STUDIES ON SOME INTERMEDIATE

REACTIONS IN BACTERIAL

PROTEIN SYNTHESIS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

ROBERT WILLIAM BERNLOHR, B. S

The Ohio State University 1958

Approved by

Department of Agricultural Biochemistry ACKNOWLEDGEMENTS

I wish to express ray appreciation to Dr. George

C. Webster for his advice and helpful suggestions during the course of this research program. The help of Dr. J. E, Varner and the other faculty members is

also greatly acknowledged.

I am indebted to the Department of Agricultural

Biochemistry for the financial assistance and facilities made available to me for the conduction of this project.

Also, the financial assistance of the Charles P.

Kettering Foundation and the Public Health Service is

acknowledged.

The encouragement, sacrifices, and help received from my wife during the past three years and in typing this manuscript are deeply appreciated.

ii CONTENTS

Page

Introduction 1

Experimental Procedures 11+

Results and Discussion

I. Whole Cell Studies on Amino Acid Incorporation 26

II* Cell-free Incorporating Systems i}2

Ill* Amino Acid Activations I4.7

IV. Intermediates in the Biosynthesis of Protein £2

Summary 68

Bibliography 72

Autobiography 77

iii LIST OF TABLES

Table Page

1. Glycine-C^+ Incorporation Into Different Tissues 27

2. Changes in RNA, DNA, and Protein 28

3. Effect of RNAase and DNAase 31+

1+. Effect of Chloramphenicol 37

$- Cell-free Amino Acid Incorporation 1+5 6. Oxygen Transfer in Amino Acid Activation 1+9

7. Chromatography of Amino Acid Hydroxamates 55

8. Distribution of AA-X 57

9. Sensitivity of AA-X to pH and Dialysis 58

10. Kinetic Uptake of Glycine-C1^ 67

iv LIST OF FIGURES

Figure Page

1. Glycine-C1^ Incorporation into RNA, DNA, and Protein 30

2. Effect of Chloramphenicol 33

3. Inhibition of RNA, DNA, and Protein Synthesis 35

h- Distribution of Glyclne-C^ in Centrifugal Fractions 39

5. Electrophoretic and Ultracentrifugal Analysis of Particulate Fractions kk 6* Chromatography of Amino Acid Hydroxamates 56

7. Spectra of the Dialyzed and Treated Supernatant Solution 60

8. Specific Activity of Components Separated by Paper Electrophoresis 62

9. Total Activity of Components Separated by Paper Electrophoresis 63

v INTRODUCTION

The elucidation of the enzymatic mechanism of protein synthesis is currently a very pressing problem, for it remains as the last major void in biochemical knowledge. Daily, hundreds of people are admitted to hospitals with conditions which Involve accelerated cell growth, such as cancer. In addition, a large number of physiological abnormalities can be traced to the occurrence of unnatural protein molecules. These two examples, to which many more could be added, indicate that the sensitive biosynthetic system responsible for the production of protein molecules is unusually prone to malfunction. Also the genetic apparatus, present in all cells, appears to be at least indirectly involved in organizing this process. It Is evident, therefore, that a knowledge of the stepwise biosynthesis of proteins could conceivably lead to an understanding of the cause of many pathological conditions. It is of considerable importance, then, to have a well-defined hypothesis at the level of reaction mechanisms in terms of chemical formulas.

In considering the possible mechanism for protein biosynthesis, several problems are encountered which are unique for this type of molecule. The first is the 1 formation of peptide bonds. Secondly, proteins are constructed from a number of different amino acids which are apparently arranged in a specific sequence and main­ tained from generation to generation. Thus differing from polysaccharides and lipids, they seem to owe this specificity to direct genetic control, and therefore, a regulating mechanism is required.

The initial activation of the constituent amino acids and model systems for peptide bond formation have received considerable attention. Prom previous studies of model systems for peptide bond formation, Lipmann (191 +5 ) discovered that during the acylation of aromatic amines, adenosine triphosphate (ATP) was utilized for carboxyl activation. Also in glutathione synthesis £Bloch (19219 )} t a similar reaction was observed in which ATP is split to form adenosine diphosphate (ADP) and inorganic ortho­ phosphate. However, a study of the synthesis of acetyl-CoA by Lipmann £t al. (1952) provided an alternative mechanism for the activation of carboxyl compounds. When the

enzymatic reaction involving acetate, ATP, and coenzyrae A

is observed, the products are acetyl-CoA, adenosine monophosphate (AMP), and inorganic pyrophosphate (PPi).

Furthermore, this reaction, representing a new type of

cleavage of ATP, is freely reversible and allows the

exchange of radioactive pyrophosphate into ATP. We are then presented with two schemes in which the phosphate bond energy of ATP could be used to drive the energy requiring synthesis of proteins*

The two possibilities for the mechanism of amino acid activation and subsequent condensation are ---

n AA + n ATP ^ (AA)n + n ADP + n Pi or

n AA + n ATP -r (AA)n + n AMP + n PPi.

The activation of amino acids was demonstrated when extracts of animal, plant, and bacterial systems were shown to catalyze an amino acid dependent exchange between ATP and inorganic pyrophosphate by Hoagland (1955)

Hoagland, Keller and Zamecnik (1955); Webster (1957);

Holley (1957); and Novelli and DeMoss (1958)* In Hoagland’s original work, hydroxylamine was also used as a trapping agent for the activated amino acