine can be oxidized to acetate.

A schematic representation of uric acid fermentation by C.
acidi-urici and C. cylindrosporum as presently understood is given
in Figure 1.

Many aspects of the purine fermentation remain to be
elucidated. The steps in the conversion of 4-amino-imidazole
to glycine are not known. It is likely that this process involves
an "active formate" which facilitates the conversion of glycine
or a glycine precursor to serine. Energy useful for synthetic
purposes may also be generated during the conversion of xan-
thine to glycine ; otherwise it is difficult to see what purpose this
process serves. It is unlikely that this reaction sequence serves
only to provide precursors of serine and pyruvate, because these
compounds cannot replace purines as fermentation substrates.
At present there is no indication of the participation of purine
or imidazole nucleotides in the breakdown of purines, but in
view of the general similarity between the mechanisms of the
fermentation and purine syntheses in animals, a possible role of
nucleotides in the purine fermentation should be investigated
more closely.

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85

H. A, BARKER

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4. Barker, H. A., and F. Lipmann, J. Biol. Chem., 779, 247 (1949).

5. Barker, H. A., Harvey Lectures, Ser. 45, 242 (1949-50).

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7. Buchanan, J. M., Phasphorus Metabolism, 2, 406 (1952).

8. Cardon, B. P., and H. A. Barker, Arch. Biochem., 12, 165 (1947).

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17. Leaver, F. W., and H. G. Wood, J. Cellular Camp. Physiology, 41, 225
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19. Lynen, F., Federation Proc, 12, 683 (1953).

20. MacGee, J., and M. Doudoroff, Bacterial. Proc, 108 (1954).

21. Mahler, H. R., Federation Proc, 12, 694 (1953).

22. Mickelson, M. N., and C. H. Werkman, J. Bacterial., 39, 709 (1940).

23. Nisman, B., Bacertiol. Revs., 18, 16 (1954).

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25. Pine, L., V. Haas, and H. A. Barker, J. Bacterial., 68, 227 (1954).

26. Rabinowitz, J. R., unpublished data.

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28. Rabinowitz, J. R., and W. E. Pricer, Jr., Federation Proc, 73, 278 (1954).

29. Racker, E., Federation Proc, 7, 180 (1948).

30. Radin, N. S., and H. A. Barker, Proc. Natl. Acad. Sci. U. S., 39, 1196
(1953).

31. Scott, D. B. M., and S. S. Cohen, Biochem. J. {London), 55, 33 (1953).

32. Stadtman, T. C, and H. A. Barker, J. Bacterial., 67, 67 (1951).

33. Stadtman, E. R., Record Chem. Progr. {Kresge-Hooker Sci. Lib.), 75, 1 (1954).

34. Wachsman, J. T., and H. A. Barker, J. Bacterial., 69, 83 (1955).

35. Wachsman, J. T., and H. A. Barker, J. Bacterial., 68, 400 (1954).

36. Werkman, C. H., R. W. Stone, and H. G. Wood, Enzymolagia, 4, 24
(1937).

37. Wieringa, K. T., Leeuwenhoek, J. Microbiol. Serai, 6, 251 (1939-40).

38. Wood, H. G., J. Biol. Chem., 194, 905 (1952).

39. Wood, H. G., J. Biol. Chem., 199, 579 (1952).

86

SOME ASPECTS OF VITAMIN AND GROWTH
FACTOR RESEARCH

ESMOND E. SNELL, Biochemical Institute, University of Texas,

Austin. Texas

The fifteen years since 1940 have been marked by an un-
precedented rate of increase in our knowledge of the nature and
metabolic role of vitamins and similar growth factors. The
perfection of chromatographic methods of purification, together
with the partial substitution of rapid microbiological assays for
the more tedious bioassay procedures with higher animals, has
contributed much to this increase. Detailed accounts of the
current status of knowledge of the vitamins have appeared
(54,57,81); here no such account will be attempted. Rather
shall we be content to emphasize a few aspects of recent nutri-
tional research (and consequently slur others) and their implica-
tions for future progress in this field.

Microbiological Techniques in Vitamin Research

Some fourteen years ago Peterson (43) and Williams (80)
emphasized the growing importance of microorganisms as tools
in nutritional research by pointing out that work with micro-
organisms had resulted in discovery of certain of the B-vitamins
(pantothenic acid, biotin) and in the first demonstration of the

87

ESMOND E. SNELL

nutritional importance of several others (/?-aminobenzoic acid,
inositol, nicotinic acid). Since 1942, as illustrated in Table I and
discussed more fully below, microorganisms (chiefly those of a
single group, the lactic acid bacteria) have played a dominant
role as test organisms in the discovery and isolation of new vita-
mins and related factors.

But the discovery, isolation, and chemical characterization
of a new vitamin represent only the more glittering facets of a
many-sided problem. How is the vitamin distributed in nature?
In what forms does it occur? What essential role does it play
in metabolism? Investigation of these and related questions
also has been immensely furthered by use of microorganisms.
Since its introduction by Snell and Strong (71) in 1939 the
technique of comparing the growth response of microorganisms
to tissue extracts and to pure vitamin as a quantitative method
for estimation of vitamins in natural materials (reviews, 2,64,65)
has become fully accepted and has increased greatly our knowl-
edge of the distribution of the vitamins in nature. Complica-
tions sometimes arise in such assays from the presence of previ-
ously unsuspected vitamin derivatives or of metabolically related
substances, and the unraveling of such difficulties can provide
information relative to the latter two questions posed above.

The study of metabolic roles of the vitamins also has been
facilitated greatly through use of artificially induced nutritional
mutants of microorganisms (reviews, 13,36) and through detailed
study of the action of inhibitors structurally related to vitamins —
inhibition analysis (reviews, 58,81). It is the thesis of this
essay that bacterial nutrition still has much to contribute to the
study of nutrition of higher animals. Some directions in which
we may look for such contributions will be pointed out in the
sequel.

Vitamins and Growth Factors Identified since 1942

A brief and incomplete review of progress in recognition of
vitamins and growth factors since the similar review by Peterson

88

VITAMIN AND GROWTH FACTOR RESEARCH

in 1941 (43) will serve to illustrate the outstanding role played by
investigations with microorganisms.

A tabulation of vitamins and growth factors discovered,
isolated, or synthesized since 1942, together with the test organ-
isms employed during their isolation or characterization, is given
in Table I. The difficulties in assigning individual names and
dates to the discovery of vitamins is obvious especially where, as
with folic acid and vitamin B12, approaches from several different
directions with different test organisms have been made, and
where the specificity of assay procedures has been improved over
a period of years by several different investigators. The popular
tendency to assign a given scientific advance to a specific individ-
ual, although sometimes justified (depending upon the magni-
tude of the advance), more often represents an injustice to other
workers whose important contributions remain thereby un-
acknowledged. So it is with discovery, isolation, and character-
ization of the vitamins — never yet a one-man job ! The names
associated with the assay methods of Table I, therefore, represent
not necessarily the original discoverers of the growth factor but
rather the devisers of the first reasonably specific assay pro-
cedures that permitted real progress toward isolation and char-
acterization of that growth factor.

PYRIDOXAL AND PYRIDOXAMINE

Discovery of these two forms of vitamin Be resulted from an
attempt to devise a quantitative microbiological method for the
determination of pyridoxine, which at that time was considered
synonymous with vitamin Be. When lactic acid bacteria were
the test organisms and pyridoxine the reference standard,
impossibly high values for the "pyridoxine" content of natural
materials were obtained (73). Obviously, substances other
than pyridoxine were promoting growth in the vitamin Bg-
deficient medium. The observation (61) that the growth-
promoting activity of pyridoxine increased upon heating with
the growth medium and that pyridoxine itself was essentially in-
active in promoting growth (61,73) suggested that closely related

89

ESMOND E. SNELL

TABLE I
Progress in Recognition of Vitamins and Growth Factors, 1942-1955

I. Substances known to serve as vitamins for both animals and microor-
ganisms.

A. Pyridoxal and pyridoxamine

1. Bioassay organisms used in detection: Streptococcus faecalis
{lactis) R: Snell, Guirard, and Williams, 1942 (73)

2. Characterization and synthesis: Snell, 1944 (62); Harris,
Heyl, and Folkers, 1944 (20)

B. Folic acid (pteroylglutamic acid) and its conjugates

1. Bioassay organisms used in isolation and characterization:
Lactobacillus casei: Snell and Peterson, 1939, 1940 (69)
Streptococcus faecalis: Mitchell, Snell, and Williams, 1941 (37)
Chicks: Hogan and Parrott, 1939 (23)

2. Isolation

Pfiffner, Binkley, Bloom, Brown, Bird, Emmett, Hogan, and

O'Dell, 1943 (45)

Hutchings, Stokstad, Bohonos, and Slobodkin, 1944 (26)

3. Characterization and synthesis
Angier and co-workers, 1946 (1)

C. Folinic acid (leucovorin) and its conjugates.

1. Bioassay organisms used in isolation and characterization:
Leuconostoc citrovorum: Sauberlich and Baumann, 1948 (56)
Streptococcus faecalis and Lactobacillus casei: Bond, Bardos, Sibley,
and Shive, 1949 (4)

2. Characterization and synthesis

Pohland, Flynn, Jones, and Shive, 1951 (47)

Cosulich, Roth, Smith, Hultquist, and Parker, 1952 (11)

3. Isolation

Kcresztesy and Silverman, 1951 (27)

D. Pantetheine — Pantethine

1 . Bioassay organism used in detection :

Lactobacillus bulgaricus: Williams, Hofr-J0rgensen, and Snell,
1949 (82)

2. Purification and characterization

Peters, Brown, Williams, and Snell, 1953 C41)
Brown, Craig, and Snell, 1950, 1953 (8)

3. Synthesis

Snell, Brown, Peters, Craig, Wittle, Moore, McGlohon, and
Bird, 1950 (72)

90

VITAMIN AND GROWTH FACTOR RESEARCH
TABLE I (Continued)

E. Biocytin

1. Bioassay organisms used in detection:

Lactobacillus arabinosus and Lactobacillus casei: Wright and
Skeggs, 1944 (85)

2. Isolation

Wright, Cresson, Skeggs, Wood, Peck, Wolf, and Folkers, 1 952
(86)

3. Characterization and synthesis

Wolf, Valiant, Peck, and Folkers, 1952 (40)

F. Vitamin B12

1 . Bioassay organisms used in detection :
Man: Castle, 1929 (10)

Lactobacillus lactis Dorner: Shorb, 1947 (59)

2. Isolation

Rickes, Brink, Koniusy, Wood, and Folkers, 1948 (53); Smith,
1948 (60)

II. Microbial growth factors of undetermined nutritional significance for
animals

A. Lipoic acid (thioctic acid)

1. Bioassay organisms used in detection

Lactobacillus casei: Guirard, Snell, and Williams, 1946 (17)

Streptococcus faecalis (enzymatic): O'Kane and Gunsalus, 1947

(38)

Tetrahymena geleii: Stokstad, Hoffman, Regan, Fordham, and

Jukes, 1949 (78)

2. Isolation

Reed, DeBusk, Gunsalus, and Hornberger, 1951 (52)
Patterson, Brockman, Day, Pierce, Macchi, Hoflfman, Fong,
Stokstad, and Jukes, 1951 (46)

3. Characterization and synthesis

Hornberger, Heitmiller, Gunsalus, Schnakenberg, and Reed,

1952 (25)

Bullock, Brockman, Patterson, Pierce, and Stokstad, 1952 (9)

HI. Bacterial growth factors of dubious significance for animal nutrition
Growth factor Test organism Investigators

A. D-Alanine Lactic acid bacteria Snell, 1945 (63)

B. Putrescine Hemophilus parainjiuenzae Herbst and Snell, 1948

m

Table continued

91

ESMOND E. SNELL

TABLE I {Continued)

C. A^-Acctylglucosamine Lactobacillus bijidus
glycosides

D. D-Lactic acid (or other Lactobacillus casei
a-hydroxy acids) (mutant)

E. /)-Hydroxybenzoic acid Escherichia coli (mutant)

F. Various peptides Lactic acid bacteria

G. Thymidine and mis-
cellaneous nucleosides
and nucleotides

H. Coprogen

and

Ferrichrome
L Biopterin

Lactic acid bacteria

Pilobolus sp.

C< Cf

Crithidia
fasciculata

Gyorgy et al., 1954

(19a)

Kuhn, 1952 (31)

Camien and Dunn,

1953 (9a)

Davis, 1950 (12)

Sprince and Woolley,

1945 (77)

Snell, 1945 (63)

Kihara and Snell, 1952,

1955(28)

Hesseltine, Pidacks,
Whitehill, Bohonos,
Hutchings, and
Williams, 1952 (21a)
Neilands, 1952 (37a)

Patterson, Broquist,
Albrecht, von Saltza,
and Stokstad, 1955
(38a)

derivatives were the active substances, and a variety of chemical
treatments combined with microbiological testing showed that
partial oxidation of pyridoxine to an aldehyde, or its amination
to an amine, yielded highly active substances (62). Synthesis
(20) of the limited number of possible structures (62) divulged
the structures of two active compounds (formulas I and II), which
were named pyridoxal and pyridoxamine (20,62,70).

CHO

(I) Pyridoxal

CH2NH2

(II) Pyridoxamine

This was the first instance in which one of the B-vitamins
had been shown to exist in more than a single simple (i.e..

92

VITAMIN AND GROWTH FACTOR RESEARCH

unconjugated) form, and many investigators still have not
adjusted themselves to the changed situation but persist in speak-
ing of the "pyridoxine content" of foodstuffs (although in many
foodstuffs no detectable pyridoxine is present, all of the vitamin
Be being present as pyridoxal and pyridoxamine (49)) or of
"pyridoxine-deficient" rations (yeast, an excellent source of
vitamin Be, is pyridoxine-deficient — i.e., it contains little or no
pyridoxine* (49)). If accuracy in nomenclature is a prerequisite
to accuracy in thought, situations such as this, which occur fre-
quently in the vitamin field (cf. vitamin A vs. vitamin A activity,
folic acid vs. folic acid activity, etc.), should receive more
attention. The matter is not entirely academic, for whereas
pyridoxine is a very stable compound in foodstuffs, pyridoxal
because of its reactive carbonyl grouping is not, and it is possible
that an inaccurate nomenclature has contributed to the delay
in recognition of the destruction of vitamin Be that may occur
in food processing.

Immediately following synthesis of pyridoxal, one coenzy-
matic form of vitamin Be was recognized by Gunsalus and co-
workers (19) to be pyridoxal phosphate (formula III); sub-
sequently pyridoxamine phosphate (formula IV) also was dis-
covered by Rabinowitz and Snell (48) . The latter product was
later found to serve as an essential growth factor for certain lactic
acid bacteria (e.g., Lactobacillus delbrueckii (34)), which cannot

CHO CH2NH2

HO [T^ CH2OPO3H2 HO |r^CH20P03H2

HgClj^J HsCl^J

(HI) Pyridoxal phosphate (IV) Pyridoxamine phosphate

* This statement may appear in conflict with the reported isolation (32)
of pyridoxine from yeast. This procedure has not been reported in detail; in
answer to a query Professor Kuhn revealed that nitric acid was used through-
out this procedure, and volunteered the opinion that the pyridoxamine
originally present probably was converted (by nitrous acid) to pyridoxine
during the course of the isolation. Most other isolations of pyridoxine were
from rice-bran extract, in which this form of vitamin Be predominates.

93

ESMOND E. SNELL

Utilize pyridoxal or pyridoxamine efficiently, and to serve also
as a coenzyme in enzymatic transamination (35).

FOLIC AND FOLINIC ACIDS

The tangled investigational threads that met in the isolation
and characterization of folic acid (formula V) have been traced

.N^ ^N,

CONHCHCOOH
I
CH2

OH ^"2

(V) Folic acid (Pteroylglutamic acid)

elsewhere (54,57,44). Although the earliest observation of an
experimental deficiency of this substance was in monkeys
(vitamin M), the discovery failed to provide a practicable assay
procedure for its isolation. Such procedures were discovered
entirely independently, with chicks (vitamin Be) as the assay
organism in one instance, and lactic acid bacteria (eluate factor,
folic acid) in the other. The bacterial assays were used at one
or another stage in all of the successful isolations.

All the organisms that respond to folic acid respond similarly
to folinic acid (formula VI). Thus, the original observations of

CONHCHCOOH

H,NrY^CH, O f'
N^^N^iH^ ^NH CH2
OH I GH2 COOH

CHO
(VI) Folinic acid

growth responses in various organisms to crude supplements
included the response to both substances. Some evidence in-
dicates that the amount of folinic acid in tissues surpasses the
amount of folic acid. To what extent the latter is an artifact,
formed from the more labile folinic acid (or the even more

94

VITAMIN AND GROWTH FACTOR RESEARCH

CONHCHCOOH

N N^ II I CH^

OH H ' COOH

(VII) Tetrahydrofolic acid

labile di- and tetrahydrofolic acids (formula VII)) during
the isolation procedure, remains to be established. Pfiffner
and co-workers (45) in their paper describing isolation of folic
acid (vitamin Be) demonstrated clearly the existence of a more
labile compound which promoted growth of Streptococcus faecalis
and which closely resembled in properties those later established
for folinic acid. However, the clear-cut differentiation of this
latter substance required a specific assay method. This was
discovered by Sauberlich and Baumann (56) who found that
Leuconostoc citrovorum (more recently identified as Pediococcus
cerevisiae) required extremely high levels of folic acid for growth,
and then grew only slowly, but grew rapidly in the presence of
small amounts of liver extracts. A second assay procedure was
independently discovered by Shive and co-workers (4), who found
that inhibition of 5". faecalis by a folic acid analogue was prevented
much more effectively by liver extracts than could be accounted
for by their folic acid content. Just as pyridoxamine and
pyridoxal were earlier produced by empirical procedures from
pyridoxine, and their structure clarified without their isolation
from natural materials, so it proved possible to form folinic acid
from folic acid by empirical procedures, isolate the active reaction
product, and establish its structure (11,47). In this instance,
however, two difliicultly separable diastereoisomeric modifications
of the growth factor were produced, and final proof of the identity
of the naturally occurring and synthetic growth factor required
separation of the diastereoisomers and comparison with the
product isolated from natural materials.

Incomplete evidence indicates that in addition to these two
substances with folic acid activity, di- and tetrahydrofolic acid

95

ESMOND E. SNELL

also may occur naturally. In addition, there appear to occur
also conjugates of each of these compounds, that contain up to
(and possibly more than) six additional glutamic acid residues
and that vary in growth activity depending upon the number of
conjugated glutamic acid residues and the test organism. Which
if any, of these compounds represents the coenzyme form of the
vitamin is not known. The difficulty presented to the analyst by
the occurrence of a vitamin in so many forms is great, and the
problem of accurate and convenient assay has not been entirely
solved.

PANTETHEINE PANTETHINE

During an attempt to culture Lactobacillus bulgaricus in a
chemically defined medium this and a number of related organ-
isms were found to require an unidentified substance for growth.
Highly purified concentrates of the substance were prepared from

CH3

I
HOCH2C-CHOHCONHCH2CH2CONHCH2GH2SH

GH3

(VIII) Pantetheine

CH3
HOCH2-G-CHOHCONHCH2CH2CONHCH2CH2S-

CH3

(IX) Pantethine

O-CH2CH-CHCHOHCH-N \

0=P-OH

^o.

G=C

/ V

O PO3H2 N^ p-^^2

I C— N

0=P-OH GH3 H

O-CH2-G-GHOHCONHGH2GH2GONHGH2GH2SH

GH3

(X) Goenzyme A

96

VITAMIN AND GROWTH FACTOR RESEARCH

fermentation residues. It was noted that these concentrates re-
placed pantothenic acid for some organisms (e.g., L. arabinosus)
but not for others (e.g., Saccharomyces carlsbergensis), and, con-
versely, very large amounts of pantothenic acid replaced the
unidentified substance as a growth factor for L. bulgaricus. Thus
the substance appeared to be a combined form of pantothenic
acid, and further work revealed its structure to be that illus-
trated in formulas VIII and IX. These findings came just as
the structure of coenzyme A (formula X) was under active
investigation, and just as Lynen had shown the acyl- transferring
role of coenzyme A to take place via a sulfhydryl grouping. The
postulate (8,72) that pantetheine represented the then chemically
unidentified portion of coenzyme A was rapidly borne out by the
enzymatic resynthesis of coenzyme A from pantetheine and
adenosine triphosphate (16). This was the first recognition of
the occurrence of the j3-mercaptoethylamine residue in nature.
The fact that the growth factor for L. bulgaricus occurs naturally
in several chromatographically distinct forms was shown to result
from the presence of a variety of mixed disulfides formed by
oxidative cross-linking between pantetheine and other sulfhydryl
compounds (5).

Just as pyridoxal or pyridoxamine and folinic acid are more
closely related to the coenzyme form of these vitamins than
pyridoxine or folic acid, so then is pantetheine intermediate
between coenzyme A and pantothenic acid. In each case, a
specific requirement for these compounds by certain organisms
appears to result from inability or a markedly lowered ability
to eflfect conversion a in the sequence :

pvTidoxine 1

folic acid > > {

pantothenic acid J

It sometimes happens that conversion b is also diflficult.
We have referred to the fact that for some lactic acid bacteria
pyridoxamine phosphate is many hundred times more active
than pyridoxamine or pyridoxal in promoting growth. Simi-
larly, for a strain of Treponema pallidum neither pantothenic acid

97

pyridoxal 1 ^ fpyridoxal phosphate

folinic acid[ > •^coenzyone F

pantetheine J (coenzyme A

ESMOND E. SNELL >

nor panthetheine serves as a growth factor; panthetheine-
4 '-phosphate (formula XI, an intermediate between pantetheine
and coenzyme A) or coenzyme A itself must be supplied to per-
mit growth (77a),

Do conditions exist in which higher animals show similar,
synthetic disabilities, where the coenzymes or their more im-
mediate (and more readily available) precursors would be more
effective therapeutic agents than the vitamin forms of commerce?
We do not know. In experimentally produced deficiency states
(usually produced in young, undiseased animals) no evidence
for this exists, but it would be unwise to overlook the possibility
while the problems of pathology of all types, of aging, etc., are
still with us.

When we say that L. bulgaricus has a specific requirement for
pantetheine because of an insufficient capacity on the part of
the cell to convert pantothenic acid to pantetheine, it is important
to understand just what is meant. Experimentally, all that one
observes is that to permit growth, pantothenic acid must be
supplied in the medium at several hundred times the concentra-
tion that suflSces when pantetheine is supplied. But with
sufficient pantothenic acid, growth does occur, showing that
some of it has been converted to coenzyme A. Thus the con-
version enzymes do occur, and can function. Is the conversion
limited because (7) insufficient of the conversion enzyme (s)
is present, or (2) because the enzyme, though present, has a
lowered affinity for its substrate, or {3) because activity of the

PO3H2 CH3
OCH2-CCHOHCONHCH2CH2CONHCH2CH2SH

CH3

(XI) Pan tetheine-4' -phosphate

CHo COOH

I I

HOCH2CCHOHCONHCH2CH2CONHCHCH2SH

CH3

(XII) Pantothenylcysteine

98

VITAMIN AND GROWTH FACTOR RESEARCH

enzyme is in some way inhibited by presence of other substances,
or (4) because pantothenic acid, in contrast to pantetheine, is not
readily absorbed by this organism? All of these are possibiUties,
and any one of them would suffice to permit the observed result.
The growth experiment by itself does not favor one explanation
over the other.

Without implying preference for any particular explanation
in this instance, it may be useful to cite evidence showing that
insufficient attention has been paid in the recent past to cell
permeability as an explanation for differing activities of closely
related growth factors. Current evidence (cf. 6,7) indicates that
conversion of pantothenic acid (or pantoic acid) to coenzyme
A occurs via the following pathway :

-2H

Pantoic
acid

Pantothenic-
acid

Pantothenyl-
cysteine

Pantetheine < Pantethine

+ 2H

j8- Alanine Cysteine -2H

+ 2H

CO5

ATP

Pantothenyl
cystine

^ . « Pantetheine-
Coenzyme A < A > U U *

^ ATP 4 -phosphate

Acetobacter suboxydans does not grow in the absence of one of
the substances shown along the main line of this conversion.
Pantoic acid and pantothenic acid have equal activity but are
much less active than pantothenylcysteine, pantetheine, 4'-
phosphopantetheine, or reduced coenzyme A. This could be
interpreted as an inability to carry out reaction b readily. How-
ever, further investigation showed that the activity of pantothenic
acid (and pantoic acid), but not of pantetheine, was greatly in-
creased by lowering the pYi of the medium; this increased
activity approximated the increased concentration of undis-
sociated pantothenic acid that resulted from the drop in pH., and
the undissociated acids very nearly equalled coenzyme A in

99

ESMOND E. SNELL

growth activity (7). Thus it appears that cells of A. suboxydans
are relatively impermeable to the anions of pantoic and panto-
thenic acids, but not to the undissociated acids. Pantothenyl-
cystine and pantethine are without activity, but the reduced
compounds, pantothenylcysteine (formula XII) and pantetheine,
show full activity. The presence of the — SH group permits
ready absorption despite presence of an ionized carboxyl group,
for the activity of pantothenylcysteine is not j&H-dependent as
is that of pantothenic acid, pantoic acid, or pantothenylglycine
(7). This indicates that the — SH group is concerned in
absorption of these compounds through the cell membrane.

Are similar phenomena of importance in animal nutrition?
We do not know, but fragmentary evidence suggests that they
may be. Thus the conversion of pantothenic acid to coenzyme
A appears at present to occur in animals by the same pathway
shown above, and cell-free extracts of rat liver convert panto-
thenylcysteine to pantetheine (22). Yet, whereas pantothenic
acid and pantetheine have equal activities in promoting growth
of rats, pantothenylcysteine has none (79) . The result is difficult
to explain except by assuming either that the biosynthetic route
is incorrect or that pantothenylcysteine does not get into the
cells. Again, the observation that relatively massive doses of
vitamin B12 given by mouth are required in the absence of in-
trinsic factor to produce remissions in pernicious anemia, whereas
very small amounts are required by injection, may indicate
difficulty in absorption of this substance through the intestinal
wall. Does the intrinsic factor, by combining with vitamin
B12, provide a "handle" by means of which receptors in the
intestinal wall can transfer this vitamin to the circulation, much
as a free — SH group in pantothenylcysteine permits efficient
absorption of this substance by A. suboxydans despite the delete-
rious effects of an ionized carboxyl group?

BIOGYTIN

When yeast extract is assayed for biotin with Lactobacillus
arabinosus, the result is much lower than that obtained with L.

100

VITAMIN AND GROWTH FACTOR RESEARCH

casei; following acid hydrolysis the value given by the former
organism rises to the constant value obtained with the latter.
This observation of Wright and Skeggs (85) demonstrated the
natural occurrence of a biotin conjugate and illustrates again
the utility of quantitative comparative bioassays in the detection
of naturally occurring vitamin derivatives. Isolation (86) and
characterization (40) of the compound, which has the structure
illustrated in formula XIII, was followed by means of this
same assay procedure.

O

II

HC CH NH2

I I I

HoC^ GHCH2CH2CH2CH2CONHCH2CH2CH2GH2CH

S I

COOH

(XIII) Biocytin

This compound replaces biotin as a growth factor for
animals, and for some but not all bacteria (84). Its metabolic
significance is not yet known, but its unusual structure and the
high proportion of the total biotin in some materials comprised by
it indicate that it is more than a metabolic oddity.

VITAMIN B12

This spectacular substance is exceptional in all ways : in the
complexity of its structure, in the variety of active forms that can
be obtained from nature, in its content of a metal ion (cobalt), in
its unusally high (even for vitamins!) biological activity, and in
the fact that one group of investigators succeeded in isolating it
through the exclusive use of man as a test animal (aided greatly
in the latter phases, undoubtedly, by the distinctive red color of
the vitamin!). Nonetheless, its isolation in this country was
greatly aided by discovery that certain lactic acid bacteria re-
quired concentrates of it for growth under some conditions — an
observation first made by Shorb (59). Because of its activity in
controlling pernicious anemia in man, in partially alleviating

101

ESMOND E. SNELL

delayed growth in children, in stimulating growth of farm
animals fed rations based chiefly or exclusively on plant products,
and, from the biochemical side, because of its relationship to
nucleic acid synthesis and other biosynthetic reactions, it has
stimulated unusual interest, and many review articles dealing
with it are available {cf. 3,15).

The structure of vitamin B12 is not yet known in detail, but
it contains an atypical porphyrin skeleton (4a) the nitrogen atoms
of which occupy four of the six coordination positions of cobalt
(formula XIV) . * The fifth is occupied by a nitrogen of a nucleo-

atypical porphyrin

CHo ^
I
HOCHCH2NH2

HCCHOHCHCHCHoOH ,

0PO3H2-''

( XIV) Vitamin 6^2 (partial formula)

tide, the nature of which can vary in vitamin B12 isolated from
different source materials. Coordination position six is occupied
typically (but not exclusively) by an anionic grouping — cyanide
hydroxide, oxalate, sulfate, nitrite, etc. — so that for any one
parent structure containing a single nucleotide, a family of
closely related compounds of slightly different properties can be
obtained. Recent results (15a) show that by incorporating any
of ten or so different nitrogen bases in the medium, Escherichia
coli 113-3 will produce vitamin Bi2-like substances containing the
corresponding nucleotides in the molecule — a finding that

* Since this was written, the probable complete structure of vitamin B12
has been clarified by a combination of chemical and x-ray diffraction
methods (D. C. Hodgkin, et al., Nature, 176, 325 (1955) and R. Bonnett,
etal., Nature, 176, 328 (1955)).

102

VITAMIN AND GROWTH FACTOR RESEARCH

multiplies indefinitely the number of possible vitamin-like struc-
tures that can be obtained. These vary in their activities for
different test microorganisms and for animals. The first com-
pound isolated, first called vitamin B12 and later cyanocobalamin,
contained dimethylbenzimidazole as the nucleotide base, and
CN~ as the anion. It is fully active in animals and all micro-
organisms so far known, and is the vitamin B12 of commerce.
Pseudovitamin B12, which is similarly constituted but contains
adenine in place of dimethylbenzimidazole, has full activity for
some (not all) microorganisms, but little or none for animals.
The significance of this variability in structure remains to be
clarified.

Similarly, the relationship of the intrinsic factor to vitamin
B12 remains to be clarified. This substance appears to be a
glycoprotein present in normal individuals but not in those with
pernicious anemia, and is necessary for the absorption of the
small amounts of vitamin B12 ordinarily present in foodstuffs.
Whether it functions by combining with the vitamin and pre-
venting its absorption by intestinal bacteria, thus conserving it
for the host, or whether it functions otherwise is not known.
The possibility that it promotes direct absorption through the
intestinal wall has been mentioned earlier (see p. 100).

LIPOIC ACID (tHIOCTIC ACId)

As early as 1937, Snell, Tatum, and Peterson (76) observed
the marked stimulating effects of acetate on growth of lactic
acid bacteria, and included this substance in the nutrient
medium for the purpose of clarifying other nutritional require-
ments of these bacteria. It was observed (75) that the substance
not only served as a buffer but played some more specific role in
growth, and that it was not required in media supplied with
yeast extract (76) . Following elucidation of several other growth
requirements of these organisms, Guirard, Snell, and Williams
(17) returned in 1941 to an investigation of this substance and
demonstrated the existence in yeast and other natural materials
of a substance that duplicated the growth-promoting effects of

103

ESMOND E. SNELL

acetate for Lactobacillus casei but was far more active on a weight
basis. This observation thus provided an assay method of known
specificity for the substance. Independently, O'Kane and
Gunsalus (38) had observed that cells of Streptococcus faecalis
grown in synthetic medium required an unidentified substance in
yeast extract to permit oxidation of pyruvate to acetate. Placed
in juxtaposition, these findings suggested identity between the
two unidentified substances, and following comparative tests of
concentrates, Snell and Broquist (68) concluded that the two
substances were identical.

In an independent line of investigation, Kidder and Dewey
had successfully sorted out the complex nutritional requirements
of a protozoan, Tetrahymena geleii, and showed it to require one
or more unidentified substances. In 1949, Stokstad and co-
workers (78) showed that this unidentified substance contained
pyridoxal, copper ions, and an unidentified growth factor that
was named protogen, all of which were necessary for growth in the
basal medium. Supplementation of this medium with the
former two substances thus permitted development of an assay
of known specificity for protogen, and the preparation of con-
centrates. Such concentrates also were highly active in pro-
moting growth of L. casei — a fact that led Snell and Broquist (68)
to conclude that the acetate-replacing factor for L. casei and
protogen were identical.

By use of these assay methods, isolation of an active crys-
talline substance was first achieved by Reed et al. (52), who

CH2
HsQ^ ^CHCHgCHsCHsCHgCOOH

HS SH

(a)

/CH2
HaC^ ^CHCHgCHaCHsCHaCOOH

s s

(6)

;XV) Lipoic acid, (a) reduced and (6) oxidized

104

VITAMIN AND GROWTH FACTOR RESEARCH

named the crystalline substance lipoic acid, and shortly there-
after by Patterson et al. (46), who later renamed the product
thioctic acid. Chemical characterization and synthesis (9,25)
showed the substance to be 6,8-dithiooctanoic acid (formula XV) ;
it can exist in both the cyclic structure and as an open-chain
dithiol, the interconversion of the two being explicitly involved in
its enzymatic role as a cofactor in the oxidative decarboxylation
of pyruvate to acetate and other a-keto acids to the corresponding
carboxylic acids containing one less carbon atom (18,51).
Such oxidative decarboxylation also requires thiamin pyro-
phosphate, and it appears an unsettled point whether the two
are combined into a single coenzyme (51).

Most nutritional investigations with higher animals have
failed to show a growth response to added lipoic acid. However,
DeBusk and Williams (14) have recently observed both increased
growth and increased efficiency of food utilization in young rats
and chicks fed trace amounts of the substance. The reasons for
the differing results are not yet known. If these results are
confirmed, this will provide another instance of a vitamin dis-
covered, isolated, and characterized as a microbial growth factor
before its role in the nutrition of higher animals was suspected.
However, in a sense it is academic whether the substance is or
is not a vitamin, i.e., required in the diet of animals. There is no
doubt that it is a biocatalyst of great importance in animal as in
microbial and plant life, and the genetic accident that determines
whether the substance can or cannot be synthesized in animals,
though it has important implications for nutrition, hardly changes
the intrinsic biochemical importance of the substance.

OTHER MICROBIAL GROWTH FACTORS

A variety of additional substances found essential for growth
of one or another microorganism during the past few years is
listed in Table I. Although they are obviously important for the
organisms that require them, little is known of their metabolic
significance. D-Alanine is required for growth of many lactic
acid bacteria, but only in the absence of vitamin Bg. It is now

105

ESMOND E. SNELL

known that this interchangeability results from the fact that vita-
min Be is required for synthesis of this amino acid; when the
latter is supplied preformed, the requirement for vitamin Be
disappears, provided each of the L-amino acids required for
growth also is supplied (24,83), This remains the only known
instance of a D-amino acid being essential for growth, although
these substances, once thought not to occur naturally, have now
been frequently encountered in various microbial products, such
as antibiotics and capsules. A closer investigation (74) shows
the D-alanine occurs chiefly in the cell wall, of which it is appar-
ently an essential component, and in soluble cell extractives.
Recent investigation of the composition of bacterial cell walls
shows them to be rich in carbohydrate materials, including
amino sugars (55). It is possible that the requirement of
Lactobacillus bifidus {or A^-acetylglucosamine derivatives (19a,31)
and of some lactobacilli, grown with pentoses, for traces of glucose
(39) may reflect a requirement for these substances as structural
materials for cell wall formation.

/?-Hydroxybenzoic acid and D-lactic acid (or its homologues)
are examples of growth requirements found so far only in artifi-
cially induced mutants; this does not lessen their importance
but is mentioned to illustrate the utility of mutant organisms in
the detection of previously unrecognized essential metabolites.
The technique has been especially valuable in permitting detec-
tion and isolation of intermediates in biosynthetic reactions —
compounds which normally have such a transient existence that
their natural occurrence would be difficult to detect in any other
way. Space limitations do not permit tabulation and discussion
of the many compounds discovered in this fashion — each of which
can serve as essential organic nutrients for certain mutant
organisms under a restricted set of conditions.

Appropriate peptides stimulate or are even essential for
growth of many bacteria, and recent investigations have begun
to throw light on the reasons these compounds show activities
not shared by their component amino acids. Three model in-
stances have so far been observed : ( 7) assimilation of one amino

106

VITAMIN AND GROWTH FACTOR RESEARCH

acid, a, may be inhibited by a second amino acid, b. Under
these circumstances, peptides of a, perhaps because of their
greater structural dissimilarity to the inhibitor, may greatly sur-
pass the free amino acid in activity. For example, D-alanine
inhibits utilization of L-alanine by Lactobacillus casei, but not that
of peptides of L-alanine. Such peptides far surpass free L-alanine
in growth-promoting activity under these conditions and may
become obligatory requirements for growth (28). Many ex-
amples of this type of behavior are now known (28). (2) An
essential free amino acid may be subject to a degradation reaction
that rapidly destroys it before much of it can be used for growth,
i.e., protein synthesis. If its peptides are not subject to the
degradation reaction, they will greatly surpass the free amino
acid in growth-promoting activity. For example, tyrosine
peptides are much more active than free tyrosine in promoting
growth of S. faecalis under conditions such that free tyrosine can
be destroyed by tyrosine decarboxylase, but peptides and
free amino acid are equally active when conditions are so ar-
ranged as to make this enzyme nonfunctional, e.g., by growth in
the absence of vitamin Be (29). {3) For reasons still unknown
absorption of one free amino acid may take place with diffi-
culty or may require the presence of unusually high concentra-
tions of a second amino acid. Under these conditions, peptides
of the amino acid may be much more effective than the free
amino acid in promoting growth. For example, Lactobacillus
delbrueckii 730 requires relatively tremendous quantities o^ histi-
dine for growth, but very small amounts of histidine peptides.
In this same organism, tyrosine peptides are much more active
than free tyrosine in promoting growth when the histidine
concentration is low, but equally active when the histidine con-
centration is raised (42), and these phenomena cannot be ac-
counted for in terms of either mechanism (7) or (2).

None of the instances cited above need be interpreted as
indicating direct utilization of the peptides without prior hydrol-
ysis; indeed some evidence suggests that hydrolysis occurs be-
fore utilization. In none of these experiments, further, is there

107

ESMOND E. SNELL

evidence for a special catalytic role of the stimulatory peptides
in metabolism, although the possibility of such a role for other
peptides is not, of course, excluded.

Variability in Nutritional Requirements with Conditions

Cursory reading of the nutritional literature, especially that
dealing with animal nutrition, leaves the impression that the
nutritional requirements of a given species for vitamins, etc.,
is a rather constant thing, that these requirements can be deter-
mined and expressed in terms of a quantitative figure for the
average daily requirement which has validity quite apart from
the conditions used in assessing the requirement.

From parts of the foregoing discussion it should be clear
that this is far from the case in microbial nutrition. Lactobacillus
casei grows well in the absence of both D-alanine and L-alanine
peptides if pyridoxal is supplied; in the absence of pyridoxal,
both D-alanine and such peptides are required, the former be-
cause it cannot be synthesized in the absence of vitamin Bg, the
latter because D-alanine inhibits utilization of free L-alanine
(which also cannot be synthesized in the absence of vitamin Be)
but not that of L-alanine peptides. Again, L. casei requires
relatively large amounts of biotin if aspartate and unsaturated
fatty acids are omitted from the medium, much less if aspartate
(but not the fatty acid) is supplied, and none at all if both prod-
ucts are supplied. Such sparing effects of one nutrient on the
requirement for another frequently indicate important metabolic
roles played by the vitamins. In the instances cited above, a
role for vitamin Be in the synthesis of d- and L-alanine is indicated,
and the necessity for biotin in the synthesis of aspartate and un-
saturated fatty acids likewise appears highly probable. Similarly,
the necessity for folic acid in the synthesis of the purine and
pyrimidine bases and serine early became evident from the fact
that in appropriate media these substances greatly reduced or
eliminated completely the requirement of L. casei and S. faecalis
for folic acid.

108

VITAMIN AND GROWTH FACTOR RESEARCH

Examples of this nature could be multiplied indefinitely,
and a more complete discussion has been presented elsewhere
(66,67). They have their basis in the fact that the vitamins are
required only to permit the functioning of enzymes that in turn
manufacture products essential for growth. Frequently, if
these products are supplied preformed, or tlie necessity for the
enzymic functioning is otherwise bypassed, need for the enzyme
(and hence for the vitamin) is reduced, and with some simple
organisms can be eliminated completely. One strongly suspects
that closer investigation would show a similar variability
(though less marked because of the greater organization and
range of metabolic reactions present) in higher animals. The
well-authenticated variability in the thiamin requirement of
animals with the proportion of the caloric intake supplied as
fat, and in the vitamin B12 requirement with the choline intake,
are straws indicating the direction research may take in these
matters.

As the number of new nutrients available for study decreases,
perhaps animal nutritionists will turn more toward the study of
such interrelationships between nutrients and their significance
for practical nutrition.

Some Future Areas for Nutrition Research

The last few years have seen a decrease in the rate of dis-
covery of new vitamins and growth factors. Some experimental
animals can now be grown on essentially synthetic diets, and it
is becoming more and more difficult to detect entirely new
bacterial growth factors. Does this mean we are approaching
the time when no more dietary factors remain to be discovered?
Although this time must eventually arrive, it appears unlikely
that it is yet here. Some of the readily available (though experi-
mentally more difficult) experimental animals have not been
grown on defined diets as yet, and there are indications of un-
identified dietary requirements for some of these (e.g., monkeys
(78a)). Similarly, little is known of the nutritional requirements

109

ESMOND E. SNELL

of thousands of microbial species. Only a dent has been made
in the problem of the nutrition of protozoa and insects.

The discovery by Fraenkel (1 5b) of vitamin B-p as an essential
growth factor for the meal worm, Tenebrio molitor, and its iden-
tification as carnitine by Carter (9b), a compound that also occurs
in higher animals but whose function is unknown, illustrates the
fruitfulness of investigations of the nutrition of the currently
neglected lower animals. Having discovered the factors required
in common by most living species, we may be on the verge of
discovering those nutrients characteristically required by a
restricted number of species (c/. D-alanine) . If such nutrients
exist, knowledge of them would be invaluable in permitting design
of antimetabolites directed against specific groups of organisms.

In bacteria, appropriate nutrients can sometimes partially
overcome the deleterious influence of certain drugs {cf. the effect
of glutamine on alcohol tolerance in some bacteria (50)).
Similar relationships should hold in animals and provide a large
area for investigation. Proper nutrition practice has already
reduced the incidence of many of the infirmities of old age.
Can further progress along these lines be made ; can, indeed, the
aging process itself be slowed?

Such questions suggest that, far from being an obsolescent
field, the future may hold triumphs for experimental nutrition
equal to those of the past.

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82. Williams, W. L., E. HoflF-J^rgensen, and E. E. Snell, J. Biol. Chem.,
177, 933 (1949).

83. Wood, W. A., and I. C. Gunsalus, J. Biol. Chem., 190, 403 (1951).

84. Wright, L. D., E. L. Cresson, K. V. Liebert, and H. R. Skeggs, J. Am.
Chem. Soc, 74, 20QA {\952).

85. Wright, L. D., and H. R. Skeggs, Proc. Soc Exptl. Biol. Med., 56, 95
(1944).

86. Wright, L. D., E. L. Cresson, H. R. Skeggs, T. R. Wood, R. L. Peck,
D. E. Wolf, and K. Folkers, J. Am. Chem. Soc, 72, 1048 (1950) ; ibid., 74,
1996 (1952).

114

THE SIGNIFICANCE OF INDUCED ENZYME

FORMATION*

S. SPIEGELMAN and A. M. CAMPBELL, f Department of
Bacteriology, University of Illinois, Urbana, Illinois

Introduction

It is no historical accident that a renewed interest in the
phenomenon of "enzymatic adaptation" came hard on the heels
of the explosive birth of modern microbial genetics. Two good
reasons can be advanced for this virtual concurrence. In the
first place, the work with Neurospora, yeast, and the bacteria
provided the genetically controlled material necessary for bio-
logically intelligent and interpretable experiments. Secondly,
from its very inception, microbial biochemical genetics posed
the gene-enzyme problem in terms of a rapidly accumulating
multitude of experimental facts which could not be ignored.
Their existence demanded an immediate search for tools which
would permit the experimental analysis of such statements as
"genes control enzymes."

There were two obvious directions which a quest of this na-
ture could, and did, take. One started with the gene and led to

* The original experiments reported here from the authors' laboratory
were supported by grants from the National Cancer Institute of the U. S.
Public Health Service and the Office of Naval Research.

t Present address: Department of Bacteriology, University of Michigan
Ann Arbor, Michigan.

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S. SPIEGELMAN AND A. M. CAMPBELL

examination of the enzymatic consequences of individual modi-
fications in the genome with the hope that the interrelations re-
vealed by their careful analysis would ultimately yield a clue to
the underlying mechanisms of genetic control. The other,
starting from the enzyme, adopted the premise that fruitful
speculation on the role of the gene was unlikely in the absence of
a relatively detailed understanding of the mechanism of enzyme
formation. Those who felt inclined toward the second ap-
proach were necessarily forced to look for a system in which the
process of enzyme synthesis could be experimentally analyzed.
It is not surprising that the attention of these workers was im-
mediately turned to the phenomenon known as "enzymatic
adaptation." There had existed in the microbiological litera-
ture since 1888 a number of instances subsumed under this desig-
nation in which the presence of a particular compound ap-
parently resulted in a well-defined change in the enzymatic
activities. However, before these or similar examples could be
accepted as systems suitable for the study of enzyme synthesis,
the following criteria had to be rigorously satisfied.

7. The changed enzymatic activity observed must not be
due to the selection of preexistent mutant types but rather to an
induced enzymatic modification against a constant genetic back-
ground.

2. The observed change in enzymatic activity must be a
reflection of the appearance of an active apoenzyme, rather than
due to the accumulation of cofactors or intermediates unique to
the metabolism of the inducing substrate.

The use of recently evolved genetic methodology adapted
to the analysis of microbial populations, and of modern en-
zymological procedures, made it possible to demonstrate in
certain instances that both criteria could be met. These re-
searches have been thoroughly summarized and discussed in
recent reviews (55,75,90) and need not be detailed again here.
Certain consequences (71,72) may, however, be noted. The-
oretically these findings possess obvious implications for the
problem of gene function. They establish that possession by a

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INDUCED ENZYME FORMATION

cell of a particular gene in its nucleus does not thereby guarantee
that the corresponding enzyme will be found in the cytoplasm in
utilizable amounts. Thus, predictive description of phenotype
from a knowledge of genotype alone is impossible, even at the
basic level of enzymatic constitution. It was necessary therefore
to revise such statements as "genes control enzyme synthesis" to
read "genes control the potentiality of enzyme synthesis."

Whereas such theoretical implications are of great interest,
it was the emergent experimental consequences which proved to
be of greater importance. In successfully meeting the two
criteria mentioned above, an unequivocal demonstration was
provided that the induced syntliesis of specific enzymes could be
attained under relatively simple and controllable conditions
against a constant genetic background.

It was evident that the system had been found and defined
which converted into experimental reality the possibility of
inquiring into the mechanism of enzyme synthesis. It is the
purpose of the present essay to summarize the information which
has emerged from the study from such systems. A complete
detailed account of the findings of the field will not be found
here. On the contrary, the prerogatives of the essayist have
been exercised to select and interpret that which is most pertinent
according to the writers' own peculiar bias. Those readers who
find themselves in agreement with both the selection and the
interpretation will label the result an example of good judgment
and analytical perception. Others will label it differently.

The very nature of the phenomenon virtually dictates the
kinds of questions which are initially asked of it. The presence
of certain agents called inducers can, in the presence of a suitable
energy supply, stimulate the formation of specific enzymes. The
use of inducible systems to elucidate the mechanism of enzyme
formation resolves itself quite naturally into attempts to provide
adequate answers to the following questions :

a. What is the nature of the precursor material which is
transformed into active enzyme molecules?

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S. SPIEGELMAN AND A. M. CAMPBELL

b. What is the nature of the enzyme-forming mechanism
which converts the precursor material into active enzyme?

c. What is the role of the inducer, the presence of which
specifically stimulates the appearance of the corresponding
enzyme?

We can now turn our attention to an estimation of the ex-
tent to which answers can be provided to the questions just
proposed.

The Nature of the Precursor

The problem of the precursor is perhaps most dramatically
exhibited by considering inductions carried out under the
simplest circumstances. It has been shown by a number of
workers with a variety of enzyme systems (55,75,90) that enzyme
synthesis can be induced in the absence of a nitrogen source in
cells suspended in a buffer solution of the inducer. That the
appearance of enzyme activity actually involves the formation of
enzyme has been established in these cases by exhibiting the
homologous enzyme in extracts prepared from the induced cells.
In such inductions, the nitrogen employed by the cell in fabricat-
ing the new enzyme molecule must come from some preexisting
nitrogenous components, and one is immediately faced with the
obvious necessity of identifying the components so employed.

Before considering the most recent experiments which have
led to a satisfactory resolution of this problem, it is of interest to
note briefly some of the earlier work which, although inconclu-
sive, nevertheless exerted a strong influence on the subsequent
development of this aspect of the problem. Monod (53), in his
classic investigation into the growth of bacteria, discovered the
existence of a severe interaction between enzyme-forming systems,
which was expressed by the fact that simultaneous synthesis of
two metabolically unrelated enzymes did not occur on exposing
cells to a mixture of the two relevant inducers. In general, only
one of the enzymes was formed at a time. A similar situation
was uncovered in the yeasts by Spiegelman and Dunn (80).

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INDUCED ENZYME FORMATION

With yeast the presence of an external nitrogen supply greatly
suppressed the severity and the extent of this interaction, and in-
deed, under certain circumstances simultaneous formation to
two otherwise interfering enzymes was made possible.

Although these interactions were discovered relatively early
in the renewed investigation of the phenomenon of enzymatic
adaptation, their detailed significance remains to be delineated.
Nevertheless, at the time, they were interpreted to suggest a
competition for some commonly required nitrogenous material
and therefore implied the existence of a precursor not as yet
specified as to its ultimate enzymatic function. To these findings
may be added those which established that the induction of
enzymes requires the participation of a functional and utilizable
energy-generating mechanism. Agents such as 2,4-dinitro-
phenol (50), sodium azide (74), and arsenate (93) which pre-
vent the utilization of energy generated by metabolism also
inhibit the induction of enzyme activity.

The importance of these early observations derived essen-
tially from the fact that they made unlikely one quite obvious and
attractive possibility of explaining the phenomenon of induced
enzyme formation. This hypothesis suggests that the mech-
anism involved is one akin to the activation of trypsinogen to
trypsin. Of course, the fact that energy is required for the induc-
tion precludes at once any simple application of this concept.
In addition, such a model would suppose the preexistence in a
cell of inactive forms of enzymes already fully determined as to
their specificity and their function. The inhibitory interactions
observed would be difficult to explain under these circumstances.
Although observations of this nature could not definitely decide
the issue, they clearly encouraged the search for a nitrogenous
precursor not as yet restricted in its specificity and potentially
convertible into more than one kind of enzym.e molecule.

In designing experiments which seek to reveal the nature of
this precursor, one can be guided by the fact that, in principle,
three mechanisms of enzyme synthesis can be written down.
They are as follows:

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S. SPIEGELMAN AND A. M. CAMPBELL

complex precursor > active enzyme (1)

complex precursor + free amino acids > active enzyme (2)

free amino acids > enzyme (3)

Reaction (1) assumes the preexistence in the noninduced cell of a
complex precursor which can be converted into active enzyme
without the involvement of the free amino acids. This property
distinguishes it from mechanisms (2) and (3) and permits an ex-
perimental decision.

Evidently what we are asking is whether it is possible for the
precursor to become active enzyme without the participation of
the free amino acid pool. Putting the question this way suggests
immediately the necessity for examining the effect on the syn-
thesis of enzyme of any experimental condition which decreases
the availability of the free amino acids. Several methods are
available and have been employed for achieving a restriction of
this nature and they may be listed as follows :

a. The use of amino acid analogues as specific agents to
prevent the incorporation of the free amino acids into protein.

b. In the case of those cells which possess an internal amino
acid pool, to examine the effect of depletion and replenishment
of this pool under conditions which would minimally disturb
other components of the cell,

c. The use of amino-acid-deficient mutants which would
make unavailable specific components.

Experiments along all these lines have been realized with
yeast and the bacteria. The following paragraphs summarize
briefly the evidence obtained.

THE EFFECT OF AMINO ACID ANALOGUES ON ENZYME SYNTHESIS

Halvorson and Spiegelman (32) carried out a study with a
series of more than 40 analogues of amino acids for their effects
on induced formation of alpha glucosidase in Saccharomyces
cerevisae. A parallelism was established between the capacity of
an analogue to inhibit net protein synthesis, as measured by
growth, and its ability to suppress enzyme synthesis. In the case

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INDUCED ENZYME FORMATION

of the effective analogues, complete and specific reversal of the
inhibition was achieved by the addition of the homologous
amino acid. The generality of these findings was extended by
the independently performed experiments of Lee and Williams
(47) who demonstrated that the administration of ethionine to
the intact rat prevented the formation of tryptophan peroxidase.
In the experiments with the yeast (32,34,36,83), it was possible
to demonstrate by direct analysis that the presence of an effective
amino acid analogue inhibits incorporation from the free amino
acid pool into the protein fraction. One interesting feature
which emerged from these experiments is that the presence of any
one of the active amino acid analogues prevents the incorpora-
tion not only of its homologue but virtually of all the other amino
acids as well. Further, no peptide fragments, unique to pools
derived from cells incubated with an amino acid analogue, could
be found.

In these studies, no evidence for an amino-acid-independent
transformation of a complex precursor into active enzyme was
obtained. The data rather led to the conclusion that the primary
pathway of the induced formation of enzyme in nondividing cells
of yeast involves the compulsory utilization of the internal free
amino acids. The fact that the utilization of nonhomologous
amino acids was blocked concurrently suggested further that the
first stable intermediate formed in the synthesis of an enzyme
molecule is of such a complexity as to demand the simultaneous
availability of a large portion of the component amino acids.

THE EFFECT OF THE AVAILABILITY OF FREE AMINO ACIDS
ON ENZYME SYNTHESIS

If the conclusions derived from the experiments with amino
acid analogues are correct, it would be expected that the ability
of cells to form enzyme should parallel the availability of free
amino acids for protein synthesis.

One striking difference between the enzyme-forming ca-
pacity of yeast as distinguished from that of many Gram-negative
bacteria receives simple explanation in these terms. Thus,

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S. SPIEGELMAN AND A. M. CAMPBELL

yeasts are able to form enzymes when suspended in nitrogen-free
solutions of inducer, whereas the Gram-negative bacteria in
general require an exogenous supply of nitrogen as a necessary
concomitant of enzyme synthesis. The work of Taylor (96)
suggests a reasonable explanation for this apparent independence
of the yeast enzyme-synthesizing mechanism. This investigator
surveyed a variety of yeast and bacteria for the presence of free
amino acids in their internal environment. Of the three yeast
types examined, all possessed detectable quantities of the five
amino acids looked for. Among the bacteria, the Gram-posi-
tives possess primarily glutamic acid and lysine. None of the
Gram-negatives included in the survey contained detectable free
amino acid by the procedures employed.

The possession of an internal pool was subsequently found to
be a universal attribute of a wide variety of yeasts (85). It
would appear that the ability of yeast to get along without an
external source of nitrogen for enzyme synthesis is due to the fact
that they have internal supply. To examine the question then
of the effect of free amino acid availability on enzyme formation
in the yeast it was necessary to devise and employ procedures
capable of modifying these pool levels both quantitatively and
qualitatively. Using such procedures, Halvorson and Spiegel-
man (33) demonstrated a strong correlation between enzyme-
forming capacity and pool level in both depletion and replenish-
ment cycles. The results obtained in these studies supported the
conclusion that free amino acids constitute the quantitatively
predominant source of nitrogen in the formation of new enzyme
molecules. Again, no evidence was uncovered which suggested
the existence of a detectable amino-acid-independent transfor-
mation of a preexisting complex prescursor into active enzyme
molecule,

ENZYME FORMATION BY AMINO ACID AUXOTROPHIC MUTANTS

The third approach mentioned which could provide rele-
vant information involves the use of the auxotrophic mutants
deficient in the ability to synthesize one or another of the amino

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INDUCED ENZYME FORMATION

acids. It was obvious that organisms such as yeast which ac-
cumulate an internal free pool would be difficult to employ in
such studies, and not unexpected difficulties arose when they
were attempted with the yeasts. Fortunately, however, this
approach was successfully applied almost simultaneously in two
laboratories, and it is interesting to note that in both instances
the organisms employed, Escherichia coli and Aerobacter aerogenes,
possess a vanishingly small internal supply of free amino acids.

One of these investigations stems from the illuminating
studies of Monod and Cohn (55) and their collaborators into the
formation of beta-galactosidase by the ML strain of E. coli. It is
interesting to note that in the course of these studies Cohn and
Torriani (19,20) had discovered the existence of an enzymati-
cally inactive protein (Pz) which was serologically related to the
beta-galactosidase. In addition to this obvious structural
relationship, they established a suggestive correlation between
the distribution of the Pz protein and the capacity of the cells to
synthesize the beta-galactosidase. Finally, they also showed
that a significant decrease in Pz occurred in cells during the in-
duced synthesis of beta-galactosidase. Although not the only
possible hypothesis entertained by the authors, it is clear that all
of these observations would receive ready explanation if Pz
were indeed the precursor of the beta-galactosidase. In any
event, taken together, the observations noted offered the most
impressive evidence existent in the literature to support the sug-
gestion that a preexistent complex specific precursor is involved
in the synthesis of a known enzyme.

There was, however, one fact that militated against the
acceptance of this view. Nitrogen-starved cells, though pos-
sessing normal amounts of Pz, showed no ability to snythesize
the beta-galactosidase unless an external source of nitrogen was
provided. It was nevertheless still possible to imagine that the
starvation procedure interfered in some way with the metabolic
step required for transformation of Pz into active enzyme.

Monod, Pappenheimer, and Cohen-Bazire (56) undertook
to investigate this question further by employing a series of

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S. SPIEGELMAN AND A. M. CAMPBELL

mutants each of which was deficient in the abihty to synthesize a
single amino acid. These mutants were subjected to a "specific
starvation" by being grown in a medium in which the required
amino acid was present in Hmiting quantities, whereas all other
compounds were in excess. Immediately upon the cessation of
growth which attended the exhaustion of the amino acid, an
inducer of the beta-galactosidase was introduced. It was found
that little or no enzyme was synthesized by cells so treated,
despite the fact that they contained normal amounts of Pz.
Such cells do, however, form enzyme immediately upon the
addition of the amino acid they are unable to synthesize. These
results made it necesssary to abandon any interpretation of the
relation between Pz and the beta-galactosidase which involves a
direct, amino-acid-independent conversion of Pz into active
enzyme. The large number of amino acid auxotrophs em-
ployed in this study would suggest further that if Pz is indeed the
precursor, a considerable number and variety of amino acids
must be added to it before it is converted into active enzyme.

Ushiba and Magasanik (98) employed essentially the same
approach in their study of the adaptive utilization of myoinositol
by mutants of Acetobacter aerogenes. The results obtained led
these authors also to the conclusion that the induced formation
of the enzymes they were studying involved extensive synthesis
from the amino acids. In a subsequent study, Rickenberg et al.
(63) reported similar experiments and results with E. coli
strain K12,

IS THERE A PREEXISTENT COMPLEX PRECURSOR?

The experiments cited thus far make unlikely any mechanism
of synthesis which presumes the conversion of a preexistent pre-
crusor into enzyme by a process which is independent of the free
amino acids. The only alternatives left are, either that there is
no preexistent complex precursor, or that one does exist but be-
comes active enzyme only after the incorporation of amino acids.
The most obvious experimental approach aimed at a choice
between these alternatives would appear to be the use of isotopic

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INDUCED ENZYME FORMATION

labels. Thus, the induction of enzyme synthesis in uniformly
labeled cells suspended in unlabeled medium should provide
the necessary data, providing the enzyme synthesized can be
isolated in a pure state and its iso topic content determined.
Rotman and Spiegelman (66), and Hogness, Cohn, and Monod
(38) independently undertook to provide data relevant to this
issue, using the beta-galactosidase system E. coli.

Rotman and Spiegelman (66) secured uniformly labeled
cells by growth in C^^ lactate. Enzyme was induced for short
periods while the cells were suspended in nonradioactive me-
dium. The beta-galactosidase synthesized was isolated and puri-
fied by means of zone ionophoresis through starch columns.
Further purification was achieved with the aid of specific
precipitation with purified antiserum. The results obtained
revealed that less than 1 per cent carbon of the newly formed
enzyme molecules could have been derived from any cellular
components existing prior to the moment of the addition of the
inducer. In the experiments of Cohn, Hogness, and Monod,
S^^ was employed as the isotopic label and similar methods were
used for the isolation of the enzyme. Identical results and con-
clusions were obtained.

These findings virtually eliminate any hypothesis which
assumes the preexistence of a complex precursor material which
is convertible into enzyme. It is evident that a mechanism
suggesting the de novo formation of enzyme from amino acids is at
present the only one which has received experimental support.

A consequence of considerable importance issuing from this
last conclusion is that induced enzyme synthesis is thereby
equated to the process of protein synthesis. It follows that data
derived from the study of enzyme induction are pertinent and
relevant to the more general problem of protein formation.
Further, the use of inducible enzymes as model systems of pro-
tein formation can, in principle and fact, confer two significant
operative advantages. In the first place, one is assured that the
synthesis of a protein is being examined — a certainty not avail-
able to experiments dependent solely on incorporation studies.

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S. SPIEGELMAN AND A. M. CAMPBELL

Secondly, the formation of as little as 0.01 ng of new protein can
be detected with ease and precision.

Precursor and the Nature of the Enzyme-Forming System

In the present discussion, the term "enzyme-forming sys-
tem," hereinafter referred to as EFS, will be used to designate
that structure in the cell which is directly and "personally"
involved in the process of fabricating the enzyme molecule.
This verbal device is employed to isolate the EFS conceptually
from all the other cellular components which can and probably
do intervene more or less indirectly in the synthetic process. It
will, of course, be noted that there is an assumption made here.
By so stating the problem we do presume the existence of such a
unique structure and, at least implicitly, ignore the possibility
that proteins and enzymes are formed by a multitude of cooper-
ating and sequential reactions.

In thinking about the possible nature of the EFS and in
designing experiments to clarify the mechanism of its functioning,
it is difficult to avoid being influenced by the results of the
investigations into the precursor question. In a sense, these
findings force an active search for EFS. The data we have
reviewed relevant to the precursor problem are satisfyingly clear-
cut, almost distressingly so. They lead compellingly to the
conclusion that in fabricating a new enzyme molecule, the cell
prefers to weave it rather than to stamp it into existence. In
this process, the simplest components are employed. Further,
we find no evidence for any stable intermediates smaller than
that requiring the presence and utilization of all the amino acids.
A stepwise formation beginning with simple peptides and pro-
ceeding through polypeptides of intermediate lengths would
appear to be eliminated. From one point of view, this is of
course a pessimistic conclusion. It suggests that a successive
approximation to an understanding of how proteins are syn-
thesized will not be achieved in terms of a gradually better in-
sight gleaned from the study of intermediate pieces of increasing
complexity as they approach the final stage of synthesis. A

126

INDUCED ENZYME FORMATION

door is thus slammed upon an extremely attractive approach for
tackling the question of protein formation. Those who accept
these conclusions obviously are faced with the necessity of find-
ing a new approach to the solution of the problem of protein
synthesis.

Adopting a mechanism of protein synthesis which involves
the simultaneous availability of the constituent amino acid leads
one quite naturally to consider a template-type mechanism. It is
unnecessary here to undertake an extensive discussion of the
recent information (3,4,12,13,21) obtained from radioactive ex-
periments with intact animals and tissues which have attempted
to decide between template and stepwise mechanisms. In
general, unequal labeling of the protein has been taken as an
argument against the template hypothesis.

A serious difficulty is introduced by the realization that
the incorporation of label compounds need not provide us with
information relevant solely to the question of total protein syn-
thesis. Exchange reactions are necessarily included in the
information obtained from such experiments. That a very real
difficulty exists is well illustrated by the recent experiments re-
ported by Gale and Folkes (25,26) in which a dissociation of net
protein synthesis from incorporation by exchange reactions has
been demonstrated. As had been shown with yeast (32), it was
found that /?-chlorophenylalanine can prevent new protein
synthesis in Staphylococcus aureus. In addition. Gale and Folkes
discovered that although the presence of this analogue effec-
tively prevents the incorporation of phenylalanine into the
protein, it has relatively little effect on the exchange incorpora-
tion of glutamic acid.

The existence of such phenomena makes it difficult to inter-
pret with certainty the significance and uniqueness of data
derived solely from incorporation studies.

On the Role of the Inducer

One of the most dramatic features of the phenomenon of
enzyme induction is the requirement for the presence of the

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S. SPIEGELMAN AND A. M. CAMPBELL

inducer and the apparent precision with which it specifically
stimulates the formation of a particular enzyme.

Monod and Cohn (55) have recently reviewed the bulk of
the published information relevant to inducer function. Much of
this knowledge we owe primarily to the efforts of these two men
and their collaborators. Certain experiments are particularly
pregnant with interesting implications for the nature of EFS and
it is these which will occupy our attention.

SPECIFICITY AND THE RELATION OF AN INDUCER TO THE
HOMOLOGOUS ENZYME

There are two possible mechanisms of inducer action which
can be formulated with sufficient exactitude to permit their
experimental elimination. These have been labeled by Monod
and Cohn (55).

7. The Functional Hypothesis. The synthesis of an
enzyme is presumed to be linked to its activity, from which
hypothesis one would conclude that effective inducers would
necessarily be substrates.

2. The Equilibrium Hypothesis. It is assumed that
inducers must form complexes with enzyme synthesized as a
sine qua non of enzyme formation.

A number of observations eliminated the first hypothesis.
Thus, Spiegelman, Reiner, and Sussman (88) demonstrated
that maltose can induce alpha-glucosidase at pli values that
preclude detectable metabolism of the maltose even by fully
induced cells. Analogues of natural substrates have been
employed for similar purposes. Thus, alpha-methyl-glucoside
can be shown (76,82) to be an inducer of maltase under con-
ditions in which utilization cannot be detected. Further,
Monod, Cohen-Bazire, and Cohn (54) have demonstrated
similarly that thio-methyl-beta-D-galactoside and melibiose,
neither of which is detectably metabolized by strain ML of
E. coli, are nevertheless inducers of beta-galactosidase.

The second hypothesis was proposed in its simplest form
first by Yudkin (103) who did so in terms of a mass action hy-
pothesis in which combination between inducer and enzyme is

128

I

INDUCED ENZYME FORMATION

assumed to drive the reaction in the direction of precursor con-
version into enzyme. This complexing concept leads to several
predictions, the experimental violation of any one of which
would require either its revision or complete abandonment.
We may list some of these as follows :

7. Substances which can complex with enzymes should be
effective inducers.

2. Substances shown to be incapable of complex formation
with a given enzyme likewise should be unable to induce this
enzyme.

3. The dissociation constant of the substance measured in
terms of its inductive effect on enzyme synthesis should be com-
parable in magnitude to the value obtained in experiments in
which the constant is derived from complexing properties of
the inducer with enzyme.

It should be noted that the term "complex formation"
employed here is meant to subsume both the specific type,
characterized by combination between an enzyme and its sub-
strate or competitive inhibitor, and the nonspecific type, repre-
sented by a complex between an enzyme and a noncompetitive
inhibitor. Thus, proof that an inducer does not form a specific
complex with enzyme does not eliminate it as an enzyme com-
plexant, since it leaves open the possibility of nonspecific com-
plex formation. There is no a priori reason for believing that
nonspecific combinations cannot function in the process of
enzyme synthesis.

It is difficult to provide convincing exceptions to the second
deduction mentioned, involving as it does only negative proper-
ties. However, violations of both (7) and {3) have been found.
Thus, Lederberg (45) exhibited a mutant of E. coli which fails to
respond to lactose as an inducer of beta-galactosidase, despite the
fact that lactose is a substrate and therefore a specific com-
plexant.

Monod, Cohen-Bazire, and Cohn (54) subjected these con-
siderations to the first systematic and thorough analysis. They
revealed that thio-phenyl-beta-D-galactoside, although a potent

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S. SPIEGELMAN AND A. M. CAMPBELL

competitive inhibitor of the beta-galactosidase, was nevertheless
incapable of inducing the formation of this enzyme. Other
specific complexants, e.g., neolactose and phenyl-beta-D-
galactoside, were similarly found devoid of inductive capacity.

An instance of an apparent violation of the third deduction
was provided by Spiegelman and Halvorson (82). The dis-
sociation constant of methyl-alpha-D-glucoside, an inducer of
alpha-glucosidase synthesis in Saccharomyces cerevisiae, was deter-
mined and compared with the constant derived from its com-
plexant properties with enzyme. The two values were found to
differ by a factor of about two hundred.

It must be emphasized that the types of hypothesis made
unlikely by the experiments cited are highly restricted in nature.
All of these experiments had in common an examination of the
effect of the inducers as enzyme complexants in terms of meas-
urable effects on enzymatic function. By their very nature
they could not preclude the possibility of combination at some
site which possessed no consequences in terms of a detectable
modification of enzyme activity.

It must further be noted that such experiments do not
eliminate the possibility that inducer serves as a coupling agent
which leads to the formation of a stable complex among enzyme,
inducer, and a third component. If the inducer would combine
with enzyme effectively only in the presence of the third com-
ponent, violation of all three predictions could obtain.

THE QUESTION OF STOICHIOMETRY OF INDUCER ACTION

Until the advent of the resourceful exploitation by Pollock
of the penicillinase-forming system of B. cereus, the question of
stoichiometry was one which could be formulated but hardly
resolved. Pollock (59-61) discovered that fleeting exposures of
cells to minute concentrations of penicillin at ° C. can lead to
the specific adsorption of enough inducer molecules to permit
penicillinase formation even subsequent to the destruction of all
unbound penicillin. With the use of S^^-labeled penicillin it
was found that specific fixation is saturated at about 80 atoms

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INDUCED ENZYME FORMATION

per cell. Having purified the penicillinase and determined the
minimum turnover number, Pollock and Torriani (cited in 62)
were able to demonstrate that each molecule of penicillin thus
fixed can cause the formation of at least ten molecules of pen-
icillinase. These observations then establish that an inducer
can act catalytically.

The unique feature of virtually irreversible adsorption of
inducer has thus far not been exhibited in other enzyme-forming
systems, and hence this type of experiment is at present feasible
only in the penicillinase-producing system.

The Relatiofiship of Inducer to EFS

The data reviewed thus far indicate that enzyme molecules
are fabricated on a template. Further, inducer molecules
appear to be specifically bound to some site concerned with
enzyme synthesis. Identification of the inducer-complexing
site with the template is the simplest and most economical hy-
pothesis at present worthy of further exploration. The question
naturally arises whether combination between template and
inducer leads immediately to full function.

The least complicated variety of such an "activator"
hypothesis would assume that a full complement of templates
preexists and that the addition and adsorption of inducer mole-
cules is all that is required to convert them to complete activity.
This relatively simple model possesses several easily testable pre-
dictions. It would, for example, suggest that, providing other
conditions are not limiting, enzyme synthesis should proceed at
its maximal rate immediately subsequent to adsorption of in-
ducer. Further, once inducer molecules are adsorbed, one
would not expect on a priori grounds to find the emergence of
any strikingly different properties of the EFS as a consequence
of the accumulation of enzyme molecules.

It is not too difficult to marshal evidence pointing up the
inadequacy of such activator mechanisms as suitable models of
induction. We may now consider the nature of this evidence

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S. SPIEGELMAN AND A. M. CAMPBELL

with a view to seeing whether it circumscribes the nature of the
induction mechanism sufficiently to serve as a useful guide in the
construction of a more suitable model.

THE KINETICS OF ENZYME SYNTHESIS
AND ITS INTERPRETATION

One of the most obvious ways of examining the adequacy of
mechanisms analogous to the activator hypothesis is in terms of
its kinetic consequences, and historically this was the first type of
data provided. The kinetics found for the induced synthesis of
maltase and galactozymase in yeast were found to be auto-
catalytic and could be accurately described by the logistic equa-
tion (71,72). Subsequently other systems were found to obey
the same kinetics (41).

The results obtained suggested that in the early phases of
the induction, enzyme-forming capacity increases as new enzyme
molecules are produced. The data are clearly inconsistent with
a simple activator hypothesis, and on the same ground the Yud-
kin mass action model (103) for inducer function was rejected
(71), since it also predicts maximal rates of enzyme formation
from the onset of induction. To explain the autocatalytic
kinetics, as well as certain data of a genetic nature to which we
will have reference below, the so-called "plasmagene" hypoth-
esis was proposed (72). In this it was assumed that the in-
ducer functions by stabilizing a complex between enzyme and
the "plasmagene" and that this complex is capable of replicat-
ing itself and producing enzyme. The widespread implica-
tions of this hypothesis and the admittedly limited experimental
support available at its formulation acted as a stimulatory
irritant. Equally plausible alternatives for explaining the
logistic kinetics were quickly offered by a number of authors.

Thus, it has been argued (53) that enzyme formation was
being followed in these studies employing metabolizable inducers
which could serve as an energy source. Under such circum-
stances, obviously the more enzyme there is present, the greater
is the rate of energy generation, and the more rapid therefore

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INDUCED ENZYME FORMATION

would be the formation of enzyme. As a consequence, the
kinetics of enzyme formation would be autocatalytic independ-
ently of the actual kinetic details of the enzyme-forming process.
Although eminently plausible, it was pointed out (76,90) that an
argument of this nature could not be used to explain away the
autocatalytic formation of hydrogenlyase, the functioning of
which could hardly yield sufficient energy for protein synthesis.
Further, this criticism turned out on subsequent investigation
(35) not to be applicable to the system against which it was
directed. An examination of the relation between the kinetics
of enzyme formation and the rate of energy supply revealed that
alpha-glucosidase synthesis is exponential only when the rate at
which energy is delivered exceeds a certain critical value. Below
this it is linear with time. Thus, if one actually performs the
induction in such a manner (e.g., anaerobically, with highly
purified maltose) as to insure that the inducer is the sole energy
source, linear and not autocatalytic kinetics are obtained for the
early phases of the induction. The original experiments were
performed aerobically under conditions in which a relatively
high endogenous respiration was the principal energy source,
the contribution of inducer metabolism being negligible.

Monod and Cohn (55) have quite correctly emphasized
that wherever possible, the complications which can attend the
utilization of an energy-generating inducer should be avoided by
the use of what they call "gratuitous induction." Conditions of
gratuity require the use of a nonutilizable inducer and a neutral
noninductive energy source. The discovery that alpha-methyl-
glucoside is a nonutilizable inducer of the alpha-glucosidase
system made it possible to reexamine the question of the kinetics
of alpha-glucosidase synthesis under gratuitous conditions.
Again exponential kinetics were obtained (35).

There exists another interesting possibility which, if it ob-
tained, would make interpretation of the kinetics of induction
difficult and uncertain. This stems from the fact that in following
the appearance of enzyme in a population cell, an over-all
average property is measured. Were the process of enzyme

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S. SPIEGELMAN AND A. M. CAMPBELL

synthesis in individual cells relatively more rapid than the time of
the experiment, and if a lag period in the onset of enzyme forma-
tion were normally distributed in the cell population, one would
obtain a cumulative curve of enzyme with time which would
have the appearance of a logistic. Benzer (8) was able to dem-
onstrate that this was not true under conditions of gratuity by
the ingenious use of bacteriophage as a tool. The virus was
employed here both to stop enzyme formation and to release the
enzyme by lysis of those cells which had accumulated enough
enzyme during the period of induction to support virus multi-
plication. Using this device he demonstrated that enzyme
synthesis is uniform in all cells in gratuitous inductions but is
heterogeneous if the inducer is the primary or sole energy
source.

This question has not been completely resolved in yeast.
An interesting attempt to do so was, however, made by Terui
and Okada (97), who photomicrographically examined the
distribution of the division times in a population of cells in which
the energy required for the division was derived from the in-
ducing substrate maltose. They come to the conclusions that
the kinetics of the over-all adaptation is explainable on the basis
of a normal distribution of lag times in the population. There
are certain features of the experiment which make for uncer-
tainty. By its very nature, it is performed under nongratuitous
conditions which, according to Benzer's results, lead to inhomo-
geneities. Further, onset of division may not be an adequate
measure of induction level. Finally, as has already been noted,
it is relatively easy to change the kinetics of enzyme formation in
the very system these authors studied from the logistic to one
which is linear with time. Whereas one can explain logistic
kinetics on the basis of a normal distribution of lag times, it is
virtually impossible to explain linear kinetics on a similar basis,
since it would involve assuming a completely unrealistic dis-
tribution.

Monod, Pappenheimer, and Cohen-Bazire (56) examined
the kinetics of beta-galactosidase formation in E. coli by the use

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INDUCED ENZYME FORMATION

of an interesting device involving a relative plot in which in-
crement in enzyme is plotted against increment in cell mass.
This procedure has many advantages for studying relative
synthetic rates, since direct comparisons can be made between
slow- and fast-growing cultures. The data provided by certain
inducers yielded linear curves when plotted in this manner.
They suggest, therefore, that their findings are not consistent with
an autocatalytic mechanism. It should be noted, however,
that whereas the relative plot is extremely useful for the study of
relative rates of synthesis, it is a comparatively insensitive pro-
cedure for the kinetic examination of the early phases of the
induction. The time interval required for a definably measur-
able increment in mass could well be long enough to make im-
possible the examination of the kinetically interesting period of
induction. That this is not a remote possibility is easily shown
with yeast which form enzyme quite a bit more slowly than bac-
teria. If the data of yeast induction are plotted relatively, a
linear phase is observed. However, the early points show an
exponential departure from linearity. Were attention confined
solely to the later points, complete agreement with the data of
Monod, Pappenheimer, and Cohen-Bazire would have been
obtained. Finally, it must be noted that recent information on
inducer fixation (15) in the beta-galactosidase system is difficult
to reconcile with linear kinetics.

A word may be interposed here about time linear kinetics
since a number of authors have taken its existence to nullify the
significance and interpretability of the autocatalytic type. It is
a fact that one can convert a synthetic reaction which is in-
herently autocatalytic to one which is linear with time, and the
possibility should neither occasion surprise nor engender con-
fusion. Thus, there is little doubt that under the usual con-
ditions the growth of bacterial populations is exponential.
Nevertheless, linear growth of a streptomycin-requiring strain
was reported by Schaefer (67). In discussing this observation
Monod (52) pointed out that a similar type of kinetics should be
obtainable artificially by establishing a constant limited supply

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S. SPIEGELMAN AND A. M. CAMPBELL

of an essential metabolite. He thus forecast his own discovery
(51) of the "bactogen" as well as the independent development
by Novick and Szilard (57) of what they call the "chemostat."

It is not surprising that whenever limitations are imposed,
whether they be of energy (35), inducer (60), or of a required
metabolite, linear rates of enzyme synthesis are observed. One
must therefore agree that enzyme synthesis need not be always
autocatalytic. However, linear and other (24) deviations from
autocatalytic kinetics may signify the presence of restrictions
which prevent the exhibition of the exponential kinetics of which
the system is capable. In any case, such deviations cannot
be accepted as compelling evidence against the existence of
exponential rates of enzyme formation.

The presence of a rising rate of enzyme synthesis means
that some self-reinforcing activation of the enzyme-forming
system is occurring during the induction. A further analysis of
the mechanism and the role of the inducer in it requires the
application of other analytical devices. One of these approaches
is genetic, and we turn now to the data derived from its use.

THE INHERITANCE OF ENZYME-FORMING CAPACITY AND
ITS SIGNIFICANCE FOR THE ROLE OF THE INDUCER

Kinetic arguments, no matter how refined, cannot of them-
selves be decisive. It was fortunately possible to examine the
question of the autocatalytic nature of enzyme formation by
completely different means and one which provided a more
subtle and surprisingly fruitful method for further probing into
the question of inducer function. The potentiality of examining
induction by essentially genetic methods was suggested by the
discovery of a new and pathological type of induction. Winge
and Roberts (102) labeled their new phenotype "long term
adapter" because it was much slower in adapting to galactose
than the normal type. They further showed that the "slow"
character was determined by an allele (g^), which was recessive
to the wild type (G).

Spiegelman, Sussman, and Pinska (89) undertook an an-

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INDUCED ENZYME FORMATION

alysis designed to uncover the mechanism underlying the
"slowness." They came up with the surprising finding that the
vast majority (99.9%) of ^s"Cell populations were noninducible.
When plated on galactose-EMB test plates they yielded small
negative clones. If the one out of a thousand positives were not
simply mutants, a situation of signal significance would be
available, for it would mean heritably different enzyme-forming
capacity against a constant genetic background. This turned
out to be the case since fluctuation tests designed to determine
the nature of the origin of the positives were inconsistent with
a mechanism involving random mutation. The Poisson variance
obtained strongly suggested that contact with galactose was
necessary and induced in a small proportion of the cells a
heritable modification leading to the ability to form the enzyme.
Once acquired, enzyme-forming ability was transmitted in-
definitely from one cell generation to the next, providing galactose
was present. However, growth of the positive cells in the
absence of the substrate resulted after six or seven generations
in a mass reversion to the original negative phenotype. The
mass reversion feature virtually eliminated any mutational
interpretation of the phenomenon.

A detailed examination of the kinetics of the reversion was
made. The data obtained were most simply explained by as-
suming that positives contained units necessary for enzyme for-
mation lacking in the negatives. These units or elements could
increase only in the presence of the inducer galactose. Growth of
positives in the absence of galactose leads therefore to a dilution
of the relevant units, and a point is finally reached at which cells
are produced possessing either none or an insuflRcient number of
these elements.

The nature of the conclusions and the potential usefulness
of the system for the study of the formation of enzyme-synthe-
sizing units warranted an attempt to obtain more critical evi-
dence. A more directly interpretable analysis of the nature of
the reversion phenomenon was undertaken (79) by the serial
isolation and characterization of single-cell progeny produced by

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S. SPIEGELMAN AND A. M. CAMPBELL

positives during division in the absence of substrate. These
single-cell analyses confirmed the earlier experiments cited, in
demonstrating that abruptly after about five generations cells of
the negative phenotype are produced with increasing frequency.
The data were uniquely amenable to a probability analysis. It
was found that a fully induced positive cell contains in the
neighborhood of a hundred particulate units and that the
minimal number of particles required for a cell to exhibit the
positive phenotype on a test plate is in the neighborhood of one.
The probability of a given particle passing into the daughter
cell was closely approximated by one half.

It proved further possible to offer evidence for the
extrachromosomal nature of the particles in this particular
instance by the comparative study of the transmission of the
enzyme-forming capacity during reductional segregation (78).
The method used was identical to that employed in a previous
study involving the inheritance of the ability to ferment meli-
biose (86), with a similar result. The effect of prior induction of
(Gg^) heterozygotes to galactose on the segregation of the
capacity to form the enzyme was studied. Noninduced hetero-
zygotes, with few exceptions, yield a 1 : 1 ratio of positive to
negative spores on galactose test plates. However, if the
heterozygotes are induced prior to segregation, and the latter
occurs in the presence of substrate, all four segregant spores
exhibit the positive phenotype in over 90 per cent of the asci
dissected. Growth in the absence of substrate for seven to
twelve divisions of four such positive spore clones, leads to a
reversion of two of them to the negative phenotype, thus re-
storing the Mendelian ratio of the phenotype. The fact that
homozygous recessives carried through the same procedure do
not yield positive spores suggests that the presence of both
the dominant gene G and the substrate leads to the uniform pro-
duction of the extrachromosomal elements which are necessary
for enzyme formation. When, in the course of the segregation,
elements are incorporated into the cytoplasm of spores carrying
the g^ allele, they are converted to the positive phenotype. The

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INDUCED ENZYME FORMATION

abrupt reversion of such positive cultures to the negative pheno-
type upon growth in the absence of inducer is in agreement with
the previous studies noted.

The autocatalytic property of the enzyme-forming system
or some necesssary portion of it is here dramatically illustrated in
terms of the transmission of enzyme-synthesizing capacity from
mother to daughter cell. At least one is needed to make more
since, if by chance a cell is produced with none, it and its progeny
are negative.

The results obtained on the inheritance of the ability to
form galactozymase parallel in many respects the prior investi-
gations of Ephrussi (23) and his co-workers into the "petite"
mutant. The data of these workers also suggest the existence of
randomly distributed particulate elements in the cytoplasm pos-
sessing autocatalytic properties and responsible for the produc-
tion of cytochrome oxidase. The autocatalytic nature is again
exhibited by the spontaneous occurrence of cells lacking the
particles which lead to the irreversible loss of the ability to form
the corresponding enzymes.

It has been possible to demonstrate (89) that the particu-
late elements involved in the two instances are distinct en-
tities, since the two enzyme-forming capacities are lost sepa-
rately. In the "petite" mutant case control of the number of
elements of the cell is apparently not achieved by variation of
inducer availability. However, cells lacking particles can be
produced by treatment with the acridine dye, euflavine. It is of
interest to note that Slonimski (69,70) has been able to demon-
strate that this compound which is so effective in preventing the
transmission of the particles from mother to daughter cells also
specifically inhibits their ability to form enzyme.

One can go beyond an independent exhibition of the
autocatalytic nature of the enzyme-forming mechanism. The
possibility of controlling active particle numbers in the case of
the slow adaption to galactose provided an experimental system
which permitted a further analysis on the role of inducer and its
relation to the active particles and enzyme.

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S. SPIEGELMAN AND A. M. CAMPBELL

Let US first consider an hypothesis (55) which would explain
the autocatalytic property in terms of enzyme function analogous
to that advanced to explain the kinetic data. Under this
hypothesis, the possession of a small amount of galactozymase can
efTect the generation of more galactozymase by virtue of the fact
that this enzyme system is involved in the energy-generating mech-
anism to the cell. The role of the galactozymase in this hy-
pothesis is confined to the supply of energy. The active particles
would then be "aggregates" of sufficient amounts of galacto-
zymase to serve as energy generators. Campbell and Spiegel-
man (11) used a respiratory deficient strain to demonstrate that
functional enzyme is lost long before the autocatalyst necessary
for further enzyme synthesis, and hence these two cannot be
identical.

The quantitative results which were obtained in the study
of the reversion from the positive to the negative state led to the
formulation of a relatively precise hypothesis concerning the
detailed nature of the reversion process. The hypothesis may
be summarized in the form of the following series of statements :
( 7) the distribution of active particles among cells is assumed to
be normal, (2) during growth in an inducer-free medium these
particles neither decrease nor increase in absolute number and
hence the average number per cell decreases exponentially,
(3) the particles are distributed at each division randomly with
equal probability to the mother cell and to the bud, and (4) the
minimal number of active particles that a cell must contain in
order to score as a positive is close to one.

These properties were derived solely from a study of the
transformation from the positive to the negative state. Anal-
ysis of the reverse process quickly led to other features which
illuminated further details of the process. Rotman and Spiegel-
man (65) were able to convert a large percentage of negatives to
the positive phenotype by treatment with fractions of yeast
extract. It was possible to show that each of the positive cells
so obtained acquired only a few active particles as a result of the
conversion. On the basis of the properties of the conversion it

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INDUCED ENZYME FORMATION

was proposed that the transformation of a negative into a posi-
tive cell does not involve the de novo formation of active particles
but rather the activation of preexistent inactive units. We thus
have two categories of particles, active and inactive. A third
type was revealed through an analysis by Campbell and Spiegel-
man (10) of the growth of active particles. This third kind is
intermediate to the active and inactive varieties and is charac-
terized by being easily converted by inducer to an active unit —
an event which is rare with the inactive particles. We will
designate this intermediate type as convertible particles. Their
presence is indicated by an abrupt rise in active particle number
when reverting positive cells are exposed to inducer. Subsequent
increase of active particles obeys an approximately exponential
law. The convertible particles occur with increasing fre-
quency as positive cells are allowed to go through dilution
growth in the absence of inducer. Further, they appear to
suffer decay, since if they are not brought into contact with in-
ducer soon after their appearance, activation by inducer is no
longer possible.

Another peculiar feature which emerges from a study of the
late portion of the reversion is an anomalous change in stability
of active particles. It was shown in one of the earlier studies
noted (89) that active particles are perfectly stable in the early
reversion divisions. However, it was found (81) that this stabil-
ity disappeared after about the seventh division in the absence of
inducer.

A Model of Enzyme-Forming System Relating
Template, Enzyme, and Inducer

In discussing the information derived from genetic ex-
periments we have deliberately used such neutral words as
particles, units, and elements.

We should now like to essay a synthesis of the biochemical,
kinetic, and genetic information we have reviewed thus far in
terms of the simplest model consistent with the known observa-
tions.

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S. SPIEGELMAN AND A. M. CAMPBELL

The model to be presented is essentially the one designed by
Campbell and Spiegelman (10) in an attempt to explain certain
paradoxical aspects of the growth kinetics of active particles in
long-term adapting stocks. Its major features are most easily
seen and developed in terms of "slow" induction. Once these
have been exposed, the scheme will be generalized and applied
to normal enzyme synthesis.

The biochemical investigation of the precursor aspect of
enzyme synthesis leads to the conclusion that a template is
involved. The kinetics of normal induction and the genetic
data obtained with the aid of "slow" strains both suggest that
induction is characterized in its early stages by an autocatalytic
activation. All the data can therefore be described in a unified
fashion by the assumption that initially the templates are rel-
atively inactive and are autocatalytically converted to full
activity during the course of the induction.

We are thus led to identify the autocatalytic active particle
defined by the genetic operations with the autocatalytically
activated template demanded by the biochemical studies.

We now inquire where in our model is the inducer likely to
fit and how it is to function. Both kinetic and genetic experi-
ments indicate that active templates increase in number during
growth in the presence of inducer. Removal of the inducer
results in complete cessation of the increase, but the number
present at the moment of removal remains constant for many
hours. In our opinion the simplest explanation which can be
offered to explain this effect is that inducer, or some derivative
of it, is irreversibly incorporated into the structure of the active
template. If one proceeds along this line of reasoning, one
difference between an active template and an inactive one
would be that only the former contains galactose. However, if
this were the only difference, exposure to galactose should con-
vert negative "slow" cells into positive ones, because in one
generation half of the templates would have been formed in the
presence of galactose. This suggests then that an active tem-
plate differs from the inactive form in some property other than

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INDUCED ENZYME FORMATION

the possession of the inducer. The possibiHty is therefore pro-
vided for a third template type, neither active nor inactive,
possessing the second property but not the galactose. A de-
scription is thus provided for the "convertible" template, which
becomes stabilized in the active form on the addition of inducer.

We now turn to a consideration of the likely nature of this
''second property" possessed in common by active and "con-
vertible" templates, but lacking in the inactive variety. For
simplicity and ease of following the argument, we may here
summarize the properties which any model constructed on the
basis of the above discussion must exhibit.

/. Each cell contains a certain number of templates
specific for the synthesis of some enzyme of the galactose system.
They may exist in "active," "inactive," or "convertible" forms.

2. A cell containing one or more active or convertible
templates will give rise within a few hours of growth in a galac-
tose medium to progeny in which most or all of the templates are
active.

3. A cell containing no active or convertible templates
may grow for many generations on galactose without any tem-
plate ever becoming active. If activation does occur it is a
rare event. It is heritable on the cellular level as required by
property (2).

4. Active templates differ from convertible ones in that
the former contain galactose. Convertible templates are readily
transformed to active ones by the addition of galactose.

5. Active templates form the enzyme mentioned under
(7) ; inactive ones do not.

From properties (2) and (J) it is clear that the presence of
some active templates in the cell greatly accelerate the activa-
tion of other templates. Whether this includes other templates
already present or only those subsequently formed is not de-
ducible from the data. The question is, however, how this
activation might take place.

The two simplest possibilities are {a) the active templates
self-duplicate, the new ones being, so to speak, descended from

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S. SPIEGELMAN AND A. M. CAMPBELL

the one originally present and (b) active templates produce some-
thing which activates inactive templates. Both may operate;
however, the functioning of the second mechanism is directly
implied by the experiments of Rotman and Spiegelman (65)
which show that inactive sites preexist and can be converted to
active ones. Under the circumstances, the self-duplicating
property can be abandoned as being superfluous for a descrip-
tion of enzyme-forming system as a protein-synthesizing machine.
What the active templates might produce which would activate
other ones is also not deducible from the data. However, the
only thing the template can be presumed to produce hetero-
catalytically is enzyme. One is then led from this model to
conjecture that the enzyme molecule itself is the activator of
inactive templates.

The picture of the mechanism emerging is clear, and we
may now generalize and formalize it. Let us denote the relevant
templates by T, inducer molecules by I, and enzyme by E.
We postulate then the following.

7. When cells have been grown for many generations in
the absence of inducer, their templates are all, or nearly all, in
the simple and inactive form T.

2. The complex T-E is unstable and can occur in either
one of two ways. In a noninduced cell each T has a small
probability of fabricating spontaneously an enzyme molecule.
Secondly an induced cell allowed to grow in the absence of in-
ducer will, when the inducer is sufficiently diluted, produce T-E
from decomposition of the inducer-T-E complex, mentioned in
the next statement.

3. The reaction

T-E + nl > T-I„-E

is rapid and relatively irreversible. Here, n is the number of
inducer molecules bound per T-E complex; n may be unity in
some cases and greater in others.

4. A population of cells grown in an inducer-containing
medium has most, or all, of its sites in the form of T-I„-E.

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INDUCED ENZYME FORMATION

5. Of the forms mentioned above, only T-I„-E can effec-
tively and rapidly catalyze enzyme synthesis.

In the nomenclature of Cohn and Monod (18) T would be
an apo-organizer, I, a co-organizer; and T-I, an organizer.
The complexes T-E and T-I„-E represent new entities not
embraced or employed by their terminology.

The reversion from positive to negative in "slow adaptors"
can be interpreted in terms of the above model in the following
manner. As soon as growth in a galactose-free medium occurs,
all newly formed templates appear as either T or T-E and con-
sequently no enzyme synthesis takes place at them. The tem-
plates which were present initially remain as T-I„-E and con-
tinue to synthesize enzyme. They are the autocatalysts or active
particles and are diluted out by growth. As the reversion
growth proceeds, one gets, as a result of the consequent dilution
of inducer, some convertible particles, T-E. The ultimate and
irreversible decay of active particles if the reversion proceeds too
long receives therefore simple explanation in terms of the in-
stability of T-E. The anomalous change in stability of active
particles (mentioned at the end of IV, B) which occurs late in the
reversion is explained simultaneously.

The conversion to the positive state occurs when a cell con-
taining one or more templates of the form T-I„-E or T-E is
placed in a medium containing inducer. Any T-E complexes
are first converted to T-I„-E and then in both cases free enzyme
is formed. By virtue of the enzyme produced other templates
can be rapidly converted to the stable T-I„-E state, provided
excess inducer molecules are present. The infrequent spon-
taneous production of positives from negatives in the "slow"
strains might be explained on the basis of the rare occurrence of
enzyme formation by unoccupied T or T-I„.

The novel function attributed to the enzyme and inducer
molecules recalls the formally similar hypothesis of the "plas-
magene theory." It should be emphasized, however, that
the present model exploits a "feed-back" feature which was ac-
tually inherent in the plasmagene model but not used. It is the

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S. SPIEGELMAN AND A. M. CAMPBELL

recognition that enzyme molecules can serve to activate un-
occupied templates that permits an explanation of autocatalysis
which does not invoke self-duplication. There are no data
existent to our knowledge which demand this latter property as
an integral part of the enzyme-forming process. It would, however,
seem necessary to retain self-duplication as a method of tem-
plate maintenance to explain the instances of cytoplasmic trans-
mission noted, particularly in the case of the "petite" mutants.

Stages in the Induced Formation of Enzyme
and Their Interpretation

The particular appeal of the model just proposed lies in the
fact that it is in principle applicable to systems other than "slow
adaptation" and permits an instructive interpretative compari-
son among them. A central feature is the assumption that the
active enzyme-synthesizing unit is the triple complex between
template, enzyme, and the inducer. The difference between
the slow adaptors and the normal variety will then be explained
in terms of the very much lower probability of an unoccupied
template of the slow type producing an enzyme molecule in the
absence or the presence of inducer. In normal varieties this
event occurs sufficiently frequently in the absence of inducer so
that at any given moment it is highly probable that there is at
least one T-E complex present per cell. This represents there-
fore the constitutive mechanism for the synthesis of the low
basal enzyme levels frequently observed in inducible strains.
In the same vein, one can explain the so-called "constitutive
mutants" (17,46) which make enzyme in inducer-free medium
even faster than the normals do in the presence of inducer.
Thus, one can suppose that "constitutive mutants" possess
templates that possess an even higher than normal probability of
spontaneously forming enzyme molecules. This has the ob-
vious consequence that constitutive enzyme formation of this
variety should more closely approximate noninduced enzyme
synthesis. Some constitutive enzyme formation is of course
referable to internal inducer production, as is beautifully shown

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INDUCED ENZYME FORMATION

by the work of Vogel and Davis (99) withA^-acetyl-ornithine
deacetylase and by Stanier's (91) and Suda's (92) skillful use of
sequential induction.

It will be noted that comparatively irreversible combina-
tions of inducer are assumed to occur only when both template
and a replica are present. This situation possesses interesting
predictive consequences which are sufficiently unique to permit
experimental examination. It is evident immediately that the
state of a noninduced cell, with the majority of its templates
unoccupied by enzyme, should be very different from one which
is induced to the condition where a considerable portion of the
templates are already in the active state, and where enzyme mole-
cules are freely available for the formation of new active com-
plexes.

In the first place, differences in response to inducer concen-
tration in noninduced as compared with partially induced cells
would not be unexpected. Induced cells would contain the
T-E complexes permitting the relatively irreversible combination
with inducer molecules. This is of course most dramatically
exhibited by the long-term adapting instance. Here, no level
of inducer concentration is capable of stimulating enzyme for-
mation in the vast majority of cells. However, once this is suc-
cessful and an induced cell is produced, normal inducer levels
are sufficient to maintain enzyme formation indefinitely. An
analogous difference can also actually be seen in normal enzyme
formation. Thus, with alpha-methyl-glucoside as an inducer it
is found that the concentration levels required to initiate induc-
tion are considerably higher than those needed to maintain
enzyme formation once it is started (77). The same observa-
tion has been made independently by Monod (cited in ref. 15)
and Spiegelman and Gilmour (81) with respect to beta-galac-
tosidase synthesis in E. coli. Here enzyme formation does not
commence at thio-methyl-D-galactoside levels of 1 X 10~^ A/
but will if the concentration is raised to 5 X 10~^ M. However,
once enzyme formation is begun, 1 X 10~^ M is perfectly ade-
quate to maintain the induction.

147

S. SPIEGELMAN AND A. M. CAMPBELL

Pollock (62) has observed interesting differences between
induced and noninduced cells in terms of sensitivity to ultra-
violet. He found that prior to the onset of penicillinase forma-
tion in the presence of inducer, enzyme synthesis is extremely
sensitive to inactivation by exposure to UV. Once, however,
enzyme formation has commenced, it becomes increasingly more
resistant to this type of inhibition. Halvorson and Jackson (31)
showed an exactly similar situation in the induced formation of
alpha-glucosidase by S. cerevisiae.

These are findings which are not unexpected if, as seems
reasonable, empty templates are more sensitive to inactivation
by UV than those combined with protein in a complex stabilized
by inducer molecules.

Finally, another difference between the induced and non-
induced state has been observed (84) in terms of response to
amino acid analogues. It has already been noted that it is pos-
sible to stop enzyme formation by means of amino acid ana-
logues. However, this inhibition is effective only if one adds the
amino acid analogue in the lag period of enzyme formation,
prior to the appearance of new enzyme molecules. If the induc-
tion is allowed to proceed to the point where enzyme is already
being formed, suppression of enzyme synthesis with the antag-
onist becomes increasingly difficult (84). We can explain this
apparently puzzling finding quite readily in terms of the model
just proposed. In noninduced cells the analogue is competing
with its homologue for an unoccupied position on the template.
It is not surprising that the competitive situation for the antag-
onist should be markedly modified when the empty templates
are filled with enzyme, for of necessity homologous amino acids
now sit bound at, or close to, the sites of competition.

Our discussion of the biochemical, kinetic, and genetic in-
formation available on enzyme induction has led us to postulate
three components of the enzyme-forming system, inducer, pro-
tein, and template. In any given case the first two can be
reasonably well defined in terms of known chemical entities.
We turn now to the question of the identification of the chemical
nature of the third member of the triad, the template.

148

\

INDUCED ENZYME FORMATION

The Chemical Nature of the Template

A template which is to serve as a device for protein syn-
thesis must be at least as complicated and as large as the molecule
which it is forming. Other than the protein molecule itself,
there are relatively few candidates one can propose which can
satisfy the two criteria of size and informational complexity.
With these restrictions in mind, the two known possibilities are
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

In the following paragraphs we will briefly examine the
available evidence for and against the possibility that one or the
other of these substances is associated with the enzyme-forming
mechanism.

DNA AS A COMPONENT OF EFS

Evidence from work with the transformation principles
(39,105) offers convincing evidence that genetic information can
be stored in and transmitted through DNA. The potentiality,
therefore, of forming any specific kind of protein molecule must
ultimately be referable to the DNA of the cell. The question,
however, which we would like to entertain at present, is whether
DNA is directly and personally involved in the synthesis of protein
or whether it effects its influence via an intermediary. De-
finitive evidence one way or the other is, at present, not available.
Presumably, an unequivocal demonstration will ultimately
emerge from experiments analogous to those being performed
in the laboratories of Brachet (9) and Mazia (40,49) with enu-
cleated fragments of ameba. At present, the best that can be
offered is a series of experiments inquiring whether a correlation
can be established between the metabolic activity or state of
DNA and the act of protein synthesis.

There exists a variety of experiments in which it is possible
to demonstrate a complete dissociation of DNA metabolism from
protein synthesis. DNA formation is known (1) to be far more
sensitive to inhibition by radiation with x-rays than is protein
formation. Baron, Spiegelman, and Quastler (5) have shown

149

S. SPIEGELMAN AND A. M. CAMPBELL

that X-ray dosages far exceeding those expected to stop the for-
mation of DNA completely permit normal enzyme formation in
yeast. Similar dissociations have been achieved with other
systems and by different means (30,58).

Kelner's (43) studies on photoreactivation of E. coli fol-
lowing exposure to ultraviolet have provided an elegant method
for a virtually complete separation of RNA and protein forma-
tion from net DNA synthesis. Halvorson and Jackson (31)
employing yeast have recently repeated and confirmed these
results. The results obtained suggest again that protein and
RNA continue to be synthesized subsequent to UV doses which
completely inhibit the DNA formation.

Cohen and Earner (16) have reported the ability of a
thymine-less mutant of E. coli which can synthesize xylose isom-
erase in the absence of an added supply of thymine. This im-
portant finding was confirmed in our laboratory (85) using the
same mutant and examining beta-galactosidase-forming capac-
ity. It was found that cells of this strain synthesized consider-
able amounts of enzyme when suspended in a synthetic medium
lacking thymine. This behavior is in striking contrast to tliat
observed with mutants possessing other metabolic deficiencies.
Thus, in our own experience and in that of others (56,58,85),
adenine-less, uracil-less, or amino-acid-deficient mutants form
little or no enzyme in the absence of the required metabolite.

An interesting apparent exception to the information cited
is that of Allfrey's (2) observation with isolated nuclei. He found
that treatment with DNase suppressed the ability of his prepara-
tions to incorporate labeled amino acids, whereas RNase had
little or no effect. The situation observed here may, however, be
a reflection of the low nuclear RNA content. If, as seems likely,
RNA is derived from DNA, destruction of the latter would elim-
inate protein synthesis in such systems.

The data cited above demonstrate that drastic interference
with DNA synthesis is often not accompanied by very striking
effects on the formation of protein. Whereas such findings can-
not eliminate DNA as an active component of the EFS, they

150

INDUCED ENZYME FORMATION

hardly lend support to the supposition that it is. The credence
assignable to such negative conclusions with respect to DNA
gains further weight from similar experiments examining RNA
metabolism which yielded strikingly different results

RNA AS A COMPONENT OF EFS

Many have postulated RNA as a key substance in protein
synthesis. Chantrenne (14) has succinctly summarized such
speculations and the supporting evidence. Here we would like
to confine our attention to the information derived from the
study of enzyme synthesis. Again, as in the case of DNA,
correlative experiments have been performed with intact cells
examining the effects on enzyme formation of agents or condi-
tions which influence RNA metabolism.

7. Experiments with Ultraviolet Light. Swenson and Giese
(94,95) demonstrated that exposure to ultraviolet dosages far
exceeding those required to stop DNA formation, results in the
inhibition of induced enzyme synthesis in yeast. Examination
of the action spectrum of the inhibition revealed that it coincided
with the absorption spectrum of nucleic acid. Halvorson and
Jackson (31) extended these interesting observations. They
examined the effects of various dosages on the synthesis of alpha-
glucosidase, the ability to use free amino acid pool components,
and the incorporation of P^^ into the nucleotides of RNA. Their
results established an excellent parallelism between the loss in
capacity to utilize the free amino acids and the ability to syn-
thesize enzyme. It was further found (85) that even slight dam-
age of RNA metabolism, as measured by ability to incorporate
P^-, had profound effects on enzyme-forming ability. Thus, at
a dose which achieved a 22 per cent inhibition of RNA metabo-
lism enzyme formation was suppressed to the extent of 95 per
cent.

2. The Effect of a Uridine Analogue on Enzyme Synthesis. One
obvious approach which could in principle yield informa-
tion pertinent to the role of RNA is to examine the effects of
various analogues of uracil and its derivatives on enzyme forma-

151

S. SPIEGELMAN AND A. M. CAMPBELL

tion. Ben-Ishai and Spiegelman (7) undertook such a study.
One of the most effective compounds found was 5-OH-uridine,
which the experiments of Roberts and Visser (64) suggest is able
to prevent the utilization of uracil for the synthesis of RNA.
The presence of as little as 5 /ig./ml. of this compound results in
virtual cessation of beta-galactosidase formation by E. coli.
Further, this inhibition can be achieved even if the OH-uridine
is introduced subsequent to the addition of inducer, at a time
when maximal rate of enzyme formation has been attained.

Several illuminating facts emerged from these experiments.
One was that the OH-uridine could effect a complete inhibition
of beta-galactosidase formation at concentrations which had no
effect on over-all protein synthesis. The apparent greater
sensitivity of the beta-galactosidase-forming system suggests that
it requires a larger effective supply of RNA precursors than other
protein-synthesizing systems. A second fact of interest is the
ability of the OH-uridine to prevent enzyme formation even
after its onset. This would suggest that continued synthesis of
RNA is required for the uninterrupted production of enzyme.
The same conclusion is derivable from the observation (56,58)
that, unlike the previously noted experience with thymine-less
mutants, uracil-less mutants cease making enzyme immediately
upon the exhaustion of externally supplied uracil.

COMPETITIVE INTERACTIONS AMONG PROTEIN SYNTHESIS
SYSTEMS FOR RNA PRECURSORS

The marked response of the beta-galactosidase-forming
systems to 5-OH-uridine and its interpretation in terms of an
elevated requirement for RNA precursors suggest other types of
experiments for exhibiting this kind of interaction. E. coli cells
growing logarithmically in a synthetic medium with ammonia
as the sole source of nitrogen do not accumulate a detectable
internal pool of amino acids. The rate of protein formation is
apparently limited by the synthesis of amino acids, since an
immediate increase in growth rate follows the addition of an
external supply of amino acids. It would be expected that the

152

INDUCED ENZYME FORMATION

sudden stimulation of protein synthesis caused by the amino
acid addition would exert an exhaustive demand on the meta-
bolic devices which supply the derivatives needed for ribonu-
cleic acid synthesis. In view of the suggested sensitivity of the
beta-galactosidase-forming system to the supply level of the
RNA precursors, the addition of amino acids might be expected
to result in an inhibition of beta-galactosidase formation. This
prediction is experimentally realized (30). Thus, the presence
of inducer fails to stimulate enzyme formation if amino acids are
added simultaneously. The suppression is virtually complete
for a period of a half hour, following which some recovery of
enzyme-forming capacity occurs. That the inhibition is re-
lated to RNA precursor supply is supported by the ability of
purine and pyrimidine bases to reverse it.

This dependence of enzyme formation on an adequate
supply of nucleic acid precursors has also been exhibited (7) in
the case of alpha-glucosidase formation in S. cerevisiae In ad-
diton to their free amino acid pool, yeasts also possess a consi-
derable internal supply of nucleotides and their polyphosphate
derivatives (68). It was found possible (7) to specifically de-
plete the nucleotide pool by incubation in the presence of an ex-
ternal supply of amino acids and energy. This treatment leads
to a loss of enzyme-forming capacity while leaving the free amino
acid pool intact. If cells are first partially induced and their
nucleotide pool then depleted, they fail to form enzyme on being
again exposed to inducer. If their nucleotide pool is, however,
replenished, enzyme synthesis proceeds normally. These ex-
periments illustrate in a different manner and with another sys-
tem the apparent requirement that RNA synthesis be possible if
enzyme formation is to continue.

EXPERIMENTS WITH SUBCELLULAR FRACTIONS

The experiments thus far described strongly implicate RNA
as the template in the process of enzyme synthesis. They cannot,
however, be taken as conclusive. It is painfully obvious that
although interesting and perhaps even ingenious experiments can

153

S. SPIEGELMAN AND A. M. CAMPBELL

be performed with intact cells, the distance between the data
and the conclusions derived fr ^m them is too great for certainty.
Definitive identification of the chemical nature and the mode of
action of the template is not likely until the latter has been phys-
ically isolated in a functional state. In vitro performance of its
function by the isolated enzyme-forming system may be sug-
gesting the impossible, since it demands even more than that
which has already been accomplished in the case of transfor-
mation in the bacteria. In the latter, genetically competent
material has been separated from other cell c 3mponents. How-
ever, the transforming principles have been asked to function
only after reinsertion into an intact living organism.

Nevertheless, that the ideal in vitro situation may be attain-
able in the not too distant future is prophetically foreshadowed
by the striking successes which have recently been recorded with
subcellular fractions. Many of these deal primarily with in-
corporations studies. To this extent it is uncertain that they
necessarily represent model systems which will permit the
further dissection of the protein-synthesizing mechanism. The
data must therefore be interpreted with caution, but their
uniqueness and potential value command consideration.

Zamecnik and Keller (104) succeeded in preparing a mi-
crosome fraction which actively incorporates amino acids when
supplemented with some component of the supernate and an
ATP-generating system. Subsequent work on the supernate
fraction by Keller and Zamecnik (42) indicated the presence of
an enzyme which generated guanosine-triphosphate, a deriva-
tive of which functions in the insertion of the amino acids into
peptide linkage. The work of Hoagland (37) and DeMoss and
Novelli (22) strongly suggests that polyphosphate derivatives of
nucleotides activate amino acids prior to their incorporation.

Lester (48) and Beljanski (6) examined the ability of
lysozyme-treated preparations of Bacillus megaterium to incor-
porate amino acids labeled amino acids. Both authors found
that treatment with RNase abolished this ability, whereas ex-
posure to DNase was stimulatory.

154

INDUCED ENZYME FORMATION

The most extensive investigation on the properties of sub-
cellular fractions has come from Gale's (27,28) laboratory. In
these studies cells of Staphylococcus aureus are disrupted by sonic
disintegration and a fraction obtained by differential centrifu-
gation which is relatively low in viable cells, and therefore pre-
sumably in intact cells. Although it is unlikely that this prep-
aration is homogeneous, it nevertheless is of the greatest in-
terest, since it is amenable to enzymatic and extractive resolu-
tion. Removal of the nucleic acid from such disrupted cell
preparations leads to a marked lowering in their ability to in-
corporate amino acids. This loss can be restored by the ad-
dition of nucleic acids from the same species, DNA being more
active than RNA on a dry-weight basis. This latter finding
may be merely a consequence of the greater stability of DNA to
isolation procedures. The data are consistent with the concept
that the RNA made from the DNA supplied is the active
agent.

A most interesting recent development has been the dis-
covery by Gale and Folkes (29) that the presence of specific
di- and trinucleotides is extremely active in promoting the in-
corporation of specific amino acids. Thus, for example, di-
nucleotides containing adenine and cytosine can completely re-
place the intact RNA in promoting the incorporation of aspartic
acid. Indeed, on an equivalent weight basis the dinucleotide is
more than a hundred times as active as the intact RNA. Inter-
pretation of these findings is yet uncertain. It may indeed be,
as suggested by Gale and Folkes (29), that these small fragments
represent that part of the RNA template which is concerned with
the insertion of the corresponding amino acid into peptide link-
age. An argument which can be raised against this assertion
stems precisely from the observed high activity of the dinucleo-
tide. It seems unlikely that nucleotide pairs are sufficient to
specify the relevant amino acids, since only 16 possibilities are
uniquely determined. At least three bases of the RNA tem-
plate would have to be involved in the specification of a given
amino acid since 20 or more choices have to be made. This rea-

155

S. SPIEGELMAN AND A. M. CAMPBELL

soning assumes, of course, that the four bases are the only com-
ponents of the code.

An alternative explanation of these findings can be offered.
It may be that the active fragments of Gale-Folkes may, by
transfer reactions, generate the nucleotide components func-
tioning in the activating mechanism suggested by the work of
Hoagland (37) and DeMoss and Novelli (22). Whatever the
interpretation, it nevertheless remains true that these results
are pregnant with many possibilities.

As distinguished from incorporation studies, the attainment
of protein synthesis has been reported in only two sorts of sub-
cellular fractions. One is the system of Gale and Folkes (28) in
which the development of "glucozymase," catalase, and the in-
ductive formation of beta-galactosidase have been demon-
strated. When these preparations are sufficiently resolved
either by removal of RNA or DNA, it is found that both DNA
and RNA stimulate the formation of both catalase and beta-
galactosidase.

Again, the relative high activity of the DNA may be a con-
sequence of greater stability to extractive procedures. No
limits to the potentialities of this system are apparent. It is
difficult to believe that its further study can fail to provide defin-
itive answers to the basic problems of template function and
specificity.

Another system which gives great promise of future fruit-
fulness are the so-called "protoplasts" of B. megaterium. Wei-
bull (100) showed that these could be prepared by treatment of
cells with lysozyme in hypertonic medium. Wiame et al. (101)
showed that these preparations were able to synthesize arabino-
kinase as demonstrated by an increased Qoj during incubation.
Simultaneously Landman and Spiegelman (44) isolated a lac-
tose positive mutant of B. megaterium and devised a stabilizing
medium for protoplasts which permits synthesis of beta-galac-
tosidase. Virtually all of the enzyme-forming capacity is re-
covered in the protoplasts. When supplemented with amino
acids, hexose-diphosphate, and inducer, they synthesize enzyme

156

INDUCED ENZYME FORMATION

at rates comparable to those of intact cells. The beta-galac-
tosidase formed has been isolated in soluble form and purified.
These preparations are amenable to enzymatic resolution, their
enzyme-forming activity being abolished by RNase. This
treatment does not destroy them physically but selectively re-
moves 80 to 90 per cent of the RNA.

It is evident that the search for a system which would per-
mit the further experimental probing of protein-synthesizing
systems is at present in an exciting but preliminary stage. There
seems little doubt, however, that a new era is being opened which
will ultimately permit a description in chemically defined terms
of the nature of the protein-synthesizing machinery.

Summary and Concluding Remarks

We have here surveyed the data which have accumulated
on the phenomenon of "enzymatic adaptation," with particular
emphasis on the eff"orts of the past decade. In view of the com-
plexity of the problem posed initially, and the difficulties which
could easily have hindered understanding or led to irrelevant
confusion, the progress which can be recorded is satisfying.

Operationally diverse disciplines have provided the data
from which a picture of the enzyme-forming mechanism has
evolved. The kinetic, biochemical, and genetic information
on induced enzyme production all lead to and can be interpreted
in terms of one model. They suggest that the enzyme-forming
system is a complex between RNA, inducer, and enzyme.

The problem has been brought to the point where further
questions must be posed in terms of the chemical structures and
reactive interrelations of the components identified. From the
experiments reviewed in the last section it would appear that the
systems needed for the experimental resolution of precisely such
questions are now well on the way to development.

It seems likely at the present writing that the next decade
will provide the necessary answers. Attention can then be
profitably turned to the problem which initiated much of the
work described, i.e., what is the nature of gene function?

157

S. SPIEGELMAN AND A. M. CAMPBELL

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INDUCED ENZYME FORMATION

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161

CERTAIN PROBLEMS IN THE BIOCHEMICAL
STUDY OF DISEASE

DeWITT STETTEN, Jr., National Institute of Arthritis and
Metabolic Diseases, National Institutes of Health, Bethesda, Maryland

The study of normal metabolic processes may be pursued
with profit at many levels of biological organization, or, more
precisely, of biological disorganization. The investigator may
elect to employ the minimally disturbed intact animal, the iso-
lated perfused organ or tissue of that animal, the sliced, teased,
or minced preparation of such a tissue, the cell-free homogenate,
or the solution of enzyme or enzymes. Each level, as here listed,
represents a further degree of disorganization of an initially highly
integrated system, and the disorganization is in each case im-
posed by the experimenter. At each successive level of dis-
organization something is gained in the form of greater simplicity,
greater control over variables, greater facility in establishing the
necessary components of the system or reaction under scrutiny.
However, at each successive level of disorganization something
of importance is also lost.

It may be worth while to consider briefly what is lost as
one proceeds with this dissection, first surgical and then chemical,
of the intact organism. As an example, the functioning of the
liver in situ is influenced by the normally continuous supply of
substrates, the normally continuous removal of products of its

162

STUDY OF DISEASE

activity. These operations depend in turn upon the activities
of remotely situated organs and tissues. Remote organs also
influence endocrinologically the function of liver in situ. In
addition, other influences, nervous, circulatory, and thermal,
remote in origin, may affect the functioning of the liver in its
normal habitat.

Isolation and perfusion of this liver at once eliminates many
of these determinants of liver function. Humoral, including
hormonal, regulation is now determined by the experimenter,
not by the animal. Nervous regulation is usually lost. If the
liver is now sliced and shaken in a bath, further loss of organiza-
tion occurs. A fraction of the cells is necessarily incised, releas-
ing normally intracellular contents to the extracellular fluid.
The remaining cells are nourished in a rather haphazard fashion,
splashing about in a vessel instead of being supplied with a steady
flow of a regulated nutrient fluid pumped past each cell through a
system of venous sinuses. The process under study is confined
by the walls of the vessel, and unless special and elaborate condi-
tions are fulfilled, the concentration of substrate continuously
declines while that of product continuously rises.

Homogenization of the tissue and disruption of the cellular
architecture is still a further insult. The microscopically visible
intracellular components, nucleus, mitochondria, nucleolus,
and microsomes, all bear, in the intact cell, geometrical relation-
ships to each other which are doubtless of functional significance
in the intact cell and which are destroyed in the process of homog-
enization. It may further be postulated that the submicro-
scopic particles, the molecules of the soluble enzymes, are likewise
in the intact cell not entirely free-swimming, but become so
when the cell is disrupted. It is possible that in the geographical
arrangement of enzymes within the cell resides the answer to
the typically polar or unidirectional character of epithelial
cells (14), and this is clearly lost in the process of homogenization.
Finally, the separation and purification of individual enzymes
from such a cell-free homogenate, though very rewarding,
inevitably divorces the enzyme from such inhibitory or excita-

163

DEWITT STETTEN, JR.

tory agents as may be closely associated with it in real life.
Whereas the determination of the equilibrium constant of an
enzyme-catalyzed reaction usually rests upon preliminary puri-
fication of the enzyme, it must be recognized that within the
cell within the liver within the peritoneal cavity of the intact
animal, most reactions of interest in metabolic studies do not
proceed to equilibrium. Indeed it is a characteristic of the living
organism that in general it operates quite remote from the equilib-
rium point.

Disease, for purposes of the present argument, may be defined
as the manifestation of alterations in metabolic processes. In
some cases, such alterations are of a qualitative nature, involving
the appearance in the organism of compounds which are appar-
ently totally lacking in the normal animal. The presence of
the abnormal hemoglobin found in sickle-cell anemia (15) or
of the Bence- Jones protein of multiple myeloma (23) are ex-
amples of such qualitative changes. In more cases, the change
appears to be of a quantitative nature, involving alterations in the
rates of normally occurring processes. The chemical change
observed in such a patient is one of a deviation from normal in the
concentration of some blood, urine, or tissue constituent, reflect-
ing a change in the rate of production or of removal of some tissue
component. The concentration of glucose or of uric acid in the
blood may be elevated, the quantity of depot lipid may be in-
creased, or the concentration of serum albumin may be lowered.

The first question which is presented by such a situation is
an analysis of the crude mechanism upon which the change in
concentration depends. Most of the tissue constituents of
interest in this regard are, in the normal subject, in a dynamic
steady state, subject to an elegantly balanced turnover wherein
the rate of generation is closely matched to the rate of destruction
or elimination. A rise in the quantity of such a constituent may
result equally readily from an increase in the synthetic rate or
from a decrease in the rate of destruction. Conversely, either an
increase in destruction or a decreased synthesis will result in a
fall in quantity. It is today possible to design experiments, in

164

STUDY OF DISEASE

certain cases, which will differentiate these two types of disturb-
ances and to show, for instance, that diabetic hyperglycemia is
a consequence chiefly of impaired glucose utilization, whereas
the hyperuricemia in certain types of gout is a consequence of
excessive production of uric acid.

Such experiments are most securely conducted on the intact
animal, and indeed, in the case of gout, the experimenter is
restricted to man, since no analogous disease in experimental
animals is available. The reasons for this preference for the
intact animal lie in the fact that the disease process is itself a
disintegration of some phase of that highly integrated system
which is the organism as a whole. Any further disorganiza-
tion imposed by the experimenter will necessarily confuse the
picture and may obscure the primary disturbance which is due
to the disease itself. Furthermore, the normal constancy of
composition of the blood with respect to glucose, uric acid, al-
bumin, etc., of the subcutaneous tissues with respect to lipid, or
of the liver with respect to glycogen, fatty acids, or cholesterol
is a resultant of many processes occurring in many tissues.
The very separation of the several tissues may result in dis-
appearance of the abnormality of rate the explanation of which
is being sought.

The foregoing argument must not be construed to mean
that only experimental results derived from the intact animal
are of use in the elucidation of disease processes. On the
contrary, all levels of biological disorganization have con-
tributed significantly to this body of knowledge. What is in-
tended is merely to point out that of such results as are secured
with isolated enzyme preparations or isolated tissues of diseased
animals, only those which are compatible with observations on
the intact diseased animal are of value in explaining the disease
process.

The contributions of the several experimental approaches
may be exemplified by an analysis of the various abnormalities
which may influence the concentration of glucose in the blood.
Glucose enters the portal blood by intestinal absorption and since

165

DEWnTT STETTEN, JR.

most of the dietary carbohydrate of mammals is polysaccharide,
this implies a preliminary digestion. Glucose enters the hepatic
vein by the hydrolysis of glucose-6-phosphate and this com-
pound may in turn arise ( 7) from glucose itself, (2) from glucose-
1 -phosphate derived from glycogen or (3) from hexosediphos-
phate arising in turn from triose phosphate, etc. These processes
undoubtedly occur also in tissues odier than the liver, notably
the kidney. Glucose may also arise hydrolytically within cells,
and as an example of this process may be cited the action of
amylo-l,6-glucosidase in muscle. This latter contribution is
probably quite small.

Glucose is utilized, so far as is known, by every mammalian
cell. For blood glucose to be utilized, it must first cross cell
membranes. It is generally held that simultaneously with or
immediately succeeding such passage, glucose undergoes hexo-
kinase-catalyzed phosphorylation. From the hexose phosphate
thus produced at least four distinct metabolic sequences may
arise.

(1) Glucose-6-phosphate — ^ glucose- 1 -phosphate -^ glucosides
(e.g., glycogen) or rearrangement products (e.g., galactose-
1 -phosphate).

(2) Glucose-6-phosphate -^ 6-phosphogluconate, initiating the
"oxidative pathway."

(3) Glucose-6-phosphate — > fructose-6-phosphate, initiating
the "glycolytic pathway."

(4) Glucose-6-phosphate — ^ glucose + inorganic orthophos-
phate.

Of interest in this regard is the question of the possible
utilization of glucose without preliminary phosphorylation.
This view rests largely upon the demonstration of a glucose
dehydrogenase activity in mammalian liver extracts which
forms gluconic acid from glucose in excellent yield (6). Attempts
to demonstrate the occurrence of this particular transformation
in the intact animal have thus far yielded negative results (20).
Provisionally this enzyme activity must be considered inoperative

166

STUDY OF DISEASE

in the intact mammal, and its presence must be regarded as an
unexplained biochemical curiosity. Whether hepatic glucose
dehydrogenase is a vestigial compound or whether it is a truly
important enzyme, the proper substrate of which has not been
identified, remains to be determined.

In addition to the foregoing fates, glucose may be lost to the
mammalian organism in the intestinal and urinary tracts. In
the intestinal tract, failure of adequate polysaccharide digestion
or excessive bacterial fermentation deprives the mammal of a
portion of ingested carbohydrate. Whereas normal sugar losses
in the urine are negligible, these become significant if either the
normal renal threshold is exceeded by an abnormal hyper-
glycemia or normal blood is circulating through kidneys with
an abnormally low glucose threshold.

In consideration of the numerous sources and fates of blood
glucose, it is certainly not surprising that there are many disease
states which result in alterations, positive or negative in sign, of
glucose concentration in the blood. Still other defects in one or
another mechanism might be anticipated to produce alterations
in blood glucose concentration but fail to do so as a consequence
of homeostatic mechanisms that are invoked. Thus simple
failure to ingest carbohydrate or failure to digest polysaccharide,
as may occur in pancreatic disease or diarrhea, will not, in
general, cause profound hypoglycemia. This is at least in part
explained by a compensatory decline in glucose utilization by
cells of the liver and is reflected in a diminished consumption of
glucose for hepatic lipogenesis in the intact animal (3) as well as
in the liver slice (24). Failure of the intestinal mucosa actively
to transport glucose from the lumen to the portal blood is seen
in sprue, and this situation also rarely leads to hypoglycemia.
The site of the defect is readily demonstrable clinically, however,
by the finding of a normal intravenous glucose tolerance in the
face of a failure of blood glucose to rise after oral administration.
It is noteworthy that the defect in sprue can be simulated by the
application of phlorhizin to the intestinal mucosa (10).

The liver normally supplements the intestinal tract as a

167

DEWITT STETTEN, JR.

source of blood glucose. The immediate hepatic source of
glucose is from the hydrolysis of glucose-6-phosphate in the
presence of a specific glucose-6-phosphatase. Glucose-6-phos-
phate arises in liver from various sources, and among these are
glycogen, via glucose- 1 -phosphate, and such noncarbohydrate
compounds as are capable of contributing organic fragments to
the several intermediates of the glycolytic sequence and the
citric acid cycle. These latter contributions derive ultimately
from the glycogenic amino acids, glycerol, and other compounds,
and the over-all process under consideration is termed glyconeo-
genesis.

Of the disease processes which influence the generation of
glucose in the liver, the best defined is glycogen storage disease
(von Gierke). In an elegant analysis of these patients (5), it has
been shown that in many instances the defect is attributable
simply to a relative or absolute deficiency of glucose-6-phospha-
tase activity in the liver. Such an abnormality makes the liver,
like normal muscle, incapable of contributing significant amounts
of glucose to the blood, but in no wise interferes with the steps of
glycogenesis. Other patients suffering from glycogen storage
disease exhibit normal glucose-6-phosphatase activity but
appear to deposit glycogen which deviates markedly from
normal liver glycogen in its branching pattern. Such glycogen
seems, in some cases, to be less readily degraded by the com-
bined action of phosphorylase and amylo-l,6-glucosidase. A
consequence of either type of glycogen storage disease is a failure
to elicit the usual hyperglycemia in response to administered
epinephrine.

Recent evidence indicates that a mode of action of epi-
nephrine, as well as of glucagon, is to favor the conversion of
inactive phosphorylase into active phosphorylase (21). Whereas
this finding, is, of itself, not a complete explanation of the mode
of action of these agents, it is highly suggestive that these stimu-
lants of glycogenolysis operate at the initial steps of glycogen
breakdown. The failure of the blood glucose to rise in response
to epinephrine in von Gierke's disease is best explained by the

168

STUDY OF DISEASE

hypothesis either that the hexose phosphate which arises is not
hydrolyzed or that, owing to its structural abnormahty, the
glycogen is not sensitive to attack even by active phosphorylase.
In this regard it is of interest that patients deficient in pancreatic
islet a-cells, the apparent source of glucagon, have recently been
described (11). Hypoglycemia was noted to occur in these
subjects.

Even assuming all the requisite enzyme systems in the liver
to be intact, clearly if there is no glycogen in the liver, glyco-
genolysis must cease to play a role as a source of blood glucose.
Virtually total depletion of hepatic glycogen ensues upon
relatively brief periods of inanition, and marked decreases in the
content of glycogen in the liver have been observed in a variety
of conditions, including exposure to low oxygen tension or
intoxication with thyroxin or thyroglobulin. It follows that
under these circumstances, hepatic glycogenolysis cannot be
relied upon as a source of blood sugar.

An apparently distinct mechanism which can be altered in
disease but which also affects the rate at which the liver con-
tributes glucose to the blood is gluconeogenesis. Profound
changes may be elicited in the experimental animal, and analo-
gous situations may be observed in clinical material wherein
variations in adrenocortical activity result in changes in the rate
at which amino acids are deaminated and glucose is formed.
In general terms, the presence of excessive amounts of the 11-
oxysteroids of the adrenal cortex results in the generation of
large amounts of glucose from noncarbohydrate precursors (22),
a rise in the total carbohydrate content of the organism, and
incidentally a rise in blood glucose concentration. Conversely,
in the adrenalectomized animal or Addisonian patient, with
glucogenesis from noncarbohydrate sources impeded, hypo-
glycemia may be encountered. In contrast to the state of
present knowledge of the mode of action of epinephrine or of
glucagon, no generally acceptable hypothesis can be offered to
account for this action of the corticosteroids.

Defects in the utilization of blood glucose are if anything

169

DEWITT STETTEN, JR.

more frequent than defects in its sources and generation. Where-
as all tissues in the mammal, so far as is known, are capable of
assimilating glucose, attention has been directed largely to the
liver and to the skeletal musculature. Although wide fluctuations
in glucose utilization may be induced in these tissues by altera-
tion of the hormonal or nutritional state, certain other tissues,
e.g., brain, myocardium, fetus, appear to be insulated against
drastic changes in rate of glucose consumption. Thus whereas
the liver or muscle is sensitive to the concentration of circulating
insulin, brain is not, although it obviously cannot tolerate
extremes of hypoglycemia.

The rate of assimilation of glucose by muscle is critically
dependent upon the activity of the islets of Langerhans, the
anterior pituitary gland, and possibly also the adrenal cortex.
Space does not permit an analysis at this time of the various
conflicting lines of evidence relating to the exact sites of action
of the several participating hormones. For purposes of the
present discussion it matters little whether the role of insulin is
to release muscle hexokinase from a physiological inhibition (16)
or to facilitate the entry of unesterified glucose from the extra-
cellular space into the intracellular compartment of muscle (9).
It will suffice, for the present argument, to select the area of
agreement between the conflicting experiments and define the
net result of insulin action as increasing the quantity of intra-
cellular glucose-6-phosphate at the expense of extracellular,
ultimately plasma, glucose. Similarly we need not concern
ourselves too much, at this time, with the question of whether
the anterior pituitary gland secretes an agent that directly
inhibits hexokinase (16) or whether it interferes in some fashion
with the operation of insulin which is bound to muscle (18).
Growth hormone, or some biological product derived therefrom,
appears to render muscle insensitive to the action of insulin in the
intact animal.

Again in the intact animal the situation is complicated by
the fact that, in addition to influencing what is happening in mus-
cle, each endocrine may exert influences upon other endocrine

170

STUDY OF DISEASE

glands. An example of this situation is the finding of an increase
in gluconeogenesis observed in alloxan-diabetic animals (7).
These animals have enlarged adrenal cortices, and it is possible
that this effect is not directly attributable to hypoinsulinism but
is rather a consequence of a secondary hyperadrenalism.

It is generally agreed today that the major effect upon blood
glucose of hypoinsulinism is a decrease in glucose utilization by
such tissues as liver and muscle. Conversely the primary effect
of hyperinsulinism is an enhancement of glucose utilization in
these tissues. Many of the complex consequences of diabetes
may be explained upon this basis alone, in that, with the genera-
tion of intracellular glucose-6-phosphate impeded, all products
derived from glucose-6-phosphate, such as lactate, pyruvate, or
CO2, will not be formed from glucose at normal rate. Other
reactions, which are coupled to those of glycolysis less directly,
such as peptide bond synthesis (8) and fatty acid synthesis
(19,4), will also be retarded. There is ample experimental
evidence that these effects are observed in the diabetic organism
and are reversed by administration of insulin.

Many diabetic patients require dosages of insulin far in
excess of what is generally considered the production of the
normal pancreas, and in various situations, most strikingly in
severe ketosis, the insulin tolerance may increase enormously.
Under these circumstances one must suppose that an "antag-
onist" to insulin is present in undue amount in the blood (12)
or tissues. Whether this antagonist is in the nature of a pituitary
hormone (2), an antibody (1), or a specific destructive enzyme
(insulinase) (13) remains to be determined.

The last fate of glucose to be discussed is urinary loss. The
normal operation of renal tubular reabsorption of glucose may
be overwhelmed by excessively high concentrations of glucose
in the blood. This may occur secondary to a variety of dis-
turbances which interfere with glucose utilization, such as
insulin deficit, or which cause excessive glucose production, such
as excessive epinephrine or excessive corticosteroid. Further,
the mechanism of tubular reabsorption may itself be defective

171

DEWITT STETTEN, JR.

and result in renal glycosuria, as is seen in Fanconi's syndrome,
or experimentally in phlorhizin poisoning.

The purpose of the above discussion has been to stress that
the concentration of glucose or of any other constituent of the

Intestine'

BLOOD
GLU COSE

Muscle

Kidney
Fig. 1.

blood is the resultant of numerous vectors. Disturbances of any
of these vectors may result in abnormalities of concentration but
may also cause secondary disturbances in other vectors. If
these secondary processes are compensatory in nature, analogous

172

STUDY OF DISEASE

to negative feedback, homeostasis results. The secondary
disturbances may, however, ampHfy the defect. As an example
may be cited the nausea, emesis, and coma of diabetic ketosis,
all leading to cessation of food intake and thereby contributing
further to the ketosis. Such a situation is analogous to positive
feedback, and if uncorrected, may lead to a fatal outcome.

The several vectors that have been discussed in relation to
blood glucose are diagrammed in Figure 1. The magnitude of
vector A is determined by the abundance of ingested carbohy-
drate, the duration and effectiveness of intestinal digestion, and
the integrity of the intestinal mucosa. It may be markedly
diminished in starvation, in disease of pancreatic acinous tissue,
or in sprue. Vector B depends upon a supply of hepatic glyco-
gen of normal constitution and an intact enzyme system to
convert it to glucose. It is exaggerated by excess epinephrine or
glucagon and is diminished in von Gierke's disease. It is like-
wise influenced by the gluconeogenic rate which is in turn under
adrenocortical regulation, is diminished in Addison's disease,
and is exalted in hyperadrenalism. Vectors C and D depend
critically upon insulin, the anterior pituitary gland, and possibly
other anti-insulin agents. Both of these vectors decrease in
diabetes and are exaggerated in hyperinsulinism. Vector E
depends upon the concentration of glucose in the blood and the
integrity of the enzyme architecture of the renal tubule.

This analysis of factors affecting the concentration of blood
glucose in health and disease is of course merely exemplary.
Given suflficient information, the deviation from normal in the
concentration of any tissue constituent might be subjected to a
similar analysis. It is the purpose of this discussion to emphasize
the complex and highly integrated nature of the normally
operating intact organism and to point up the difficulties in-
herent in the elucidation of the disturbances which are en-
countered in clinical medicine.

Much has been learned about diabetes from comparative
studies of liv^er slices (4) or enzyme solutions (17) derived from
normal and from diabetic animals. The ability of such prepara-

173

DEWITT STETTEN, JR.

tions to synthesize fatty acids is far less if they are derived from
diabetic rather than normal animals, and these findings are
compatible with what is known to happen in the intact animal
(19). It is probably incorrect, however, to refer to such prepara-
tions as "diabetic liver slices." If by "diabetes" is meant the
relative or absolute deficiency of insulin, then all liver slices,
regardless of source, are necessarily diabetic. The difference
between isolated livers from diabetic and nondiabetic animals
is chronological, not diagnostic. Slices prepared from livers of
pancreatectomized or alloxanized animals have been deprived
of their source of insulin, have been "diabetic," for a somewhat
longer period of time than have similar slices prepared from
tissues of a normal animal.

References

1. Bernstein, C, Jr., J. B. Kirsner, and W. J. Turner, J. Lab. Clin. Med.,
23, 938 (1938).

2. Bornstein, J., and C. R. Park, J. Biol. Chem., 205, 503 (1953).

3. Boxer, G. E., and D. Stetten, Jr., J. Biol. Chem., 153, 607 (1944).

4. Chernick, S. S., I. L. Chaikoff, E. J. Masoro, and E. Isaeff, J. Biol. Chem..
186, 527 (1950).

5. Cori, G. T., Harvey Lectures, Ser. 48, 145 (1954).

6. Harrison, D. C, Biochem. J. {London), 26, 1295 (1932).

7. Ingle, D. J., E. H. Morley, and D. Stetten, Jr., Am. J. Physiol., 182,
263 (1955).

8. Krahl, M. E., J. Biol. Chem., 200, 99 (1953).

9. Levine, R., M. S. Goldstein, B. Huddlestun, and S. P. Klein, Am. J.
Physiol., 163,70 (1950).

10. Lundsgaard, E., Biochem. Z., 264, 209 (1933).

11. McQuarrie, I., E. T. Bell, B. Zimmermann, and W. S. Wright, Federation
Proc, 9, 337 (1950).

12. Marsh, J. B., and N. Haugaard, J. Clin. Invest., 31, 107 (1952).

13. Mirsky, I. A., R. H. Broh-Kahn, G. Perisutti, and J. Brand, Arch. Bio-
chem., 20, 1 (1949).

14. Patterson, W. B., and D. Stetten, Jr., Science, 109, 256 (1949).

15. Pauling, L., Proc. Am. Phil. Soc, 96, 556 (1952).

16. Price, W. H., C. F. Cori, and S. P. Colowick, J. Biol. Chem., 160, 633
(1945).

17. Shaw, W., and S. Gurin, Arch. Biochem. andBiophys., 47, 220 (1953).

174

STUDY OF DISEASE

18. Stadie, W. C, N. Haugaard, and M. Vaughan, J. Biol. Chem., 200, 745
(1953).

19. Stetten, D., Jr., and G. E. Boxer, J. Biol. Chem., 156, 271 (1944).

20. Stetten, M. R., and D. Stetten, Jr., J. Biol. Chem., 187, 241 (1950).

21. Sutherland, E. W., and C. F. Cori, J. Biol. Chem., 188, 531 (1951).

22. Welt, I. D., D. Stetten, Jr., D. J. Ingle, and E. H. Morley, J. Biol. Chem.,
197, 57 (1952).

23. Wilson, D. W., J. Biol. Chem., 56, 203 (1923).

24. Wyshak, G. H., and I. L. Chaikoff, J. Biol. Chem., 200, 851 (1953).

175

THE HORMONES, THEIR PRESENT
SIGNIFICANCE, THEIR FUTURE

GREGORY PINCUS, The Worcester Foundation for Experimental
Biology, Shrewsbury, Massachusetts

Introduction

With the accelerating increase of our knowledge of the
biochemistry of the hormones, any summary assessment of their
role as biologically active substances is fated to be overmiserly
in description and hazardous in extrapolation. The descrip-
tion is overmiserly not merely because of the necessity for con-
densation, but also because the acquaintance of any single in-
dividual with the diverse and heterogeneous fields of investi-
gation involved is bound to be limited. The extrapolation is
hazardous because one is in the position of a mathematician
attempting to write the equation of an S-shaped curve which has
not yet exhibited its inflection point. To compensate to some
extent for inadequate acquaintance with all the data, this writer
will confine his observations to animal hormones and chiefly to
those which are active in mammals, and as a substitute for pre-
cise extrapolation he will offer speculations as well based as pos-
sible on established fact.

To indicate the diversities which confront any student of
hormonology I have compiled Table I, which lists those sub-
stances having fairly established claims to being active internal
secretions in animals. A number of additional substances

176

HORMONES

TABLE I

A List of Animal Hormones, Their Chemical Nature, and Some of Their

Biological Activities

Name

Tissue of
origin

Chemical
nature

Growth and de- Corpus cardia- Unknown

velopment (GD) cum, ring
hormone gland,

prothoracic
gland
JuvenUe hormone Corpus allatum, Unknown

ring gland
Stimulant of GD Brain Unknown

secretion

Insect gonad stim- Corpus allatum Unknown
ulant

Voltinism hor-
mone

Stimulants of
chromatophores

Subesophageal
ganglion

X-organ, sinus
gland, central
nervous system

Unknown

Intermedin-like
polypeptides

Retinal pigment- Central nervous Unknown
stimulating hor- system

mone

Molting inhibitor X-organ and

sinus gland

Crustacean gonad X-organ and

stimulant sinus gland

Secretin

Pancreozymin

Cholecystokinin

Enterogastrone

Intestinal mu-
cosa

Intestinal mu-
cosa

Intestinal mu-
cosa

Intestinal mu-
cosa

Unknown
Unknown

Polypeptide

Unknown
Unknown
Unknown

Principal

known
biological
activities

Facilitates metamor-
phosis in insects

Inhibits adult differ-
entiation in insects

Induces GD hor-
mone secretion in
insects

Stimulates female
gonad activity

Causes diapausing

eggs

Induce lightening or
darkening of pig-
mented end or-
gans in Crustacea

Stimulates contrac-
tion and disper-
sion of retinal pig-
ment granules in
Crustacea

Prevents molting in
crustaceans

Stimulates gonad ac-
tivity in crusta-
ceans

Stimulates secretion
of pancreatic juice
and bile

Enhances secretin ef-
fect

Stimulates gall blad-
der contraction

Inhibits gastric mo-
tility

Continued

177

GREGORY PINCUS

TABLE I {Continued)

Principal

known

Tissue of

Chemical

biological

Name

origin

nature

activities

Vasopressin

Posterior pitui-

Polypeptide

Antidiuretic, periph-

tary

eral vasocon-
strictor

Oxytocin

Posterior pitui-

Polypeptide

Stimulates uterine

tary

muscle contrac-
tion

Insulin

Pancreas /3-cells

Protein

Hypoglycemic, gly-
cogenic

Glucagon

Pancreas a-cells

Protein (?)

Hyperglycemic, gly-
cogenolytic

Thyroid hormone

Thyroid gland

Tetraiodothyro-

Increases tissue oxy-

nine in thyro-

gen consumption

globulin

Parathyroid hor-

Parathyroid

Unknown (per-

Regulates calcium

mone

gland

haps one or

and phosphate

more polypep-

metabolism

tides)

Epinephrine

Adrenal medulla

Catechol amine

Vasoconstrictor, gly-
cogenolytic

Norepinephrine

Adrenal medulla

Catechol amine

Vasoconstrictor,
neurohumoral

Cortisol

Adrenal cortex

11,17,21-Hydroxy-

Glycogenetic, anti-

lated progester-

phlogistic, thymo-

one

lytic, protein cat-
abolic

Corticosterone

Adrenal cortex

11,21-Hydroxy-

Glycogenetic, thy-

lated progester-

molytic, protein

one

catabolic

Aldosterone

Adrenal cortex

11,21-Hydroxy-

Sodium-retaining,

lated, 18 alde-

weakly thymolytic

hydic progester-

one

Deoxycorticoster-

Adrenal cortex

2 1 -Hydroxylated

Sodium-retaining,

one

progesterone

potassium-excret-
ing

Hydroxyandros-

Adrenal cortex

11 -Hydroxylated

Weakly androgenic

tenedione

A*-androstene-

and protein ana-

3,17-dione

bolic

Testosterone

Testis

1 7-hydroxylated-

Androgenic, protein

A*-androstene-

anabolic

3-one

178

HORMONES

TABLE I {Continued)

Principal

known

Tissue of

Chemical

biological

Name

origin

nature

activities

Androstenedione

Testis and

A^-Androstene-

Weakly androgenic,

adrenal cortex

3,17-dione

protein anabolic

Estradiol

Ovary, testis,

Estratriene-3,17-

Estrogenic, protein

adrenal cortex,

diol

anabolic in fe-

placenta (?)

males, thymolytic,
growth-stimulat-
ing

Estriol

Placenta

16-Hydroxy estra-
diol

Estrogenic

Progesterone

Ovary, adrenal

A^-Pregnene-3,20-

Progestational, ovu-

cortex, pla-

dione

lation-inhibiting

centa

Relaxin

Ovary

Unknown

Relaxes pubic liga-
ments

Adrenocorticotro-

Anterior pitui-

Polypeptide

Stimulates adrenal

phin

tary

cortex secretion

Growth hormone

Anterior pitui-

Protein

Protein anabolic.

tary

stimulates bone
growth

Follicle-stimulat-

Anterior pitui-

Glycoprotein

Stimulates ovarian

ing hormone

tary

follicle

(FSH)

Intermedin

Anterior pitui-

Polypeptide

Disperses chromato-

tary

phore granules in
skin

Luteinizing hor-

Anterior pitui-

Protein

Induces ovulation

mone (LH)

tary

and gonadal hor-
mone secretion

Prolactin

Anterior pitui-

Protein

Stimulates milk pro-

tary

duction and ma-
ternal behavior

Thyrotrophin

Anterior pitui-

Protein

Stimulates thyroid

(TSH)

tary

hormone produc-
tion

might well have been included, but the listing clearly indicates
the chemical variety of hormones and their broad range of
activities. The latter, however, are inadequately indicated,
since only the chief activities are listed. There is, in fact, no

179

GREGORY PINCUS

tissue in the mammalian body which is exempt from some sort of
hormonal influence either in the course of its development or in
its functional activities. Investigations of hormone physiology
have quite properly been centered on the more specific actions of
these substances, but it becomes more and more evident that the
spheres of action are often extremely broad, and frequently far
beyond the narrow limits suggested by the tissue of origin and
its known interrelationships with other organs and tissues.
Thus the expected action of ovarian estrogen as a promoter of
female reproductive tract growth and of estrous behavior is ac-
companied by multitudinous activities quite outside the repro-
ductive realm; estrogens are also hair and bone growth reg-
ulators, thymolytic, euphoria-inducing in the menopause,
mitogenetic in the epidermis, enzyme inhibitory in the adrenal
cortex, phagocyte-stimulating, alkalosis-inducing, antiathero-
genic, tumorigenic, antigoitrogenic, antihyperglycemic, en-
hancers of cortisone-induced weight loss and glycosuria, hypho-
thalamic threshold lowering, anemia-inducing in dogs, etc.,
etc. Similar multiplicities of action may be cited for most
hormones. Out of this welter of activities there arises the con-
cept of an array of hormonal substances circulating to the
various tissues of the body and interacting in a vast complex of
functional processes.

It is not necessary to labor the point that the hormones are
chemically a heterogeneous lot and biologically ubiquitously
active. The only thing that unites them is a definition of them
as internal secretions. How then may one consider them as
biochemical entities? I suggest that for any and every single
hormone there are five fundamental questions to be answered:
(7) What is its chemical structure? (2) How does it origi-
nate? (3) What does it do? (4) How does it do it? (5)
What is its fate? In the case of no single hormone do we have a
complete answer to all these questions. An inspection of Table
I indicates that the chemical structures of most hormonal sub-
stances are either unknown or only partially defined. If we
consider those with known chemical structure we find that even

180

HORMONES

in the best instances the answers to questions (2), (3), (4), and
(5) are either incomplete or lacking. Let us consider an out-
standing example.

Cortisol as a Hormone Prototype

Cortisol is firmly established as an adrenocortical hormone.
Because of its medical utility, Cortisol, and, interchangeably, its
transformation product cortisone, have been during the past
five years objects of extraordinarily intensive investigation.
How does it originate? The best information available suggests
that its biosynthesis as a specific adrenocortical product is ac-
complished by at least two major biogenetic pathways (7,10).
These are depicted in Figure 1. Adrenocortical enzyme sys-
tems responsible for steps (</), (^), (/), and {g) have been de-
scribed and partially characterized. The transformations in-
volved in (a), the nature of X, and the events occurring in {h)
and iji) are unknown. The scission of the side chain of choles-
terol to yield A^-pregnenolone (reaction (c)) is known to be
expedited by adrenocorticotrophic hormone (ACTH), but (A) is
apparently AGTH-independent. It is not certain that these
two biosynthetic pathways (one via cholesterol, the other choles-
terol-independent) are the only ones occurring in the adrenal
cortex.

When we ask ourselves what does Cortisol do we are con-
fronted with a plethora of data. As in the case of the estrogens
a great diversity of physiological effects are known. Cortisol
is not only glycogenetic, antiphlogistic, protein catabolic, it is
also eosinopenic, lymphopenic, an inhibitor of estrogen action
on the uterus, edematogenic, glycosuric, hypertensive, aborti-
facient, an adjuvant in fat mobilization, a maintainer of vas-
cular tone and vasomotion in the capillary bed, antianaphylac-
tic, diabetogenic, muscle-work sustaining. It is not possible to
ascribe these diverse effects of Cortisol to a single basic action,
e.g., its neoglycogenetic activity. Nor can we as yet define a
circumscribed set of actions as fundamental, although there have

181

GREGORY PINCUS

been attempts to do so {cf. Russell and Wilhelmi (13)). Thus
the therapeutic effects of Cortisol do not correlate with its meta-
bolic effects; indeed several of the latter have been called

GH3COOH

HO

HO

A^-Pregnenolone

11-Deoxycortisol

(e)

Progesterone 1 7-OH-Progesterone

Fig. 1. A representation of the biosynthesis of Cortisol.

"side effects." Though the antiphlogistic (or "anti-inflamma-
tory") effects appear to be primary to therapeutic effects, the
ameliorations observed in a wide variety of diseases lead one to

182

HORMONES

suspect as responsible a combination of actions, some of them
tissue-specific, others nonspecific. Even in the fairly simple
muscle-work test, the increase of efficiency caused by Cortisol is
in part due to its making glucose available to the muscle, but
there is also a component ascribable to a direct effect of Cortisol
on the muscle itself.

In considering how Cortisol exerts its characteristic effects the
problem we face is its biochemical mode of action. Here we are
confronted with a rather specific puzzle and a general dilemma.
The specific issue rests on the fact that Cortisol given orally
to men and animals is very nearly as effective as when it is given
by the parenteral route. In being absorbed from the gut Cor-
tisol passes through the liver, in which it is subjected to extraor-
dinarily rapid chemical transformation (6). Is it possible
then that one or more transformation products are the agents
responsible for the various effects of Cortisol, and that we must
search for these if we are to investigate the substance truly
active biochemically? May a variety of metabolites in fact be
the basis of the diversity of effect? A number of attempts to
obtain the characteristic lymphocytopenic effect of Cortisol by
incubating it with lymphocytes in vitro have met with failure.
Is this because a liver-formed metabolite is indeed the effective
agent? Even in those instances where direct local action has
been demonstrated, e.g., on direct intra-articular or intradermal
injection, there is evidence of rapid transformation of Cortisol to
subsidiary products (12). This possibility of "active" hormone
being something else at the site of its action applies to most if not
all of the hormones.

The general dilemma is that which involves all who are
interested in the mechanism of hormone action. It is first of all
the problem of disentangling direct from indirect effects. Thus
Cortisol is extraordinarily potent in effecting glycogen deposition
in the liver of adrenalectomized animals. Yet this effect cannot
be obtained by incubating Cortisol in vitro with liver tissue.
Glycogen deposition represents the end result of a series of events
in which Cortisol plays a role; the best evidence available sug-

183

GREGORY PINCUS

gests that liver glycogen deposition results from glucose pro-
duced from certain tissue protein catabolized under the in-
fluence of Cortisol (13). An examination of the mechanism of
Cortisol action must therefore involve not liver glycogenesis but
gluconeogenesis from protein. Since the disentanglement of
indirect effects has been in itself a task of major proportions, it is
perhaps not surprising that studies of the mechanism of Cortisol
effects have lagged. The fact is that we do not have an ex-
planation of how Cortisol exerts its direct specific effects, nor in-
deed do we have this for any of the hormones.

A number of possible hypotheses have been advanced to
explain the mode of Cortisol action. It is interesting that these
hypotheses might apply to the discussion of any hormone action.
They may be summarized as follows: (7) Cortisol acts as a co-
factor to an enzyme system controlling the rate of a specific
process ; (2) Cortisol acts to affect the permeability of the cell to
substances necessary for certain enzymatic processes; (3)
Cortisol acts upon the processes controlling the movement of
electrolytes in such a way that the significant concentrations of
these electrolytes, both intra- and extracellular, are the rate-
controlling factors (9). Thus far every attempt to demonstrate
that Cortisol is a component of an identifiable enzyme system
has failed. A meaningful characterization of the cellular per-
meability alterations that may be ascribed to Cortisol has not
been made. Although certain aspects of the general metab-
olism of K and Na as influenced by Cortisol have been well de-
scribed, the specific effects of their alterations upon cortisol-
labile metabolic systems have not been established. If we
find ourselves with no explanation of the mechanism of Cortisol
action, it is not completely because we lack biochemical re-
actions in which Cortisol must play a fairly direct role. Gluco-
neogenesis from protein, the effect on muscle-work, the in-
teresting cooperative role played by Cortisol with nor-adrenalin
in the maintenance of vasomotion in the capillary bed, its func-
tion as an antihyaluronidase, its actions on connective tissue
elements and the release of effector substances in inflammatory

184

HORMONES

reactions — all these offer the investigator opportunities for
analytic dissection. Sooner or later by the study of these or other
reactions a definite role for Cortisol will be assigned. That
none has been found as yet is perhaps not too surprising. Out-
standing metabolic effects of insulin have been known for over
thirty years, but the means whereby these effects are exerted are
still not definitively established ; similarly, no biochemical mech-
anisms have been elucidated for the long-known actions of
epinephrine, thyroxin, estrogens, and androgens.

Our final question concerning Cortisol— what is its fate? —
may be partially answered. Its metabolism has been studied in
men and animals, in vivo and in vitro. Certain chemical trans-
formations which it undergoes have been determined, and the
enzyme systems responsible for these transformations have been
at least partially characterized (4). The chief known pathways
of Cortisol catabolism are presented in Figure 2. The reactions
indicated are based principally upon studies of urinary products
obtained after the administration of Cortisol, but these data are
also in accord with in vitro studies involving Cortisol incubation
with liver slices or homogenates. Quantitatively major trans-
formations are indicated by heavy arrows, minor ones by the
lighter arrows. Compounds II to XIV are products charac-
teristic of Cortisol metabolism in human subjects, compounds
XV to XVI appear in guinea pig urine following Cortisol ad-
ministration (1) and have not been seen in the urines of other
species, whereas II to XIV, if they exist in guinea pig urine,
have not yet been identified therein. Actually in man the 11-
oxygenated 17-ketosteroids (compounds VI, VIII, XI, and XIV)
represent 2% to 3% of administered Cortisol and compounds V
and VI are the major urinary transformation products, ac-
counting for approximately 30%. In man, therefore, some
two-thirds of administered Cortisol is unaccounted for in terms of
identifiable metabolites. In the guinea pig compounds XV to
XVII account for approximately 4% of administered Cortisol,
with the probability that a larger percentage may be repre-
sented by compounds similar to V to VIII, although it should be

185

GREGORY PINCUS

186

HORMONES

o

o
o

KH O

C
O

■ i-H
4-»

a

G

O

+-•

bi)

187

GREGORY PINCUS

noted that in the guinea pig the reaction I^II does not appear
to occur to an appreciable extent, whereas II— >I occurs readily
(2). In all species examined thus far a considerable pro-
portion of the transformation products of Cortisol are unknown.
In recent studies with C^ ^-labeled Cortisol there have been in-
dications that a variety of products may occur, some of which
may be nonsteroidal. Furthermore, there are indications of
species differences in the mode of metabolism ; thus in the rat and
mouse there is considerable excretion of fecal metabolites,
whereas in man up to 80% of the 4- G^ ^-Cortisol radioactivity has
been excreted into the urine (5).

In the case of the steroid hormones the search for metab-
olites generally has paralleled what has been described for
Cortisol. No complete balance sheet is possible but a variety of
transformation products are well established. When we con-
sider the hormones the chemical nature of which is less well es-
tablished, our knowledge of their fate in the body is meager
indeed. Thus it is known that certain anterior pituitary hor-
mones disappear extremely rapidly from the blood stream, but
where they go and what their chemical fate is, remains a mys-
tery.

Having discussed in summary fashion the major biochemi-
cal problems of a fairly intensively investigated hormone, I
think I have made evident some of the hiatuses in our knowledge
concerning it. Similar considerations apply to other hormonal
substances. What they do is known to a greater or lesser extent,
but it may be safely stated that there is no complete knowledge of
all the actions of any single hormone. How they do it is for the
most part a mystery, at least in the ultimate sense of mechanism
of action. And the biochemical fate of most of the hormones is
unknown, and for those concerning which we have some specific
information a complete balance sheet has not been established.

Synergism and Antagonism

There are other aspects of hormone biochemistry not en-
compassed by our five leading questions. For example, sub-

188

HORMONES

sidiary perhaps to the problem of mechanism of action are the
problems of inhibition and synergism in hormone action. Ap-
parently straightforward examples of hormone antagonism may
be cited : the inhibition of androgens by estrogens, the antago-
nism between pituitary growth hormone and AGTH in a num-
ber of their effects, the anti-insulin effect of certain cortico-
steroids, diuretic and antidiuretic hormones. Synergism is less
easy to exemplify — estrogen and progestin seem to synergize in
certain effects, not in others; and augmentation of androgen
stimulation has been observed with thyroxin administration; a
horse pituitary extract in itself inactive appears to sensitize the
adrenal to ACTH action. When one examines these types of
hormone interaction somewhat more closely, it becomes difficult
to be certain that the same process is affected by the interacting
hormones. Thus androgen and estrogen both stimulate pros-
tate growth ; the former acts upon the secretory tissue, the latter
on the utricle ; yet estrogen is employed in prostatic disease as an
androgen antagonist. Estrogen sensitizes the uterus to pro-
gestin action and is thus synergistic, but large estrogen doses may
actually inhibit pseudopregnant proliferation due to proges-
terone. The crux of the matter lies in the identification of the
specific chemical processes labile to the various interacting hor-
mones, and this, as we have said before, still eludes us.

Hormone Therapy

I have scarcely remarked on the therapeutic actions of the
hormones, particularly their role in affecting various pathologi-
cal processes. The earliest uses of hormones in therapy were for
replacement in obvious cases of deficit. The wide usage of
estrogen in the menopause is considered replacement therapy,
as is insulin for the control of diabetes, thyroid in hypothyroid-
ism, and corticosteroid in Addison's disease. With the advent
of new and more potent preparations of various types and partic-
ularly with the discovery of the antiphlogistic actions of ACTH
and corticosteroids, experimental investigations of hormone ef-

189

GREGORY PINCUS

fects in practically every known disease have multiplied. At the
moment it is hard to see the forest for the trees, but a concept of
"regulatory" therapy appears dimly. The most vocal protago-
nist of such a concept has been Professor Hans Selye, whose
various publications on "diseases of adaptation" suggest a dis-
turbance of the normal pituitary-adrenal relationship as one
etiologic factor in a great variety of diseases (14). It is impos-
sible to summarize adequately the voluminous discussion and the
shifts and changes which have marked this general theory. The
endocrine basis has been a notion of imbalance between mineralo-
corticoid and glycocorticoid hormones of the adrenal cortex
which in turn involves imbalances in mineralocorticotrophic and
glycocorticotrophic pituitary hormones. Thus far no mineralo-
corticotrophin has been established as a distinct entity, al-
though Selye believes that pituitary growth hormone serves that
function. ACTH acts as the glycocorticotrophin. The theory
demands that continued stress induces an alteration in adreno-
cortical output of such a nature that mineralocorticoids tend
under chronic stress to preponderate among the secretory
products. Certain recent findings are worth mentioning.
First of all, the discovery of aldosterone as an extremely potent
adrenal mineralocorticoid (15) suggests that this substance may
be the prophlogistic adrenal hormone. Secondly, the excretion
of aldosterone-like sodium-retaining activity in human urine
appears to be increased by growth hormone administration and
unaffected by ACTH administration (17). Thirdly, chronic
ACTH administration, the presumed equivalent of chronic
stress, significantly alters the nature of adrenocortical secretion;
in the rabbit, primarily a secretor of corticosterone, chronic
ACTH treatment tends to increase Cortisol and decrease corti-
costerone secretion (8). Since corticosterone is more of a
mineralocorticoid than Cortisol (the typical glycocorticoid),
ACTH, in the rabbit at least, tends to alter adrenocortical se-
cretion toward prophlogistic hormone output. A detailed dem-
onstration of the nature of aldosterone biogenesis and of the
nature of adrenal secretion in chronic stress is clearly needed.

190

HORMONES

Hormones and the Future

In describing certain aspects of the present biochemical
status of the hormones I think I have indicated by inference
some of their future prospects. Gaps in our knowledge con-
cerning them are most obvious, and these will one day be filled.
Instead of examining specific deficiences and what their cor-
rection will imply, it may be worth while to take a broad look
ahead.

Basic to this broad look is the expectation that one specific
set of deficiencies must and will be corrected — our knowledge of
the chemical nature of the hormones must be advanced so that
either the chemical structure of known hormones will be es-
tablished, or that, in the case of complex substances, chemically
pure preparations will be available. Much of the controversy
and confusion in hormone research may be traced to the prop-
erties of impure hormone-concentrating extracts. Thus the
paradoxical hyperglycemic action of insulin has been established
as due to the presence of glucagon in insulin concentrates.
Much controversy concerning the salt-retaining activities of
adrenal extract appears to be resolved by the isolation and
identification of aldosterone. A number of mysteries in thyroid
hormone biochemistry have been solved with the establishment
of triiodothyronine as an active principle. Many more such
instances could be cited. The chemical nature of the hormones
listed as unknown in Table I should be known, and among those
known many should be better known.

Essential also to a broad look is a realization of the per-
vasiveness of the hormones in body economy. If we consider
the known products of the glands of internal secretion and their
known active metabolites, it may be calculated that over 50
biologically active substances circulate continuously in the blood
of mammals as hormones. There is no vital process which is
exempt from their influence, and yet, with few exceptions, these
substances are not essential for life. In the rat, for example,
neither thyroidectomy, nor gonadectomy, nor hypophysectomy,

191

GREGORY PINCUS

nor adrenalectomy is fatal. What happens in these events is
that the rates of certain processes are reduced to a minimum.
When the need arises for a speeding up of these rates it cannot be
fulfilled, for the sources of the prime accelerators are gone.
The margins of safety afforded by hormone-paced homeostasis
are reduced to a minimum. But we are indeed concerned with
more than safety alone ; the physical and mental dullness of the
hypothyroid individual, the asthenia of the Addisonian, repre-
sent deficits in an optimal rate of living. One of the extraor-
dinary features of adequate hormone balance is the mental and
physical vigor of the organism, its adaptability, its drive.

One of the questions to be asked therefore is whether op-
timal rates of living are a function of a specific balance of hor-
monal secretion. Must this large conflux of circulating effector
substances contain precise quotas of each component? What is
the long-run effect of even minor excesses and deficits? We have
developed a tolerable understanding of the daily requirements
for a good number of vitamins. Are there similar daily hor-
mone requirements? Are there individual as well as average
requirements? One obvious result of our assessment of vitamin
requirements has been the administration of adequate amounts
to aging individuals. Somewhat less certainly geiiatric hor-
mone administration is practised. But even before the obvious
deficiencies of age are apparent, may there not be subtler im-
balances contributing to inadequacies and inefficiencies of
general metabolism, of nervous function? In a recent study of
steroid excretion in over 600 men and women of various ages
(11), we were struck by the remarkable consistency of individual
secretion patterns. Each individual has a characteristic quanti-
tative output of certain steroids and therefore a characteristic
ratio of one type to another type. This consistency of personal
patterns has been demonstrated in fine chemical detail for the
individual metabolites in the urinary ketosteroid array by Dob-
riner and his colleagues (3). What is its significance? Broadly
speaking there is an implication of set secretory activity by
steroidogenic organs along with fixed modes of metabolism of

192

HORMONES

the hormones by extraglandular tissue. How do these patterns
become fixed?

There is at present no vahd answer to this question. One
reason why an answer evades us is our inabiHty to assess in even
approximate quantitative fashion the degree of interdependence
of the various hormonogenic systems of the body. From
laboriously attained bits and pieces of evidence we have the
beginnings of an understanding of such interdependencies. We
know, for example, that adequate estrogen action requires
adequate thyroid hormone, that the secretion of ACTH may be
suppressed by various corticosteroids, that pituitary gonado-
trophin release depends in the rat on an estrogen-progestin
balance. Is there a key pacemaker to this system of checks and
balances? Or are we dealing with a complex servel mech-
anism? Are there endogenous antihormones which play a role
in the complex hormone equilibrium? Certainly in any con-
sideration of the effectiveness of hormone secretion it is important
to evaluate those influences which tend to inactivate the hor-
mones. For every known hormone there exists in vivo one or
more inactivating mechanisms. These are enzymatic systems
which oxidize, reduce, detoxify. What is their quantitative and
qualitative contribution to the endogenous hormone pattern?
What regulates their rate of functioning? We have recently ex-
amined a blood-borne enzyme system that inactivates ACTH.
Its concentration in the blood of certain individuals is negligible,
whereas in the blood of others it is so high as to effect ex-
tremely rapid loss of ACTH activity. What is the biochemical
basis of this large individual difference?

We have indicated that both the rate of secretion and the
rate of destruction are key processes in the setting of hormone
balance. One other functional phenomenon still scarcely ex-
plored requires mention — the phenomenon of action of the end
organ. One characteristic of the biological response of or-
ganisms to hormones is variability. Certain individuals show
very low response thresholds, others very high ones, and there is
a characteristic distribution in between, exemplified by the

193

GREGORY PINCUS

well-known dosage-response curve. In intact organisms this
may be reflective of quantitative variations in hormone-in-
activating systems; but one may observe this variability in
response in isolated tissues or cells, e.g., in the glycogenolytic
action of glucagon or epinephrine on liver cells in vitro, in the
effects of insulin on glycogenesis of the isolated diaphragm.
What is the basis of this variable effectiveness, which in certain
instances may amount to nearly complete ineffectiveness? One
may of course postulate hormone-inactivating mechanisms of
varying efficiency present in the end organs themselves. An-
other possibility inheres in the quantitative variations of sub-
strates or cofactors of the hormone-labile systems. A third, and
most intriguing, possibility is the existence in the end-organ
tissue of native hormone analogues as inhibitors. I may cite
as an example the recent demonstration by Velardo and his
collaborators (16) of the pacemaking action of estriol. When
estradiol, which is a much more potent uterus-stimulating es-
trogen than estriol, is administered along with estriol, the degree
of stimulation over a considerable dosage range is determined by
the estriol, not by estradiol. It is as if estriol has an estradiol-
displacing action at the effector sites. Is it possible that for
many, most, or even all of the hormones endogenous antagonists
are produced either as extraglandular metabolites of the active
compounds or by the secretory tissues themselves? One of the
most interesting phases of the adaptation syndrome is the so-
called stage of adaptation. Effects characteristic of the initial
stage of alarm cannot be duplicated at this time by identical
stresses. May not the stage of adaptation be characterized by a
shift to secretion of, for example, corticoid antagonistic sub-
stances which saturate the sites of action at the end organs and
render the corticoids ineffective.

Thus far in attempting a broad look at the future of the
hormones we have apparently been occupied in asking questions.
But I feel that these questions are keys to the future. In the case
of any individual hormone we must continue to ask our five
leading questions. But this is not the limit of our inquiry. We

194

HORMONES

Still have the larger questions of hormone interaction. There
are complex balance sheets to be set up, monograms, and in-
dividual accountings. We must account for pathological and
personal variations in function and for declines in function at-
tributed to "normal" aging. If this seems a gigantic task, I
think its magnitude is exceeded by its fascination.

In our discussion we have considered the hormones as
regulators of a great variety of vital processes. I should like to
emphasize that these processes may vary from intracellular
chemical reactions to complex behavior. Androgens affect
protein anabolism, but also the pecking order of cockerels.
Cortisone inhibits protein synthesis, induces euphoria, and is,
under certain circumstances, psychotogenic. Progesterone pre-
vents water intoxication and induces broodiness in hens. We
are concerned not only with intimate biochemical events but
also with the organism as a whole. In their practical applica-
tion, therefore, the hormones must be considered not only as the
agents of specific biochemical effects but also as governors of
behavior and perhaps even ideation. Hormonal treatment as
now practised for premenstrual tension and menopausal vaso-
motor symptoms is designed not only to enable women to func-
tion better but to feel and behave better. Perhaps this is just
another way of stating that hormones have considerable effects
on the central nervous system.

If, then, we visualize the hormones as continuing to con-
tribute more and more to the metabolic efficiency of the or-
ganism in health and disease, to its adaptability to stress, to its
resistance to involution, to its emotional well-being, to its better
prenatal and postnatal development and growth, have we come
to the end of our prophecy? I suspect not. It may not be
entirely pertinent to a biochemical essay, but I cannot forbear
mentioning certain contributions to the livestock industry.
Hormonal caponization has become a standard practise in
poultry husbandry. I dare say that fairly simple hormone
treatments will shortly be used to improve the quality of beef,
pork, mutton. It is possible that control of the sex ratio in

195

GREGORY PINCUS

animals by hormonal means may one day be attained. Cer-
tainly hormone therapy in veterinary medicine will be increas-
ingly practised. Then there is anthropology. The measure-
ment of physical and emotional differences between the so-called
races of men has disclosed differences that may have rather
profound endocrine bases. What about endocrine mechanisms
in the fat metabolism of the Eskimo, pituitary and androgen
function in the Pigmy, adrenal function in ceremonial rites?
Are there endocrine bases to personality and behavioral tribal
characteristics?

I have thus far limited this discourse to known animal hor-
mones. I should like to remark finally and briefly on prospects
of discovery. Mammalian endocrinology has been the most
intensively exploited field, particularly because of medical im-
plications. At this writing the probability of the discovery of
many new mammalian hormones appears limited. I am not
unmindful of the need for further exploration of pituitary factors
(adipokinin may be established as the latest) and of gastro-
intestinal tract hormones and of the parathyroid mystery and of
prospects of new neurohumors. But I do believe that our
knowledge of significant hormones in the lower vertebrates and
invertebrates is at best elementary. A few faithful and ex-
traodinarily able investigators have discovered fascinating
hormonal mechanisms in insects and Crustacea, with the result
that the problems that they have uncovered can keep many
people busy for a long time. Comparative endocrinology as a
science is not even an infant, it is a conceptus. If you would like
to be the discoverer of a new animal hormone, apply your bio-
chemistry to any one of innumerable species of nonmammalian
forms. There lies the untouched treasure.

I hope it is evident that the trajectory of the curve de-
scribing hormone research has not yet reached its inflection
point. Certainly evident to the scientific pioneer are the pros-
pects of wide-open spaces, of intriguing new phenomena, and of
mysterious natural events. There is no stage in individual de-
velopment from conception to senility which is exempt from

196

HORMONES

hormonal influence. There is even dying itself. Moreover,
there is a diversity of problems which is unparalleled. In the
study of any single hormone the resources of organic chemistry,
of cellular and general physiology, of enzyme biochemistry,
and even of neuropharmacology and psychology may be called
into action. As biologically active substances the hormones
have an often baffling ubiquity. For a long time they will
continue to be inciters of curiosity and stimulants to investiga-
tion. To judge by the scientific rewards which have been
reaped, there are greater rewards to come.

References

1. Burstein, S., and R. I. Dorfman, J. Biol. C/um., 213, 581 (1955).

2. Burstein, S., and R. I. Dorfman, personal communication.

3. Dobriner, K., J. Clin. Invest., 10, 950 (1953).

4. Dorfman, R. I., and F. Ungar, Metabolism of Steroid Hormones. Burgess,
Minneapolis, 1953.

5. Gallagher, T. F., H. L. Bradlow, D. K. Fukushima, C. Beer, T. H.
Kritchevsky, M. Stokem, M. L. Eidinoff, L. Hellman, and K. Dobriner,
Recent Progr. Hormone Research, 9, 41 1 (1954).

6. Hechter, O., Ciba Foundation Colloquia on Endocrinol., 7, 272 (1953).

7. Hechter, O., and G. Pincus, Physiol. Revs., 34, 459 (1954).

8. Kass, E. H., O. Hechter, I. A. Macchi, and T. W. Mow, Proc. Soc. Exptl.
Biol. Med., 85, 583 (1954).

9. Mechanism of Corticosteroid Action in Disease Processes, Annals A^. Y.
Acad. Sci., 56, 623 (1954).

10. Pincus, G., in Paul Kallos, ed., Progress in Allergy. Little, Brown,
Boston, 1955.

11. Pincus, G., L. P. Romanoff, and J. Cado, J. Gerontology, 9, 113 (1954).

12. Pincus, G., E. B. Romanoff, and L. P. Romanoff, Ciba Foundation Colloquia
on Endocrinol, 7, 240 (1953).

13. Russell, J. A., and A. E. Wilhelmi, in F. D. W. Lukens, ed., Medical
Uses of Cortisone, Vol. 1 . Blakiston, New York, 1 954.

14. Selye, H., The Story of the Adaptation Syndrome. Acta Montreal, 1952.

15. Simpson, S. A., J. E. Tait, A. Wettstein, R. Neher, J. von Euw, O.
Schindler, and T. Reichstein, Helv. Chim. Acta, 37, 1 163 (1954).

16. Velardo, J. T., F. C. Hisaw, and C. M. Goolsby, Federation Proc, 12,
68 (1953).

17. Venning, E. H., Ciba Foundation Colloquia on Endocrinol., 8, 190 (1955).

197

PROBLEMS OF CELLULAR BIOCHEMISTRY

CARL F. CORI, Department of Biological Chemistry, Washington
University School of Medicine, St. Louis, Missouri

If the present era of biochemistry needs a general charac-
terization, it might be said that the central problems are those
concerned with enzyme action. The reason for this is based on
the recognition, in the last fifty years, that most of the chemical
reactions which occur in living organisms are enzyme-catalyzed
and that synthesis and degradation of organic molecules gen-
erally involve a series of enzymes acting in sequence. From this
would follow the present trend of obtaining as much relevant
information as possible about each enzymatic reaction a par-
ticular organism or tissue is capable of carrying out. When one
considers the whole array of living forms and the great diversity
of organic molecules which occur in nature, it seems clear that
work along these lines will progress actively for a long time to
come, subject only to the limitation of available methods.

The level of significance, the depth to which one can
penetrate into a problem, is severely limited by methods. It can
be clearly seen how recent methodological advances have led to
solutions of problems which were previously unattainable.
Among others, the use of isotopes, of chromatography, and of
precision instruments for physical measurements come to mind.

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CELLULAR BIOCHEMISTRY

The proposition that most biological problems, if one probes
deeply enough, can be reduced to enzyme problems, would
seem to justify a certain amount of optimism about the possible
attainments of the future. Each scientific era sets up its own
criteria of what is regarded as significant. We would regard as
deeply significant even the partial solution of such problems as
that of nucleic acid and protein synthesis, which are in turn con-
nected widi the much larger problem of self-duplication of
genetic material and its somatic expression through tlie control
of the formation of specific enzymes.

The example just given indicates the degree to which the
boundaries between various disciplines, once sharply delineated,
have been blurred. The widening territory in which it is pos-
sible to operate with biochemical methods enhances the feeling of
the unity of science and facilitates cross-fertilization between
diflfercnt disciplines; it also has the effect of counteracting the
much decried tendency to overspecialization. Remarkable as
the advances are in the elucidation of cellular structure through
the use of the electron microscope, biochemists would not have
paid much attention if there had not been present an awareness
of the importance of structural elements for biochemical events
in intact cells. Here one needs refer only to investigations on
the role of mitochondria for the energy metabolism of the cell.
The study of the biochemical potentialities of these and other
cellular particles is important in relation to the activity of the
cell as a whole.

Cellular Organization

The rapidly increasing information about enzymatic re-
actions is accompanied by attempts to apply this knowledge to
the intact cell, the ultimate goal being an understanding of the
processes which determine the integration of enzymatic ac-
tivities at the cellular or even organismic level of organization.
Biochemistry is moving but slowly in this direction, because
there are many difficulties, but there can be no doubt that such

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CARL F. CORI

understanding will have far-reaching consequences for biology
as a whole and that it will be of great benefit in the treatment of
disease. In a sense the problem has been worked on extensively
in the past on a variety of unicellular organisms. There is also
available a large amount of information which is based on work
on the intact animal, with a variety of methods, including more
recently the use of isotopes. The study of regulatory mech-
anisms and the endocrine control of metabolism fall into this
category.

In this article an attempt will be made to outline some of
the problems which arise in this field, and to point out certain
areas where additional information is needed. It is not in-
tended to cope with a vast, quite heterogeneous, and widely
scattered literature or to treat any given problem exhaustively.
The field will be narrowed down further by considering mainly
mammalian cells.

Rate of Penetration of Sugars into Cells

One of the first questions which arises is that of rate of
penetration of various substances through the cell membrane.
For example, is it the rate of entrance of metabolites, more or
less common to all cells, such as sugars, amino acids, fatty acids
and their keto derivatives, which determines the rate of metab-
olism within the cell, or are the rate-limiting steps the enzy-
matic reactions in which these metabolites are used as sub-
strates? In the first case the cell membrane would have a con-
trolling influence over the rate of metabolism ; in the second case
the effective enzyme concentration would be decisive. In
spite of a very large literature on the subject of permeability,
information on this question is very scanty.

Work carried out recently in this laboratory by Drs. Field,
Heimberg, and Crane and not as yet reported has given a defi-
nite answer to this question for the Ehrlich ascites tumor cells
metabolizing various sugars. These washed, single-cell prep-
arations show one of the highest rates of glucose catabolism

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CELLULAR BIOCHEMISTRY

known for mammalian cells (anaerobic lactic acid production
equivalent to 25 per cent of dry weight of cells per hour at 37°
G.) ; their rate of lactic acid production remains linear for
many hours under anaerobic conditions, and there is little if
any lag period when glucose is introduced at zero time, and ini-
tial acid production is measured titrimetrically with a glass elec-
trode assembly. The final distribution of various sugars (some
of which were utilizable and some of which were not), based on
the aqueous phase of medium and cells, was close to unity, and
equilibrium was reached within one minute at 37 ° G. over a
wide range of sugar concentrations. Only by lowering the
temperature to 20 ° G. could the actual rate of penetration be
measured, which was found to be rapid enough, even at that
temperature, to sustain the observed rate of lactic acid formation
at 37 ° C. Furthermore, rates of lactic acid formation, deter-
mined at different concentrations of glucose, gave typical Line-
weaver-Burk plots and K^ values which were of the same order
as iT^ determined on hexokinase extracted from the cells (A'^ was
close to 1 X \0~^ M per liter in both cases). These and other
observations make it quite certain that the rate of sugar metab-
olism of these cells is determined by the rate of activity of the
intracellular enzymes and not by the rate of penetration of the
sugars through the cell membrane.

It is possible to decrease the rate of lactic acid formation in
these cells by means of sugars which, while not being phos-
phorylated, have sufficient affinity for a common transport sys-
tem to act as competitive inhibitors. Thus galactose and 3-
methyl glucose inhibit the rate of lactic acid formation from glu-
cose, fructose, and mannose, and the degree of inhibition, at
different concentrations, is determined by the relative affinities
of these sugars for the transport system.

The question of the rate of penetration of sugars into the
tissues of the intact animal is not so easily analyzed. In a study
carried out some twenty years ago on rats it was noted that the
ratio, fermentable tissue sugar/plasma sugar, was lowest for
skeletal muscle, intermediate for diaphragm, and highest for

201

CARL F. CORI

heart muscle. Following glucose injection, heart muscle
proved to be much more permeable than skeletal muscle. This
was attributed to the difference in the number of open capil-
laries in the active cardiac muscle as compared to resting
skeletal muscle. In heart, after glucose injection, the ratios,
tissue sugar/plasma sugar, ranged from 0.4 to 0.6. Had the
sugar been present only in the extracellular fluid space, the
ratio would have been about 0.2. Part of the sugar was there-
fore intracellular, and the increase in the intracellular concen-
tration with rising plasma concentration indicated that the rate
of penetration was greater than the rate of utilization. When
skeletal muscle was stimulated, thereby imitating heart muscle,
an intracellular distribution of sugar could be demonstrated (6).

Following the intravenous injection of a nonutilizable sugar
into a nephrectomized animal, to take relatively simple ex-
perimental conditions, the following factors come into play in the
distribution of the sugar in the body: (7) rate of penetration
through capillary wall; (2) rate of distribution in the extra-
cellular fluid space ; (3) rate of penetration into different tissue
cells ; and (4) selectivity of various cell membranes. In general,
(7) is a very rapid process, to judge from the initial rate of dis-
appearance of the sugar from the blood. The rate of (2) and
hence of (3) will depend on the cross section of open capillaries,
on blood pressure, and on rate of blood flow; for this reason
different tissues will attain a state of equilibrium at different
rates.

In regard to (4), definite information is available with re-
spect to erythrocytes, small intestine, kidney tubules, and
peritoneal cavity. The latter shows no selectivity with respect
to the rate of absorption of various hexoses and pentoses, whereas
intestine and kidney are highly selective. The capillaries of the
choroid plexus (representing the so-called blood-brain barrier)
are also selective, whereas the glomerular capillaries allow the
passage even of substances of relatively large molecular weight,
such as inulin.

Whether the mechanism of uptake of sugars by tissue cells

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CELLULAR BIOCHEMISTRY

is similar to that in intestine and kidney is an open question.
In intestine the more rapid absorption of galactose and glucose
as compared with pentoses is attributed to an "active" process,
specifically phosphorylation of the hexoses, whereas the absorp-
tion of the pentoses is attributed to a simple process of dif-
fusion. This phosphorylation (and by implication) -dephos-
phorylation mechanism is also supposed to operate in the prox-
imal convoluted tubules of the kidney. Without attempting to
examine the evidence for this hypothesis in detail, certain diffi-
culties may be pointed out at this time. Carefully controlled
experiments carried out by Sols (18) with extracts of rat intes-
tinal mucosa have failed to show the presence of enzymes capable
of phosphorylation of galactose and 3-methyl glucose (contrary
to statements found in the literature). Yet these two sugars
are absorbed as rapidly as glucose. A hexokinase capable of
phosphorylating glucose, fructose, and mannose and having
a specificity similar to that of brain hexokinase was always found
in these extracts. On the dephosphorylation side there is the
diflSculty that glucose-6-phosphatase is missing in the kidney of
children with severe glycogen storage disease of liver and kidney,
but these children do not have glycosuria (5).

Keston (14) has recently described the occurrence of a
mutarotase in kidney, an enzyme which has previously been
found in molds and which is specific for certain sugars. On the
assumption that a form of low abundance, presumably an open-
chain form of sugar, penetrates preferentially into cells, a role is
ascribed to this enzyme in sugar transport in all tissues. Muta-
rotase, according to this theory, would control an important
rate-limiting step in carbohydrate metabolism, the rate at
which sugar enters the cell, and would also be implicated in
diabetes and the action of insulin. Levine's (16) theory of
insulin action is also based on the assumption that the rate of
glucose utilization in tissues is controlled by the rate of sugar
transport across the cell membrane. An alternative hypothesis
is that the rate of glucose metabolism is controlled by intracel-
lular enzymes. Important as a decision between these two

203

CARL F. CORI

alternatives may be, rigorous proof for either concept has not
been obtained so far.

Compartments of the Cell

The control of rates of metabolism by intracellular enzymes
may be illustrated in the following examples. When isolated
frog muscle is compared at rest and during tetanic contraction,
a more than 1 00-fold difference in the rate of lactic acid forma-
tion can be demonstrated. The mechanism underlying this
increase in enzymatic rates has not been explained. Recent
work (9) indicates that at 0° C, even if there is no demon-
strable splitting of ATP or phosphocreatine during contraction,
there occurs nevertheless an increase in the concentration of
inorganic phosphate which may arise from an unknown precur-
sor. The concentration of inorganic phosphate in resting muscle
is high enough to saturate phosphorylase, the first enzyme in the
chain of reactions from glycogen to lactic acid. If the reaction
catalyzed by this enzyme were the rate-limiting step in glycolysis
in resting muscle, and if it were then accelerated 1 00-fold by the
formation of inorganic phosphate during contraction, one would
have to make the additional assumption that at rest the enzyme
is almost completely separated from inorganic phosphate. A
determination of intermediates of glycolysis in resting muscle
shows that hexosemonophosphate is present in a concentration
which would saturate phosphofructokinase. During contrac-
tion hexosemonophosphate increases (8), and in order to make
phosphofructokinase the rate-limiting step, the same additional
assumption would have to be made as in the case of phosphory-
lase — a compartment of the muscle cell which does not permit
contact between the enzyme and its substrate — a barrier which
is broken down during contraction and is re-established during
rest. Because of the possible existence of such barriers, it may
be misleading to draw conclusions from steady-state concentra-
tions of intermediates. For example, exposure of isolated frog
muscle to epinephrine causes an approximate doubling of the

204

CELLULAR BIOCHEMISTRY

hexosemonophosphate concentration, but this is associated with
a minimal increase in the rate of lactic acid formation (11).
Instead of envisaging a "barrier," one could also assume that
certain muscle enzymes exist as inactive precursors in resting
muscle and are changed to active forms during contraction. In
fact, epinephrine has such an effect on muscle phosphorylase,
which consists in the conversion of an inactive to an active form
(see below). For want of a better one, the term "compartment"
will be used to describe these deviations from the usual kinetics
of enzymes as obtained in cell-free systems, and it will be under-
stood that explanations for this phenomenon are highly specu-
lative.

Compartments can also be invoked to explain glycogen
formation in the liver. The equilibria of the enzymes involved
are such that if the steady-state concentration of inorganic
phosphate in contact with phosphorylase were 1 fxM. per gram
liver, the steady-state concentration of glucose-6-phosphate
would have to be about 7 fxWL for glycogen synthesis to occur.
This is a much higher concentration than is found in the liver.
Even if one assumes that inorganic phosphate is not in contact
with phosphorylase so that the concentration of glucose-6-phos-
phate could be lower, one is still faced with the problem of how
glucose-6-phosphate formed by hexokinase escapes the action of
glucose-6-phosphatase. On the other hand, when glycogen is
being broken down, the glucose-6-phosphate formed is accessible
to the phosphatase. It is as if glycogen synthesis and degrada-
tion were completely separated in the liver cell, in spite of the fact
that there is a common intermediate step, catalyzed by phospho-
glucomutase. We are at present completely in the dark as to
how such cellular operations are accomplished. The examples
could be multiplied, since many other intermediates are known
which can enter different metabolic pathways.

To judge from work carried out with isolated mitochon-
dria the complexity is very great even on the subcellular level.
Factors of permeability are encountered owing to the presence of
a membrane and of internal subdivisions as revealed by electron

205

CARL F. CORI

microscopy. Several enzymes have been shown to be latent in
mitochondria ; this implies either that the enzymes were present
in an inactive form or, if active, that they were not in contact
with their substrates. Furthermore, the metabolic activity of
the mitochondria must be integrated with that of the cell as a
whole. The relationship is such that metabolites, e.g., pyruvic
acid, prepared by other enzyme systems must be supplied to the
mitochondria as oxidizable substrates, while the cell must in
turn be supplied with ATP which is being regenerated in the
mitochondria. The functioning of the cell depends to a large
extent on this interrelationship.

The complexity of the system consists not only in the large
number of diverse chemical operations which are carried on
simultaneously in the same cell but also in the control of the
speed of these operations. One might speculate that the neces-
sary catalysts for a particular operation are associated into a
unit within the cell. For example, hexokinase, phosphogluco-
mutase, and phosphorylase in loose combination, perhaps in a
separate compartment in the cell, would be a unit for glycogen
synthesis, while other molecules of phosphorylase and phospho-
glucomutase in combination with glucose-6-phosphatase would
form a unit for glycogen degradation in the liver. Glucose-6-
phosphatase, according to DeDuve et al. (7), is bound to the
microsomes. Although phosphorylase and phosphoglucomu-
tase appear to be present in a soluble form in a liver homogenate,
this does not exclude loose combinations which would be
broken up by present methods used for the disintegration of
cells. Crane and Sols (4) have shown that over 90 per cent of
the hexokinase in a brain homogenate is bound to particles
which sediment at relatively low centrifugal speeds and which
resemble mitochondria, whereas homogenates of other tissues
contain a considerable part of hexokinase in soluble form. No
information is available at the present time as to whether these
findings have any physiological meaning. It would seem that
new methods will be needed to explore this territory.

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CELLULAR BIOCHEMISTRY

Complexities of Organization

Even if it is granted that the different chemical operations
of the cell are carried out as if they were spatially separated, one
has dealt with only a small part of the complexity of the system.
The inherent capacity of the cell to duplicate itself and to exert
a seemingly purposeful control over its function has made it
difficult to assert that the ordinary laws of physics and chem-
istry apply — a difficulty which the microcosm shares with the
macrocosm. One may take as an example the following
dilemma. The accretion of material of exactly the same molec-
ular structure prior to division of the chromosomes and the
control exerted by this structure on the formation of specific
enzymes starts an unending causal chain, where one enzyme is
necessary for the formation of another enzyme and so on. In
order to break this chain, it is necessary to introduce a non-
enzymatic step, either in the process of self-duplication of
genetic material or in the formation of the first enzyme of a
chain. When faced with such complexities, it is not surprising
that some biologists prefer to invoke the operation of special
vital forces which they believe are peculiar to living matter.
This point of view also finds its expression in statements to the
effect that enzymes studied in vitro after extraction from the cells
are not the same entities which act in the intact cell. Granted
that this may be true in certain cases, one would rather investi-
gate the reason for such deviations than be discouraged about
the validity of the methods that enzyme chemists have used.

The higher forms of organization in multicellular organ-
isms are characterized by the increasing complexity of regulatory
mechanisms on the evolutionary scale. One finds specialization
of chemical operations according to cell types assembled in
organs, increasing control through the central nervous system, im-
proved transport of materials to and from the cells through the
development of the circulatory system, and finally the elabora-
tion of highly active and specific substances, the hormones,
which exert a control over enzymatic processes. A remarkably
constant internal environment has been achieved through the

207

CARL F. CORI

development of regulatory systems and the organism has gained
in performance, but at the same time the different parts of the
organism have become highly interdependent.

Hormone Effects

As a general outline, the following factors are known which
enter into a regulatory system such as that of the blood sugar
level: (7) specialized tissues which respond to changes in the
blood sugar concentrations, e.g., the sympathetic centers in the
hypothalamic region and the islet tissue of the pancreas; (2)
effectors, which are partly nervous, partly humoral; and {3)
targets, which are the enzymatic reactions specifically influenced
by the effectors. It is on the last point that a few additional
comments may be made. Although phosphorylase has been
implicated in the metabolic action of epinephrine, hexokinase
in that of insulin, and oxidative phosphorylation in that of
thyroxin, none of these systems is as yet sufficiently well under-
stood to make one confident that the results obtained so far ex-
plain the actions of these hormones in the intact animal.

Epinephrine and Glucagon

The characteristic metabolic action of epinephrine in the
intact animal consists in a rapid breakdown of glycogen in liver
and muscle, even when at the time of injection the reaction is
proceeding in the opposite direction. Thus, epinephrine not
only causes increased phosphorylase activity but also has an
effect on the direction in which phosphorylase activity is pro-
ceeding.

Phosphorylase occurs in liver and muscle in an active and
inactive form, and there are enzymes present which can convert
one form into the other with great rapidity. The active form in
muscle, phosphorylase a, is a dimer of the inactive form, phos-
phorylase b (13). Although phosphorylase b can be activated

208

CELLULAR BIOCHEMISTRY

by adenylic acid, the concentration needed (1 X 10"^ M) is
higher than that found in muscle. Epinephrine, when added to
liver slices or to diaphragm muscle incubated aerobically in
phosphate-Ringer's solution, causes a rapid increase in the con-
centration of active phosphorylase and at the same time the
glucose output of the liver slices and the lactic acid production
by the diaphragm are increased (19), No effect of epinephrine
has so far been described in a cell-free system. In order to
measure the relative amounts of the two forms of phosphorylase
in resting muscle, special precautions are necessary. The tech-
nique recently used involved freezing the gastrocnemius muscle
in situ in amytalized rats. The muscle was rapidly homogenized
in the cold in a Waring Blendor in a solution containing 0.001
M versene and 0.02 M fluoride, which inhibits the intercon-
version enzymes but has no effect on phosphorylase activity.
Under these conditions rat muscle was found to contain 30 to
50 per cent phosphorylase a, the remainder being phosphory-
lase b.* Following the subcutaneous or intravenous injection
of epinephrine the phosphorylase a content of muscle rose to
nearly 100 per cent within a few minutes, whereas the total
phosphorylase content remained unchanged. In isolated rat
diaphragm Sutherland (19) observed an increase in phos-
phorylase a content within 4 minutes after the addition of epi-
nephrine. Unexplained is the mechanism of the reversal of phos-
phorylase action. This occurs in an isolated rat diaphragm
which is actively synthesizing glycogen from the glucose in the
medium. As soon as epinephrine is added, glycogen break-
down exceeds glycogen synthesis so that there is either no gain
or an actual loss of glycogen (20). The hexosemonophosphate

* Similar values were obtained when muscle not frozen in situ was homog-
enized within a few seconds after excision from the animal. When fluoride
was omitted from the solution used for homogenization, almost all of the
phosphorylase was present in the inactive or b form. An amount of homogenate
corresponding to 4 to 5 mg. of muscle was used in the phosphorylase tests.
The activity in a test without adenylic acid, expressed as per cent of the
activity with adenylic acid, corresponds to the per cent of phosphorylase a.

209

CARL F. CORI

content of muscle is increased by epinephrine, which should
favor glycogen synthesis rather than breakdown. No indication
has been found for an increase in inorganic phosphate which
would favor breakdown. One could refer here again to the
compartments of the cell, but this is not a satisfactory explana-
tion since it is not amenable, at the present time, to experimental
investigation.

Glucagon, a protein recently isolated from the pancreas in
crystalline form, increases the phosphorylase activity of the liver
in the same way as epinephrine (19). In contrast to epi-
nephrine, glucagon does not cause an increase in blood lactic acid
in the intact animal, and it also has no action on the phosphory-
lase a content of the isolated diaphragm. It is possible that
glucagon cannot penetrate into the muscle cell. What the com-
mon denominator, if any, might be between epinephrine and
glucagon in their action on the liver is unknown; possibly
glucagon causes the liberation of a sympathomimetic amine in
the liver. In conclusion it might be pointed out that epi-
nephrine and glucagon probably act on the enzymes which main-
tain a balance between active and inactive phosphorylase rather
than on phosphorylase itself, but this awaits verification in a
cell-free enzyme system.

Muscular activity also influences the relative amounts of
active and inactive phosphorylase in muscle. Continuously
active muscles, such as heart and diaphragm, contain a higher
percentage of phosphorylase a than resting skeletal muscle.
Stimulation of isolated frog gastrocnemius at a rate of 60 single
shocks per minute for 1 to 2 minutes, i.e., conditions which per-
mit work at a steady state without development of fatigue,
resulted in a marked increase in the percentage of phosphorylase
a. On the other hand, repeated tetanic stimulation during 1
to 2 minutes until the muscle showed fatigue, resulted in an
almost complete disappearance of phosphorylase a. These ex-
periments, which have not as yet been reported, illustrate again
the dynamic balance which exists in muscle between inactive
and active phosphorylase.

210

CELLULAR BIOCHEMISTRY

Insulin

Less definite statements can be made about the mechanism
of insulin action than about that of epinephrine. There is in-
direct evidence, obtained by a variety of methods on isolated
tissues and on intact animals, that the first step in glucose utili-
zation is accelerated by insulin. The same step presumably is
inhibited by a substance of pituitary origin which is present in
the serum of diabetic animals, which disappears after hypoph-
ysectomy and reappears again when the diabetic hypoph-
ysectomized rats are injected with growth hormone plus
cortisone (3). The diabetic serum was tested on isolated rat
diaphragm and the inhibition of glucose uptake could be
counteracted by the addition of insulin. A lipoprotein fraction
prepared from diabetic plasma and from anterior pituitary pro-
duced a strong inhibition of the hexokinase reaction in a cell-
free system, but the reversal of this inhibition by insulin was
incomplete and did not have the desired degree of reproduci-
bility (15). In isolated rat diaphragm the inhibition of glucose
uptake produced by a lipoprotein fraction from serum of dia-
betic rats was completely reversed by insulin. It should be
pointed out that the effect of injection of these lipoprotein frac-
tions in intact animals has not so far been reported.

The severe disturbance in fat metabolism in the liver of the
diabetic animal is now regarded as secondary to the inhibited
glucose utilization (10). In the reversible series of reactions
represented by acetyl CoA^fatty acid, there are two reductive
steps in the direction to the right, for each C2 fragment which is
added in the lengthening of the fatty acid chain (17). These
reductive steps require DPNH which can be supplied by gly-
colysis. Whether the DPNH formed during the operations of
the Krebs cycle is available for fatty acid synthesis is uncertain.
In the absence of sufficient glucose utilization in a liver depleted
of its glycogen reserves, the steady-state concentration of DPNH
diminishes, and this shifts the equilibrium to the left, i.e., fatty
acids are now broken down and ketosis results (12). It should
be mentioned that the liver in ketosis responds very sluggishly

211

CARL F. CORI

to insulin, since it takes some time until secondary changes are
repaired. That the normal liver responds rapidly to insulin, as
do other tissues, follows from observations made on normal and
diabetic subjects with the hepatic vein catheterization technique
(2). On injection of insulin the hepatic output of glucose dimin-
ishes within a short time. Another secondary disturbance is an
increased glucose-6-phosphatase activity in the liver, which re-
sults in increased dephosphorylations (1). This nullifies to some
extent the beneficial effect of fructose in diabetes. This sugar is
phosphorylated to fructose- 1 -phosphate by a separate enzyme
and so is able to bypass the metabolic block at the glucose level,
but in its metabolic transformations fructose- 1 -phosphate is in
equilibrium with glucose-6-phosphate, and thus is converted to
glucose by the increased activity of the phosphatases.

There are some cells which are not influenced by insulin.
The ascites tumor cells which were discussed earlier have been
tested under a great variety of conditions, but a clear-cut effect
of insulin on the rate of sugar uptake could not be shown. The
conditions which make a cell respond to insulin are therefore
not clearly defined. In general, an enzymatic step, such as that
mediated by phosphorylase or hexokinase, which is at the be-
ginning of a series of consecutive reactions, cannot, by being
speeded up, increase the rate of the over-all reaction unless it is
the rate-limiting step. It has been shown by several methods
that phosphorylase is the rate-limiting step for glucose formation
in the liver (19). The first step may be rate-limiting under the
following conditions which, if changed, will permit an increase
in the over-all rate. (7) Part of the enzyme is present in an
inactive form. This is the condition under which epinephrine
is able to exert its eflfect, by converting an inactive form of phos-
phorylase to an active form. (2) The substrate concentration is
below that which saturates the enzyme. (3) Product inhibition
occurs because a subsequent enzymatic step in a reversible chain
of reactions is relatively slow. (4) The enzyme is not fully active
because it is combined with an inhibitor. Conditions (2) and
{4) have each been suggested as the condition under which

212

CELLULAR BIOCHEMISTRY

insulin is able to exert its effect, i.e., by increasing permeability
of the cell membrane for glucose or by displacing an inhibitor.
Animal hexokinase, in contrast to that of yeast, is noncompeti-
tively inhibited by glucose-6-phosphate, and competitively
inhibited (with respect to ATP) by ADP (4). If (3) were the
necessary condition, insulin would accelerate an enzymatic step
other than the first, or in some other way counteract the product
inhibition.

Thyroxin

The effect of thyroxin and triiodothyronine in uncoupling
aerobic phosphorylation in isolated mitochondria is obtained not
only with dinitrophenol but also with a number of other unre-
lated substances, none of which can replace thyroxin in thyroid
deficiency. It may be difficult to show that this is the primary
effect in the intact animal. The greatly increased oxygen con-
sumption in the hyperthyroid animal may well more than com-
pensate for the decreased efficiency of aerobic phosphorylation
and could be the primary effect.

This discussion about the action of epinephrine, insulin, and
thyroxin makes it clear how difficult a problem it is to unravel
the mechanism of their actions, not to speak of a number of other
hormones the study of which has just begun. The task is made
somewhat easier if one adopts the point of view that hormones
are specific in their action, that they influence only one kind of
reaction, and that the apparent multiplicity of their effects is
secondary to a primary effect. The metabolic hormones which
have been most extensively studied from a biochemical point of
view have more or less conformed to this pattern.

References

1. Ashomore, I., A. B. Hastings, and F. B. Nesbitt, Proc. Natl. Acad. Sci.
U.S., 40, 673 (1954).

2. Beam, A. B., B. H. Billing, and S. Sherlock, Cib a Foundation Colloquia on
Endocrinol., VI, 250 (1953).

213

CARL F. CORI

3. Bornstein, J., and G. R. Park, J. Biol. Chem., 205, 503 (1953); Bornstein,
J., J. Biol. Chem., 205, 513 (1953).

4. Crane, R. K., and A. Sols, J. Biol. Chem., 203, 273 (1953) ; ibid., 206, 925
(1954).

5. Cori, G. T., Harvey Lectures, Ser. 48, 145 (1952-53).

6. Gori, G. T., J. O. Gloss, and G. F. Gori, J. Biol. Chem., 103, 13 (1933).

7. DeDuve, G., J. Berthet, H. G. Hers, and L. Dupret, Bull. soc. chim. biol.,
37, 1242 (1949).

8. Fischer, R. E., and G. T. Gori, Am. J. Physiol., 772, 5 (1935).

9. Fleckenstein, A., J. Janke, R. E. Davies, and H. A. Krebs, Nature, 774,
1081 (1954).

10. Gurin, S., in V. A. Najjar, ed., Fat Metabolism. Johns Hopkins Press,
Baltimore, 1954.

11. Hegnauer, A. H., and G. T. Gori, J. Biol. Chem., 705, 691 (1934).

12. Helmreich, E., H. Holzer, W. Lamprecht, and S. Goldschmidt, Z.
physiol. Chem., 297, 113 (1954).

13. Keller, P. J., and G. T. Gori, Biochim. et Biophys. Acta, 72, 235 (1953).

14. Keston, A. S., Science, 720, 355 (1954).

15. Krahl, M. E., and J. Bornstein, Nature, 773, 949 (1954).

16. Levine, R., and M. S. Goldstein, Brookhaven Symposia in Biology, 5, 73
(1952).

17. Lynen, F., Federation Proc, 72, 683 (1953); Mahler, H. R., Federation
Proc, 72, 694 (1953).

18. Sols, A., Third International Congress of Biochemistry, Summaries of Communica-
tions, p. 53, Brussels, 1955; Biochim. et Biophys. Acta, 79, 144 (1956).

19. Sutherland, E. W., and C. F. Gori, J. Biol. Chem., 788, 531 (1951);
Sutherland, E. W., Phosphorus Metabolism, 7, 53 (1951).

20. Walaas, O., and E. Walaas, J. Biol. Chem., 787, 769 (1950).

214

ENZYMES AS REAGENTS

EFRAIM RACKER, Division of Nutrition and Physiology, The Public
Health Research Institute of the City of New York, Inc., New York

end to which our currents tend

Inevitable sea
To which we flow, what do we know

What shall we guess of thee?

Arthur Hugh Clough

During the past ten years our concept of enzymes has
undergone a noteworthy change. Enzymes are no longer the
mysterious catalysts of a decade ago. They have become the
mysterious reactants of today. Fifty years ago, kinetic experi-
ments were performed which suggested the formation of enzyme-
substrate intermediates during catalysis, but only in recent years
has a direct demonstration of their existence been achieved.
Now, an extensive search for these enzyme-substrate compounds
is under way in various laboratories. The enzymologist of
yesterday who isolated a crystalline enzyme and recorded a few
of its properties considered his work a task well done. The
enzymologist of today lacks this satisfaction. He realizes that
with the isolation of the enzyme his task has just begun, and,
depending on his own background and direction of interest, he
will approach the purified enzyme as a protein of unknown
chemical structure, or as a catalyst with specific kinetic proper-
ties, or as a reactant which combines with the substrate. As a
protein chemist, he will determine the amino acid composition;
he will attempt a study of the amino acid sequence; he will
modify the protein and search for a relationship between its

215

EFRAIM RACKER

Structure and biological activity. As "kineticist," he will study
the process of substrate activation; he will explore the con-
ditions governing the efficiency of catalytic performance; and
he will derive equations compatible with his data which might
give him clues to the mechanism of enzyme action. As a
progressive enzyme "reactionist," he will use the enzyme as a
stoichiometric reactant ; he will explore the active center of the
enzyme and the points of enzyme-substrate interaction.

Some of these investigations require rather large quantities
of highly purified enzymes, and it is not surprising that those
enzymes which are readily available in large amounts have been
studied most extensively. For example, crystalline glyceralde-
hyde-3-phosphate dehydrogenase (TDH) can be prepared in
gram quantities either from rabbit muscle or from yeast in the
course of a few days. During the past years it has become
the subject of study in numerous laboratories all over the world.
Since these studies have been conducted with a relatively pure
protein, and because of the multifunctional activities of TDH,
this enzyme will be frequently cited as an example in the
following pages.

Enzymes as Proteins

The biosynthesis of proteins, their molecular structure, and
the relationship of structure to biological activity are among the
unsolved and most challenging problems in biochemistry.
Enzymes are particularly suited for such studies because their
biological activity can be rapidly and accurately determined by
very sensitive methods. The specificity of these methods permits
the detection of a particular enzyme in a mass of other proteins.
It was possible in this way to detect the formation of new enzyme
proteins in cell-free systems, although little increment in total
protein nitrogen had occurred (10).

Studies on the composition and sequence of amino acids of
various enzymes having the same action but obtained from
different sources may represent the beginning of a new kind of
comparative biochemistry. A remarkable similarity in amino

216

ENZYMES AS REAGENTS

acid composition, molecular weight, DPN binding, turnover
number, and other catalytic properties is exhibited by prepa-
rations of glyceraldehyde-3-phosphate dehydrogenase from
yeast and from rabbit muscle (50). An unidentified crystalline
protein from papaya latex was "diagnosed" as lysozyme because
of a resemblance in amino acid composition to the egg white
enzyme (42). It is difficult to conceive that these similarities are
coincidental. Although some differences in the biological and
chemical properties of these related proteins exist, these seem
minor compared to the great similarities, particularly in view of
the fact that even enzyme preparations from a single source
reveal microheterogeneity on rigid examination (6). For
example, TDH from yeast was fractionated into proteins of
different electrophoretic behavior with apparently identical
biological activity (22). Such minor variations actually serve
to make us more fully aware of the remarkable precision of the
cellular machinery responsible for the production of these
complex polymers. It seems quite logical therefore to assume
that the specific assembly of amino acids takes place with tlie
aid of a structural guide.

There are, on the other hand, enzymes from different
sources, for example, alcohol dehydrogenase from yeast and liver,
which have vastly different properties although they catalyze
the same reaction. It would be interesting to learn whether a
discrepancy in catalytic efficiency (alcohol dehydrogenase from
yeast is over 50 times as active as the liver enzyme) is mirrored
in pronounced differences of amino acid composition or sequence.

The relation between protein structure and biological
activity of enzymes has been studied mainly by (7) chemical
alterations of the protein; (2) partial proteolytic degradation
of enzymes; (3) construction of enzyme models; and (4)
investigations of the active centers of the enzymes.

MODIFICATION OF PROTEINS BY CHEMICAL ALTERATIONS

Essentiality or nonessentiality of certain groups for enzy-
matic activity has been deduced from chemical alterations, such^

217 ' ■ ^

~f

— Vv\ <5

I 2^ i ^- ! ?f. *^ .A 5^ Y

EFRAIM RACKER

as acetylations, oxidations, reductions. The workers in this field
have been fully aware of the diffculties of this approach and have
emphasized the importance of demonstrating the purity of the
new protein derivative and, if possible, the reversibility of the
induced modification. Extensive acetylation of pepsin (13)
resulted in loss of enzyme activity which was fully restored after
removal of the acetyl groups. Among other types of modifica-
tions, two of more recent data may be quoted. Diisopropyl
fluorophosphate (DFP) inhibits certain esterases as well as
proteolytic enzymes with esterase activity, whereas other
enzymes appear unaffected {cf. ref. lb). Cholinesterase and
chymotrypsin are completely inactivated after reacting with one
mole of inhibitor per mole of enzyme. From the inactive
proteins, 0-phosphoryl serine was isolated after hydrolysis (40).
Serine is therefore strongly implicated as part of the active site,
provided steric hindrance, which will be discussed below, can
be ruled out. Since an acyl shift from nitrogen to the hydroxyl
group of serine may have occurred secondarily during the pre-
parative procedure, the hydroxyl group may not be the primary
reactor {cf. ref. 6a), It is significant that no phosphothreonine
was found in the hydrolyzates. What determines the specificity
of the interaction between DFP and enzyme-serine is unknown.
Free serine or enzyme-threonine does not react with DFP.
Perhaps sequence analysis of peptides containing the radioactive
phosphate of DFP^^^ obtained after partial hydrolysis, may yield
information on this point. The biological reactivity of hydroxy
amino acids has been recently demonstrated by an entirely
different approach. A rapid incorporation of labeled inorganic
phosphate (P^^) was shown to take place into the phospho-
protein fractions from which phosphorylated hydroxy amino
acids were isolated (1,18).

The susceptibility of TDH to iodoacetate (lAA) has been
known for many years. Since it has been shown (23) that TDH
contains firmly bound glutathione (GSH), which can be liberated
by proteolytic enzymes, the interaction with lAA was reinvesti-
gated (24). A very rapid reaction was found to take place

218

ENZYMES AS REAGENTS

between lAA and enzyme-bound GSH, which was dependent
on the presence of DPN. By comparison, the interaction between
lAA and free GSH was quite slow. With three equivalents of
lAA, enzymatic activity of TDH was completely blocked within
a few minutes, and after proteolytic digestion of the modified
protein, no unreacted glutathione was liberated. Furthermore,
addition of acetyl phosphate to the enzyme led to the formation
of acyl enzyme, from which a thiol ester was liberated by
proteolytic digestion. Formation of acyl enzyme did not take
place after treatment with lAA, but if acetyl phosphate was
added prior to the inhibitor, the alkylation of the enzyme by
lAA was delayed. These findings with lAA and similar experi-
ments with A^-ethyl maleimide quite conclusively identified
GSH as part of the active center of TDH, and ruled out the
possibility that lAA inhibits enzymatic activity by steric hin-
drance of enzyme-substrate interaction.

In the case of inhibitors of enzymes which act on large
molecular substrates, steric hindrance may, however, play an
important role. Some of the immunological antienzymes
probably act in this manner. The inhibition of trypsin by
ovomucoid inhibitor is dependent on groups which are non-
essential for proteolysis. This was shown by the fact that
acetylated trypsin, which is enzymatically active, is not readily
inhibited by ovomucoid inhibitor (9). It seems reasonable to
assume that steric hindrance plays a major role in the inhibition
of trypsin activity on proteins by ovomucoid, particularly since
both inhibitor and substrate are rather large molecular sub-
stances (2a).

It is possible, on theoretical grounds, to differentiate three
types of chemical modifications which may result in altered
biological activity: (7) interaction with the active center; (2)
interaction with sites in the vicinity* of the active center leading
to steric hindrance (if the inhibitor is a large molecule, the site

* In a coiled protein structure of the enzyme, a site in the vicinity of
the active center may be far removed in terms of the amino acid sequence of the
extended protein.

219

EFRAIM RACKER

of interaction may actually be far removed) ; and (3) partial
inhibition due to interaction with sites removed from the active
center, "activating groups" in the sense of Langenbeck (27).
The fact that only a limited number of specific blocking agents
is available which can be used without leading to protein
denaturation has seriously hindered progress in this area of in-
vestigation.

MODIFICATIONS OF PROTEINS BY PARTIAL DIGESTION

The effect of partial proteolytic digestion on the activity of
enzymes has not been extensively explored. It has been apparent
for many years that, in some instances, biological activity of
proteins may be retained after fragmentation, as was first
demonstrated in the case of proteolytic digestion of diphtheria
antitoxin (cf. 35). More recently (17), phosphorylase was
cleaved into two inactive fragments and the lost activity was
restored by addition of adenosine-5-phosphate. After exposure
to proteolytic enzymes, TDH lost its activity, which was restored
by adding SH compounds (23). ATPase activity of myosin was
retained after proteolytic digestion (11). Ribonuclease, in-
cubated with carboxypeptidase, contained over 70% of its
original activity, although 20% of its nitrogen was split off.
Further exposure to carboxypeptidase had no effect (29, cf. also
la and 15a). The activation of proenzymes such as chymo-
trypsinogen is another example of proteolytic modification which
has yielded information about the active center. The splitting of
a single peptide bond between arginine and isoleucine appears to
suflfice for the activation of chymotrypsinogen (6b) . Of special
interest is the demonstration of enzymatic activity in dialyzable
fragments of autodigested pepsin (34). These small "enzymes"
were found to contain only 3% of the specific proteolytic activity
of native pepsin, but to retain 64% of its catalytic activity toward
the synthetic substrate acetyl-L-phenylalanyldiiodo-L-tyrosine.
Elucidation of the structure and amino acid sequence of the active
fragments may yield significant information about the active

220

ENZYMES AS REAGENTS

center as well as about the role of the activating groups in the
native protein. This knowledge may also lead to the synthesis
of the enzyme models not too far removed from biological reality.

ENZYME MODELS

From the earliest organic enzyme model recognized by
Liebig (30), to the recent models of specific esterase activity
exhibited by SH compounds (33) and tlie polyfunctional
catalysts of mutarotation (47), a very large number of enzyme
models have been studied {cf. 27). Some of them, e.g., the
carboxylase models of Langenbeck, show remarkable similarity
to enzymes in regard to certain kinetic properties, product
inhibition, and inactivation of the catalyst. The polyfunctional
catalysts of Swain and Brown exhibit a high substrate specificity
and form substrate complexes resembling those formed by
enzymes.

Study of model reactions has often clarified our thinking
about the active center of enzymes and their mechanism of
action, but a word of caution should be added concerning the
indiscriminate use of model reactions. For example, the
chemical oxidation of aldehydes in the presence of phosphates
has been used as a model of enzymatic dehydrogenation of
aldehydes (52), in spite of the fact that the course and products of
the chemical and enzymatic oxidation are quite different.

Enzymes as Catalysts

The basic concepts of Michaelis concerning enzyme-sub-
strate interactions have stood the test of time, although the simple
original formulation has undergone some essential alterations.
Among the more important factors which required consideration
were: the presence of more than one active center, multiple
points of substrate attachment, the occurrence of more than one
enzyme-substrate intermediate, the participation of multiple
substrates, including cofactors and water, and the effect of
specific buffers and of pYi on the active center as well as on
activating groups.

221

EFRAIM RACKER

The kinetic complexities of metabolic processes, such as
glycolysis and the citric acid cycle, which are catalyzed by
multienzyme systems, have frequently been pointed out. In
order to analyze the individual enzymatic steps, it was necessary
to separate the catalysts from each other. Now it slowly becomes
apparent that the surface of the single enzyme may mirror some
of the complexities encountered in multienzyme systems. The
fact that a reaction catalyzed by a single enzyme includes the
formation of several enzyme-substrate intermediates, required
the introduction of the "Michaelis compound" as the rate-
limiting step (4,5). It may be confusing at first to learn that
pure crystalline glyceraldehyde-3-phosphate dehydrogenase
can catalyze five apparently different reactions {cf. 37). One
can explain oxidation-reduction, acyl transfer, phosphorolysis,
and perhaps even the phosphatase activity exhibited by this
enzyme in terms of the over-all process of aldehyde oxidation
coupled to phosphorylation. However, there is at present no
plausible explanation for the destruction of DPNH in the pres-
ence of the enzyme (36). It is very difficult to obtain accurate
kinetic data for the individual steps catalyzed by TDH, since
some of the "side reactions" interfere with the measurements.
Moreover, small modifications in the protein molecule, such as
the blocking of the SH groups, may change the ratios of the
different catalytic activities. Since oxidation of SH groups
occurs during the purification of TDH, a microheterogeneity is
introduced which is more treacherous for kinetic studies than
gross impurities with unrelated proteins. This difficulty cannot
be overcome by the addition of cysteine or GSH, which not only
reduce the enzyme incompletely but also interact with the
aldehyde substrate. However, fully reduced enzyme, suitable
for kinetic studies, can be isolated from rabbit muscle with the
aid of ethylenediamine tetraacetate (23),

A change in pYL may determine which of a number of
possible products is formed by an enzyme. In the case of papain
and the cathepsins (15) a decrease in the hydrogen ion con-
centration alters the enzyme-catalyzed reaction in such a

222

ENZYMES AS REAGENTS

manner that the transfer of an acyl group to an acceptor other
than water predominates. The kinetics of an enzyme-catalyzed
reaction may also be altered by pH because of a shift to a new
rate-limiting Michaelis compound (43).

SUBSTRATE-ENZYME INTERACTIONS

In the enzyme-catalyzed reaction:

E + S . ' ^ ES '-^ E + product

Km =

'■m

1^2 ~r ^3
^1

the dissociation constant Kj) for the enzyme-substrate complex
(ES) is equal to the Michaelis constant iT^ only when ^3 is very
small compared to hi', then K^ = k-i/ki = K^. Experimental
data are accumulating which demonstrate that Kj) cannot be
equated to Kj^. It was shown for some peroxidases {cf. 4,5) and
for succinic dehydrogenase (41) that ^3 is considerably larger than
k'l, so that Kd may differ from Kj^ by several orders of magnitude.
Similar discrepancies have been recorded for enzyme-coenzyme
reactions (49). It appears necessary therefore to evaluate the
different rate constants independently. Ingenious methods for
the direct (4,5) and indirect (41) determination of the velocity
constants have been devised.

In a theoretical paper by Foster and Niemann (8), it was
pointed out that in multifunctional catalysis, which involves
the formation of several reactive intermediates, the experimental
values for kz may not represent the velocity constant for the
decomposition of the enzyme-substrate complex.

Various experimental approaches have been made to the
problem of multiple points of substrate attachment. The use of
isotopes in the case of symmetrical substrates, the effect of
modified substrates, of inhibitors, and of high substrate con-
centrations on rate of enzyme activity are among the best known
examples. If the enzyme contains two spatially independent
points of interaction with the substrate, as shown in Figure 1,

223

EFRAIM RACKER

the presence of substrate excess may lead to the formation of
inactive enzyme. A modified MichaeUs-Menten equation was
derived for this case by Foster, McRae, and Bonner (7) :

Ve. (S)
V =

k: + (S) + (S)7C

in which K^ and V^^ are analogous to K^ and F^^ of the
Michaelis-Menten equation. The term {S)^/C is an index of
the probability of two substrate molecules combining with the

ACTIVE COMPLEX INACTIVE COMPLEX

Figure 1.

enzyme, resulting in the formation of inactive enzyme substrate
complex. At low concentrations of substrate the term (S)VC'
is negligible ; with increasing substrate concentrations it becomes
appreciable, the velocity v decreases. When the concentration
of substrate reaches the value of C, the velocity becomes half
of maximal ; thus one half of the enzyme molecules is in the form
of the inactive, oversaturated enzyme complex. Experimental
data on the effect of indoleacetic acid concentration on the
growth of oat coleoptile sections fit in a remarkable manner the
theoretical curve for a two-point attachment. Indeed, studies on
the effectiveness of various indoleacetic acid analogues revealed
that two specific groupings on the molecule are required for
biological activity.

A similar interpretation has been given to the inhibition of
acetylcholinesterase at high substrate concentration (cf. 55).
An extensive analysis of the mechanism of action of this enzyme
has been carried out by Nachmansohn, Wilson, and their
collaborators (31,54). They investigated the formation of the
enzyme-substrate complex with the aid of ionizable and non-
ionizable substrates and inhibitors at various pH values. These
kinetic studies revealed some interesting properties of the two
sites of the enzyme, which are referred to as the anionic and

224

ENZYMES AS REAGENTS

esteratic sites, according" to their interactions witli the quaternary
ammonium and the ester moiety of the substrate. A theory of a
two-step mechanism of action was proposed which included
the rate-Hmiting formation of an acyl enzyme as an intermediate.
The acyl group can be hydrolyzed or transferred to an acceptor
other than water, e.g., hydroxylamine or alcohol. The in-
activation of acetylcholinesterase by inhibitory phosphate esters,
such as tetra alkyl pyrophosphate, was shown to be an enzymatic
process reversible by suitable nucleophilic replacing agents.

SUBSTRATE-SUBSTR.\TE INTERACTIONS

Theories of the mode of action of glyceraldehyde-3-phos-
phate dehydrogenase and glutamic dehydrogenase which assume
interaction between substrates prior to enzyme action have been
widely accepted, although no experimental evidence for chemical
addition products as intermediates is available. In the case of
glyceraldehyde-3-phosphate, it was assumed that phosphate
adds to the aldehyde prior to its oxidation to an acyl phosphate
{cf. 52). A useful kinetic approach to this problem has been
made by Biicher and Garbade (3). The experimental data on
the effect of phosphate or arsenate on the apparent Kr„ value for
the substrate (glyceraldehyde or glyceraldehyde-3-phosphate)
seem to rule out the formation of a glyceraldehyde diphosphate
as intermediate. Strecker has presented (45) evidence of a
similar nature against the chemical formation of the a-imino
compound as intermediate in the reductive amination of a-keto-
glutarate to glutamate.

Enzymes as Analytical Tools

Analysis of biochemical events is dependent on the avail-
ability of analytical tools. Isolation procedures and chemical
determinations often lack accuracy and specificity. Enzymolo-
gists have naturally turned to enzymes as an aid in assays of
intermediary metabolites. Kjeldahl, over 70 years ago (19),
was probably the first to propose the use of an enzyme as an
analytical tool (invertase for sucrose deterixiination). Since
then, numerous enzymes have been used for the assay of sub-
strates, coenzymes, and enzymes. The obstacle of an unfavor-

225

EFRAIM RACKER

able equilibrium, which leads to incomplete utilization of the
substrate, can be overcome by the removal of products with
secondary enzymes. The availability of spectrophotometers has
fostered the development of many sensitive and rapid assay
methods. If an enzyme-catalyzed reaction results in no changes
in light absorption, it is usually possible to link it to a reaction
with suitable spectral properties. Occasionally several enzymes
have to be added in excess, while the enzyme to be assayed is
added in limiting amounts. The number of complications due
to side reactions increases, however, with the number of auxiliary
enzymes used.

"enzymatic purity" of enzymes

In the course of these studies the enzymologist became aware
that a new kind of purity is required for his analytical enzyme
tools, since the criteria of chemical purity had little meaning in
this case. An enzyme preparation though homogeneous accord-
ing to the most rigid physicochemical measurements may con-
tain an enzyme impurity which represents only a fraction of 1 %
of the total protein. The preparation may be worthless for his
analysis if the contaminating protein happens to be (and fre-
quently is) a very active enzyme which catalyzes a side reaction
in his assay system. On the other hand, another preparation
less pure according to physicochemical determinations may be
quite suitable as analytical reagent. Thus, the presence or ab-
sence of contaminating enzymes which give rise to side reactions
with substrate, product, or coenzyme determines whether or not
an enzyme is usable. Therefore, new criteria of "enzymatic
purity" must be established for each enzyme reagent.

An example encountered in our laboratory may serve to
illustrate the complexities of some of these assays. A preparation
of glucose-6-phosphate dehydrogenase from brewers' yeast (20)
has been widely used for the quantitative determination of
glucose-6-phosphate and of TPN. The enzyme can be used
to regenerate TPNH in TPN-linked oxidation-reductions. In
combination with hexokinase and glucose it is used for the de-

226

ENZYMES AS REAGENTS

termination of ATP. It can be used in an assay for hexokinase
or phosphohexose isomerase. In the course of several years
during which this enzyme served as a useful reagent in many
laboratories, differences in the properties of various preparations
probably due to minor variations in the purification procedure
were encountered. Thus, some preparations contained traces
of a TPNH oxidase or of phosphogluconic dehydrogenase which
prevented the achievement of a sharp end point at the time of
the complete reaction. Variable amounts of hexokinase, phos-
phohexose isomerase, adenylate kinase, and glutathione reduc-
tase were found in these preparations. Accurate values for ATP
could not be obtained in the presence of adenylate kinase, and
an analysis of glucose-6-phosphate or ATP in a sample which
contained some oxidized glutathione gave erroneous values owing
to glutathione reductase. Occasionally it is possible to make a
preparation suitable as a reagent by the use of an inhibitor.
For example, treatment with A^-ethyl maleimide has been found
to eliminate adenylate kinase activity without inactivating glu-
cose-6-phosphate dehydrogenase (25).

The suitability of an enzyme for quantitative substrate de-
termination is dependent on an appropriate substrate affinity
and absence of inhibitory side reactions. Preparations of glu-
cose-6-phosphate dehydrogenase from Torula yeast, although of
much higher specific activity than the preparations from brew-
ers' yeast, were found to react much more sluggishly at low glu-
cose-6-phosphate concentrations and were inadequate for assay
purposes (44).

ANALYSIS OF MULTIENZYME SYSTEMS

The determination of an enzyme in crude tissue extracts in
which complex chains of metabolically linked reactions take
place often poses great difficulties. Measurements of formation
of the product or of disappearance of the substrate are compli-
cated by the presence of the other members of the multienzyme
systems which act on the same compounds. Although blocking
of secondary reactions may be successfully used in some instances,
in other instances new complications, e.g., "product inhibition,"

227

EFRAIM RACKER

may arise. Perhaps the most reUable of all the methods of en-
zyme assay in crude systems is one used least frequently, since
it requires the availability of auxiliary enzymes of "enzymatic
purity." The method consists of adding a large excess of the
other enzymes of a multistep assay system to allow a more ac-
curate determination of the rate-limiting enzyme.

Perhaps one should not conclude the discussion of these "prac-
tical" aspects of enzymes as reagents without mentioning their
usefulness as analytical tools in the determination of chemical
structures. This approach, clearly envisaged by Emil Fischer,
has served as the key method in the determination of the struc-
ture of many coenzymes and is increasingly being used in the
elucidation of the structure of proteins and other biological
polymers.

Enzymes as Reactants

In the early years of enzymology, the oxidation of an alde-
hyde by a DPN-linked dehydrogenase was presented as:

A 1 1 1 1 Enzyme ... ,.-.

Aldehyde > Acid (1)

^ DPN

When DPN became available in gram quantities and was
widely used as an hydrogen acceptor instead of as a catalyst, the
reaction was written :

Aldehyde + DPN+ ^"''^"' Acid + DPNH + H+ (2)

Now that an aldehyde-oxidizing enzyme, glyceraldehyde-3-
phosphate dehydrogenase, has become available in gram quanti-
ties and the participation of its SH group in the catalysis of alde-
hyde oxidation has been demonstrated, the first stages of the
reaction sequence can be written as:

Aldehyde + DPN+ + SH-enzyme >

Acyl-S-enzyme + DPNH + H+ (3)

From the physiological point of view, expressions (2) and
(3) must be considered as representing Beckman cell artifacts

228

ENZYMES AS REAGENTS

far removed from true cellular reality. But the enzymologist
has learned a great deal about the mechanism of enzyme action
from studies with coenzymes and enzymes as reactants.

METHODS OF ANALYSIS OF ENZYMES AS REACTANTS

In most instances, enzyme-substrate compounds appear to be
unstable. The first direct demonstrations of their existence de-
pended on the use of rapid spectrophotometric methods. The
appearance of new absorption bands in the case of peroxidases
(4) and the disappearance of the DPN-enzyme absorption band
of glyceraldehyde-3-phosphate dehydrogenase (38) on addition
of the respective substrates, are examples of such studies. On
the other hand, the few enzyme-coenzyme compounds investi-
gated so far appear to be quite stable (38,49,50). Inhibitors
such as /?-chloromercuribenzoate, iodoacetate, and hydroxyl-
amine have been most useful for the analysis of enzyme-substrate
and enzyme-coenzyme interactions (16,38,49). Isotopically
labeled substrates have also been valuable tools in these studies.
With C^'*-labeled acetyl phosphate, radioactive acyl glyceralde-
hyde-3-phosphate dehydrogenase was prepared. Hydrolysis
with proteolytic enzymes resulted in the release of a thiol ester
with the properties of acetyl glutathione (24). Formation of a
phospho-enzyme in the case of phosphoglucomutase was first
suggested by studies with P^^ (14). The use of isotopically
labeled substrates or cofactors in incomplete systems (omitting
either a substrate or a cofactor), which frequently results in an
enzyme-catalyzed redistribution of the label, helps to elucidate
reaction sequences. Emphasis should be placed, however, on
the use of pure systems for such studies so that exchange reac-
tions, due to either contaminating coenzymes or contaminating
enzymes, can be ruled out. Important conclusions regarding
the mode of enzyme action have been deduced from studying the
point of cleavage with isotopically labeled substrates {cf. 21).

Finally, valuable information has been obtained from kinetic
investigations of the complexes formed between enzymes, co-
enzymes, and substrates. It has made feasible a bold study of

229

EFRAIM RACKER

these specific interactions in intact cells (5). This investigation
represents a new and important approach to the steady-state
kinetics of intracellular metabolism.

THE MICROCYCLES OF ENZYME-SUBSTRATE INTERACTIONS

Catalysis may be looked upon, in many instances, as a cyclic
process in which the catalyst undergoes reversible changes.
Evidence for this thesis has been obtained in the case of the few
enzymes which have been closely examined from this point of
view. The enzymologist, hardened by his experiences with com-
plex metabolic cycles, now begins to turn to the exploration of
enzymes as "microcycles."

6-l-P E-P E G-l,6-P

G-6-R

Figure 2.

Phosphoglucomutase catalyzes the reversible transformation
of glucose- 1 -phosphate (G-l-p) to glucose-6-phosphate (G-6-p).
The reaction was first written as G-l-p<=^G-6-p. When the func-
tion of glucose-l,6-diphosphate (G-l,6-p) as a coenzyme was
discovered (28), the reaction was written as: G-l-p + G-l,6-p«=^
G-1 ,6-p + G-6-p. With the elucidation of the role of the enzyme
protein as phosphate acceptor (32), the reaction sequence can be
represented by a cyclic process which includes all reactants, as
shown in Figure 2. The enzyme occurs in muscle extracts as
phosphoenzyme (E-p). In the presence of the substrate (either
G-l-p or G-6-p), the phosphate group is transferred and glu-
cose- 1,6-diphosphate is formed. The latter then returns the
phosphate to the enzyme and forms the product (G-6-p or
G-l-p).

230

ENZYMES AS REAGENTS

Another example of a microcyclic sequence of interactions is
catalyzed by giyceraldehyde-3-phosphate dehydrogenase and
its pyridine nucleotide coenzyme (Figure 3).

DPN combines with the SH-enzyme to form a DPN-enzyme
complex (DPN-E) which can be measured by its absorption
band at 360 lUfx. The aldehyde interacts with the DPN-enzyme
to yield acyl enzyme and reduced DPN. In the presence of
phosphate, the acyl enzyme is phosphorolyzed to acyl phosphate
and SH-enzyme, which combines again with DPN to regenerate
the DPN-enzyme complex (38).

ALDEHYDE DPN-E

DP N H + H*

l+ACCEPTOR)

+ PHOSPHATE

A C Y L- P

Figure 3.

The enzyme crystallizes from rabbit muscle in the presence
of ethylenediamine tetraacetate as the DPN-enzyme complex
with 3 moles of DPN per mole. After removal of DPN by
treatment with charcoal, the enzyme is somewhat less stable and
crystallizes only with difficulty. Exposure of the charcoal-
treated enzyme to acetyl phosphate results in the formation of
acyl enzyme. Crystallization of the protein from this mixture
occurs again quite readily and the crystalline material contains
the acetyl enzyme: It gives a positive hydroxylamine test and
oxidizes DPNH, thereby forming free acetaldehyde from the
acetyl group. With 1,3-diphosphoglycerate, the formation of a
phosphoglyceryl enzyme has been demonstrated (26).

231

EFRAIM RACKER

Thus, every one of the intermediates of the niicrocycles of
glyceraldehyde-3-phosphate dehydrogenase and phosphogluco-
mutase can be isolated. It should be pointed out that in both
instances this feat has been made possible by the artificial inter-
ruption of the cyclic process of catalysis, either by removal of the
coenzymes, as with glyceraldehyde-3-phosphate dehydrogenase,
or by omission of a phosphate acceptor, as with phosphogluco-
mutase. With many enzymes, however, neither of these two
approaches is feasible, and, in order to change the course of
events, alternative methods, such as the use of inhibitors, must be
explored.

INHIBITORS

An outstanding biochemist once remarked that only un-
inhibited investigators use inhibitors in complex biological sys-
tems. Pitfalls similar to those encountered in inhibitor studies
of multienzyme systems also confront the investigator of a micro-
cycle which is catalyzed by a single enzyme. On the other hand,
unambiguous studies with inhibitors such as sodium fluoride and
iodoacetate have been landmarks in the history of the biochemis-
try of glycolysis and of muscular contraction. Inhibitors, used
cautiously, have also been valuable tools in the study of enzyme-
coenzyme-substrate interactions.

The protective action of coenzymes or substrates against in-
hibitors has yielded important information about the mode of
their interaction with the enzyme. Early studies of Rapkine on
TDH with several SH inhibitors indicated an interaction be-
tween the SH groups of the enzyme and DPN. Alcohol dehy-
drogenase from yeast can be protected against lAA by DPN or
ethyl alcohol (c/. 37) ; testosterone dehydrogenase can be pro-
tected by DPN against /^-chloromercuribenzoate (48). It should
be pointed out that more complex effects may be encountered.
Thus, DPN, which protects TDH against iV-ethyl maleimide,
increases markedly the susceptibility of the enzyme to lAA.
Glyceraldehyde-3-phosphate protects TDH against lAA, but so
do some other phosphorylated compounds (e.g., 3-phospho-

232

ENZYMES AS REAGENTS

glycerate) which are not substrates and do not react directly
with SH groups (24).

The inhibitor may block a partial reaction of the cyclic proc-
ess of enzyme catalysis and permit a functional separation of a
multistep system. For example, iodoacetate inhibits the oxido-
reduction catalyzed by TDH, but arsenolysis of the acyl enzyme
can still take place (38). Potassium cyanide stabilizes acyl
enzyme even in the presence of DPN, without interfering with
arsenolysis (26). Tetra alkyl pyrophosphate has been shown
(54) to inhibit cholinesterase by forming a phospho-enzyme in-
stead of an acyl enzyme. In contrast to the acyl enzyme, the
phospho-enzyme is not readily hydrolyzed, and nucleophiiic re-
placement agents must be used to reactivate the active center of
the enzyme.

Oxidized (S-S) TDH, which is devoid of dehydrogenase
activity, has been shown to hydrolyze acetyl phosphate (12). A
stimulation of the phosphatase action of TDH has also been
noted after treatment with iodoacetate (26). An inhibitor can,
therefore, not only block a cyclic process but channel it into a
side reaction, Iodoacetate alters the properties of this catalyst
so fundamentally that instead of acting as an acyl transfer agent,
it becomes a catalyst of hydrolysis. This exhibition of new
catalytic activities in the presence of inhibitors requires most
cautious interpretation of observations obtained with enzymes
which have been exposed to maltreatment by enzymologists or
histochemists. The activation of unspecific phosphatases by
iodoacetate or by maleate, reported in the older literature {cf.
53), may represent a similar alteration of functional SH groups
resulting in a change in catalytic activity.

EXCHANGE REACTIONS

The use of isotopically labeled compounds in biochemical
research has led to the discovery of a new group of reactions
often referred to as exchange reactions. They may be defined
as reactions in which an isotopic label is incorporated into a
compound, although no synthesis of the latter has occurred.

233

EFRAIM RACKER

There appear to be at least two different kinds of enzyme cata-
lyzed exchange reactions ; these will be referred to as ( 7) transfer-
exchange reactions, and (2) metabolic exchange reactions.

( 7) The transfer-exchange reactions represent a special case
of a general reaction, usually referred to as a transfer reaction :

AB + G ' AC + B

These transfer reactions are catalyzed by enzymes which are
widely distributed in nature. Transaminases, transglycosidases,
transketolase, and transaldolase are a few examples of this type
of reaction. These enzymes are assumed to form a complex with
the substrate and then catalyze the transfer of a portion of the
substrate to another acceptor molecule. For example, the amino
group of glutamate is transferred to oxaloacetate by a trans-
aminase. If, in the above equation, free B can substitute for G
as an acceptor, we are dealing with a transfer-exchange reaction
which results in no synthesis but can be detected by incorporation
of a labeled compound. The exchange takes place at the same
atom of the donor molecule A, in contrast to the metabolic ex-
change reactions discussed below.

(2) The metabolic exchange reactions occur in the course
of a cyclic process and are due to the peculiarity of certain reac-
tion mechanisms that illustrate a special aspect of enzymes as
reactants. The cyclic process of enzyme catalysis may proceed
in such a manner that an exchange of atoms takes place between
the substrate and the catalyst or coenzyme. For example, a
phosphate exchange reaction is catalyzed by phosphoglucomu-
tase (see Figure 2). In experiments with P^^-labeled glucose-1-
phosphate, the coenzyme G-l,6-diphosphate becomes labeled
(46) because the phosphate is transferred from C-1 of G-1, 6-p to
C-6 of the substrate G-l-p, which becomes the coenzyme. An
analogous action takes place with oxaloacetate, which acts as a
catalyst in oxidation of acetyl CoA in the Krebs cycle. The
acetyl group of acetyl GoA condenses with one end of OAA, while
the other end is actually oxidized in one turn of the cycle. This
is not necessarily an essential feature of the cyclic process but is

234

ENZYMES AS REAGENTS

due to the peculiarity of the aconitase enzyme, which dehydrates
the oxaloacetate-end of citrate rather than its acetate-end.

PHYSIOLOGICAL SIGNIFICANCE OF EXCHANGE REACTIONS

It is apparent from the above that one must differentiate
between exchange reactions and incorporation due to the net
synthesis of AB*:

A + B* > AB*

Carbon 14 from carboxyl-labeled acetate will appear in oxalo-
acetate which on decarboxylation can yield pyruvate or phos-
phoenol pyruvate. From these three-carbon compounds, glyco-
gen containing the carbons of acetate will be formed. It has been
known to physiologists for many years that acetate cannot be
used for the biosynthesis of glycogen. We are dealing here with
an exchange reaction. It is also apparent that it is impossible
to draw conclusions about quantitative aspects of biosynthetic
pathways from incorporation studies with isotopes only, unless
exchange reactions can be ruled out.

Although no net synthesis of a peptide bond, for example,
can be achieved by transfer exchange reactions, these reactions
may participate in biosynthetic pathways. It is very probable
that transaminases and transpeptidases contribute to the bio-
synthesis of amino acids and peptides. It is nature's privilege
to delegate the specific function of reductive amination to glu-
tamic dehydrogenase. The glutamate formed by this enzyme
from ammonia and a-ketoglutarzte acts as the key middleman
for numerous transfer reactions to other keto acids which lead to
new amino acids.

The physiological role of the metabolic exchange reactions
is not as apparent. Since in the course of these curious reactions
a spatial redistribution of certain groups (e.g., acetyl or phos-
phate) takes place, it might be conceived that reactions of this
type play a role in the transportation of groups and compounds
across the cell membrane or from one intracellular structure to
another. If work is to be accomplished, the exchange must be
catalyzed by two different kinds of reactions as shown in Figure

235

EFRAIM RACKER

4, (a) and (b), e.g., phosphorylation with ATP and dephos-
phorylation by hydrolysis.

This brings us to the general problem of enzymes participat-
ing as reactants in specific physiological functions. Interaction of
aldehydes with proteins in the process of vision (51), interaction

b

-> AA+A

Figure 4.

of nucleotides with the proteins involved in muscular contrac-
tion (39), and the role of cholinesterase in the process of trans-
mission of nerve impulses (31) may represent examples of the
participation of enzymes as reactants in physiological processes.

Concluding Remarks

We can look upon enzymes as active centers embedded in a
protein matrix. The function of the protein molecule is to pro-
vide activating groups which increase the rate of catalysis
and permit specificity of interaction between the enzyme and its
substrate. Perhaps the most striking illustration for this differ-
entiation between the active center and the activating group of
the protein is the example of the "little enzymes" obtained from
hydrolyzed pepsin. These dialyzable fragments catalyze effi-
ciently the hydrolysis of a small molecular substrate, but have
lost most of their activity with proteins as substrate. Thus, the
activating groups of the native enzyme are required for the spe-
cific and efficient hydrolysis of the large molecular substrate.

Perhaps another function of the protein is to provide the cata-
lytic center with the anchors and the biochemical stability which
is needed for intracellular localization.

Kinetic investigations have received a new impetus during
the past ten years. The concept of the polyaffinity of substrates,

236

ENZYMES AS REAGENTS

first clearly formulated by Bergmann (2), the kinetic ramifica-
tions of the polyaffinity concept of a stepwise interaction be-
tween substrate and enzyme, the re-evaluation of the velocity
constants, and the existence of complex multiheaded enzymes
with multifunctional catalytic activities are among the major
developments in this area. They have stimulated new experi-
mental approaches and at the same time contributed to a more
timid approach regarding the interpretation of kinetic data. It
is self-evident from the above considerations that it is very diffi-
cult to interpret unambiguously kinetic data obtained with crude
enzyme preparations which may contain inhibitors or interfering
enzymes. Moreover, the meaning of painstaking studies, even
with highly purified enzymes, may be obscured by the presence
of side reactions. For example, several times recrystallized
pyruvic kinase contains adenylic kinase, and triose phosphate
isomerase may be found in several times recrystallized aldolase.
Even pure enzymes with multiple heads, such as glyceraldehyde-
3-phosphate dehydrogenase, catalyze side reactions which com-
plicate the life of the kineticist.

Enzymes have become accepted as analytical tools. Some
enzymatic methods used for determination of metabolic inter-
mediates (e.g., the various hexose phosphates) have a degree of
specificity rarely attained by colorimetric and isolation proce-
dures. However, the drawbacks of these enzymatic methods are
by no means negligible because of the rigid criteria of purity
which have to be applied to the reagents used.

In recent years a few crystalline enzymes have become avail-
able in large quantities and have permitted an investigation of
their participation as reactants in the reactions they catalyze.
It has been possible to show that a cyclic process takes place in
which enzyme, coenzyme, and substrate interact to produce a
sequence of new intermediates. Glyceraldehyde-3-phosphate
dehydrogenase can be isolated as an acyl enzyme and phospho-
glucomutase as a phosphorylated enzyme.

These are some of the currents and undercurrents in present
enzymology. What are the perspectives? Hundreds of enzymes

237

EFRAIM RACKER

have been described which await further study; many more
enzymes will be discovered in the future; nearly endless seems
the number of enzymes which might be induced by adaptive
processes or might be selected in microorganisms fished from the
mud of California. It is clear that the availability of enzymes will
not be the limiting factor in the future of enzymology. The
purification of enzymes is gradually becoming a routine pro-
cedure and biochemists are beginning to attach to it the
stigma of a necessary evil. The protein chemists are turning to
sequence analysis and to a search of the chemical properties of
the active centers and their neighborning groups. The kineti-
cists, after collecting essential data on the interaction between
the substrate and various intracellular and surface enzymes, are
beginning to return to more complex multienzyme systems and
even approach the kinetics of the intact cell. Their studies may
bring new clues to the great physiological mystery of the mode
of substrate entry into the cell. Enzymes as analytical tools will
undoubtedly infiltrate further into the laboratories at the ex-
treme wings of biochemistry: physical chemistry and medical
diagnostics. Many more enzymes will be investigated as reac-
tants, and cycle after cycle will appear on the surface of the deep
waters of metabolism.

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240

ATTEMPTS AT THE FORMULATION OF SOME
BASIC BIOCHEMICAL QUESTIONS

FRITZ LIPMANN, Biochemical Research Laboratory, Massachusetts
General Hospital and the Department of Biological Chemistry, Harvard Medical

School, Boston, Massachusetts

The fogginess in which tJie physical-chemical aspects of the
inner working of the living organisms had in the past been
shrouded, inevitably invited mystification of one kind or another.
In counterreaction, the past generation of biochemists was
almost religiously concerned with the task of demystifying life.
Through their pioneering, the fog lifted during the twenties and
thirties. But now we witness the development of a new phase,
in which the hesitant, skeptical, and largely analytical approach
is being discarded. The depth to which the understanding of
organismic methodology has reached demands reorientation.
At this stage it seems most important to ask the right questions.
I will not attempt much more than to try to raise a few such
relatively naive questions and even these in not too explicit a
manner.

The approach toward clarification appears to be most
hopeful by way of defining those distinctive attributes of the
living state which have been thought to indicate a novel and
specifically "living" phenomenon. We will first consider
briefly the energy problem, which is relatively the farthest ad-
vanced, and then try problems specifically related to duplication.

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The Energy Problem

The recognition of the relative simplicity of the energy
supply system of the living organism has made it possible to
understand a large number of biosynthetic mechanisms (15).
We now feel justified to state with confidence that the energy
problem can be related to a boundary condition of the living
system as a whole and does not represent an intrinsic character-
istic of the life process except that it is a necessary premise for its
existence. The living organism is to be defined as a system with
a boundary condition of a constant influx of energy from the
outside. Such a system, however, in no way transcends the laws
of thermodynamics. In the past, a suspicion often crept in that
need might arise for a modification of the second law. Recently
a tendency developed to build into a definition of life the con-
cept of a negative entropy (20) which, however, is not a charac-
teristic of life but rather of the environmental conditions which
permitted life to develop. The earth is indeed, if isolated and
considered as a quasi-autonomous system, appropriately repre-
sented by including the incidental radiation energy as a boundary
condition, abstracting it, as we mostly tend to do anyway, from
the solar system and the universe. Although, in its elaboration of
metabolic work, the living organism acts in the capacity of a
refinery, the essential boundary condition almost always refers
back to the influx of energy from the outside into the earth
sphere.

Patternization

Much emphasis is generally placed on our recently ac-
quired capacity to understand the biochemical mechanisms for
building up structures such as steroids, carotenoids, and similar
complex molecules, which had previously been elucidated
through organic chemical analysis. Although these complex
molecules have their important significance for organismic life,
the most important problem is the problem of the building up of
specific and unique structures by fusing a number of otherwise

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unlike units through a uniform Hnking mechanism. This
creates unique and specific surface and structure patterns which
we call proteins, nucleic acids, and mucopolysaccharides. The
primary determinants are a fixed sequential arrangement.

So far this type of problem has not occurred in man-made
chemistry and it is rather puzzling. Energy is needed first
for the joining of the common links in the backbone. Secondly,
the lining up of a defined sequential arrangement, the patterni-
zation, represents a structural energy equivalent or rather, an
increase in entropy (Maxwell's "demon"), which is not quite
easily defined with our present notions. Patternization, I feel,
is dominant in duplication, replication, and mutation. It is
possibly important to reiterate that the energy requirement for
patternization may be divided into (7) a well-defined caloric
equivalent for the joining of the links in the respective backbone
structures and (2) an "energy of position," the directing into a
specific order. The latter is biologically the most important.
We meet here a novel situation, the biochemical definition of
which has no well-understood examples to fall back upon. It
has become the meeting ground between biochemistry and
genetics. To define the problem in all its facets, a fusion be-
tween biochemical and genetic principles will be needed.

It is most likely not mere coincidence that metabolic chem-
istry has come to a point where the problem of energy distribu-
tion apparently is entering a new stage. The great successes of
the past twenty years were obtained by a methodology which
aimed at extracting enzymes, coenzymes, and such from the cell.
In this manner, rather complex systems could be separated and
reunited successfully as, for example, the glycolytic system. The
limitation, however, of this approach becomes apparent now.
The old methodology of cleaving and separating seems to fall
short because the complex and more intrinsic functions of the cell
appear to depend on macromolecular arrangements of greater
specificity. Therefore, from the analytical point of view, the
intrinsic involvement of structure in certain metabolic systems
requires new approaches which are slowly developing. It is in

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part the previous success of the methodology of cleavage into
the smallest possible units which now acts almost like a mental
block.

The common denominator in many of our difficulties seems
our inability to understand structure in biochemical terms.
This is particularly evident in the approach to protein synthesis.
With the analysis of protein synthesis, the energy problem now
tangibly merges with the propagation problem. This is ob-
vious if one realizes that many forms of specificity are an ex-
pression of a particular sequential arrangement of the amino
acids, a sequential arrangement into chains of various shapes,
together with an interlocking through interchain links such as,
e.g., disulfide links.

A lively discussion of the manner in which these sequences
are built up has recently sprung up, motivated in particular
through ingenious models for deoxynucleic acids (23). These
nucleic acids appear to carry a directing force, often referred to
as the code of replication. Metabolically speaking, there is
still a link missing from predetermined nucleic acid chain se-
quences leading to determination of amino acid chain sequences,
although dependence of protein synthesis on nucleic acid struc-
tures appears to be quite real. In spite of still meager facts,
there is a need to start discussion in this biochemical vacuum.

Before embarking on particulars, it seems important to
realize that the synthesis of a fixed sequence can be effected
methodologically either by a predominant space pattern, a
template, or by a timing device analogous to the assembly line.
In reality, it most likely will be effected by a mixture of both.
It is my impression that presently template processes have been
in the foreground of discussions, including my own (16), because
of the greater ease in devising template models. The assembly
line procedure, however, is really met with mostly in biochemical
systems, including a few cases with sequential arrangement on a
relatively small scale (10,21).

These problems are now in the foreground because it is here
that genetics and biochemistry meet, namely, where the trans-

244

BASIC BIOCHEMICAL QUESTIONS

mission of the code laid down in the genetic material is trans-
lated into chemical mechanisms. A chemical methodology of
code transmission, it appears, consists of arranging specific
sequences through standard links in the line-up. Possibly a
master methodology may exist for a specific sequential arrange-
ment of unlike units through a common linkage system which
forms the backbone structure of the resulting compound. How-
ever that may be, patternization or sequential arrangement in-
cludes, but largely transcends, what we have been mostly con-
cerned with so far, namely, mere mechanisms of linking.

Synthesis of Peptidic Links

It has at times appeared that the main problem in under-
standing protein synthesis is to tackle the mechanism of peptide
bond formation. However, to understand protein synthesis
truly, the understanding of peptide bond formation is a premise
rather than an end in itself. It will be wise, nevertheless, first
to obtain a background by reviewing our present understanding
of the mechanism of peptide bond formation. It seems that
uniformly, peptide linking starts with the activation of the
carboxyi group rather than of the amino group. The primary
source of energy appears to be the energy-rich phosphate bonds
of ATP, the energy currency. Unfortunately, the finer mech-
anism of the conversion of phosphate into active acyl grouping is
still only imperfectly understood. There are different subtypes
which are of considerable interest. The conversion of phos-
phoryl to active acyl may occur (7) by a pyrophosphate elimina-
tion from ATP, directly or possibly through other nucleoside
triphosphates (NTP) or (2) by phosphorolysis of the terminal
phosphate of ATP or other NTP's. These two mechanisms at
first looked like interchangeable types of procedure. On close
inspection, however, they might represent two distinct and rather
different mechanisms. The pyrophosphate elimination, as it
seems to develop more and more clearly, leads to a linking be-
tween the acyl grouping and die nucleotide part of the NTP (2)

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FRITZ LIPMANN

with the elimination of a molecule of pyrophosphate (1,3,12,18),
while phosphorolysis apparently leads in some manner to the
formation of acyl phosphate with elimination of nucleoside
diphosphate (NDP) (5,7,13,21). In other words, the pyro-
phosphate elimination represents an activation through linking
the carboxyl to the nucleoside by way of a monophosphate bridge,
forming an NMP^^acyl. On the other hand, phosphorolysis
seems to represent a discarding of the nucleotidic part of the
energy carrier and a more or less direct linking of the carboxyl to
the phosphate (5,13). Such a pyrophosphate elimination scheme
was first observed in a CoA-linked acyl activation. A similar
mechanism appears to apply to the synthesis of the peptidic bond
in pantothenic acid (18). But more important, an analogous
mechanism has now been found to operate in the activation of
normal amino acids (11). In both cases, the acyl '^phosphonu-
cleotide seems not to be freely diffusible but rather tightly
linked to the activating protein. This interpretation is mainly
based on isotope exchange reactions and should rather be con-
sidered as somewhat preliminary. Nevertheless, it may serve as
background for a discussion of the mechanism of peptide bond
formation in protein synthesis.

Protein Synthesis

With this still sketchy information on mechanisms for
peptide bond synthesis, we now proceed much less securely to
possible mechanisms of lining up the different amino acids into a
specific protein pattern. Sequential arrangement of the amino
acids obviously is the dominant problem before us. If the
apparatus is available to line up a predetermined sequence of
amino acids most of which will be quite long, twenty, forty, or
more links, the final shaping of the molecule might be expected
to be wholly or partly a spontaneous one. In any case, this
phase of final shaping will be neglected at the present, and our
focus will be exclusively on sequential arrangement as a back-
ground for specificity, even though this almost certainly is a
somewhat precarious simplification.

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BASIC BIOCHEMICAL QUESTIONS

There are good indications for the assumption that the
carboxy-activated amino acids enter directly into the mech-
anism of sequential arrangement (8,22,24). Furthermore, the
amino acids may enter as an amino acid'^P-nucleoside. The
activated amino acid seems, however, to approach the assembly
line not isolated but still fixed to the amino acid specific enzyme
protein (11). As a first approximation to a mechanism, we then
consider a specific enzyme for each amino acid which as a war-
head carries the amino acid linked to the phosphoryl of a mono-
nucleotide, eventually to effect selective linking into chains.

There is, on the other hand, a certain likelihood that the
amino acid chain is deposited and grows along on ribonucleic
acid. The incorporation of radioactive amino acids takes place
in special microsomes rich in nucleic acid (4,24). Recent ob-
servations (14) have indicated that the microsome carrying out
amino acid synthesis contains approximately equal amounts of
nucleic acid and protein. This observation seems to counter-
indicate a nucleoprotein as responsible for the direction of the
amino acids into sequences. The amount of protein would be
too small to permit such an interpretation. But the particular
activated amino acid approaches the assembly line in association
with its specific enzyme. An enzyme-mononucleotidc'^amino
acid might react with a specific nucleic acid which by its own
sequential arrangement of nucleotides is supposed to direct the
sequential patternization of a protein.

One process of progressive chain elongation has been suc-
cessfully analyzed, namely, fatty acid synthesis (17). It ap-
peared in that case that chain elongation always occurred be-
tween two activated molecules. But the energy of only one
energy-rich link is utilized, namely, the thioester bond to the
carboxyl which enters the • COCH2 • link. To compare this
methyl-carboxyl linking to the amino-carboxyl linking in a pep-
tide is not too far fetched, since it has been found (6) that the
methylene-carbonyl bond is hydrolyzed by chymotrypsin, which
makes the biochemical comparison between • CO — CH2 • and
• GO — NH • linking rather plausible. The importance of this

247

FRITZ LIPMANN

consideration lies in the fact that here we have a case where chain
growth is terminal and is maintained in such a manner that each
addition prepares for the following by permitting reaction only
between two activated carboxyl derivatives, the energy of only
one of which is used in the process. The terminal activated
carboxyl remains intact for the next step of chain elongation.
Furthermore, although fatty acid synthesis impresses as a homo-
geneous process where long, straight, uniform chains are
synthesized from indentical two-carbon fragments, obviously
during the chain elongation the growing chain is changing con-
stantly. At least three different enzymes participate with
different specificity for activation of short, intermediate, and long
fatty acids (19). The process, therefore, is not a homogeneous
process, but a handling of a changing molecule which changes
through the process of elongation itself. Although a long-
chain fatty acid is homogeneous as compared to a polypeptide
chain, the two processes of chain elongation may have common
features. It is possible, though no indication has appeared so
far, that chain elongation of the polypeptide chain proceeds
similarly through linking of an activated carboxyl waiting at the
polypeptide terminal for a newly arriving, likewise activated
amino acid.

However that may be, such considerations do not help much
in solving the cardinal question of how activated amino acids
are lined up in the specific sequence particular for the special
protein or enzyme. In all present considerations the mech-
anism of the linking process still presses into the foreground be-
cause there is some information present. The mechanism of
directing into proper sequence must be determined through
some kind of coding mechanism which seems to involve a trans-
lation, in Gamow's terminology (9), of specific nucleotide se-
quential into amino acid sequences.

Conclusion

The understanding of the basic mechanisms of joining mole-
cules together has opened the possibility of approaching the

248

BASIC BIOCHEMICAL QUESTIONS

problems of duplication, reproduction, and individualization
which I like to bring together under the term patternization.
This means that essentially they all may be reduced to the
problem of joining building stones into a specific reproducible
sequence or pattern. With the energy problem more or less
out of the way, it becomes more apparent that patternization is
distinct from, although superimposed upon, the process of link-
ing.

It is astonishing to realize that the more one proceeds with
the understanding of the workings of the organism, the more one
becomes concerned with methodological problems. Strangely,
the prying into the mystery of life reduces more and more to an
unraveling of a sometimes rather unusual and unexpected
methodology of the cell. On the other hand, it is significant
that the complexity and methodology of man-made instruments
seem to converge increasingly towards the complexity of or-
ganismic methodology and instrumentation.

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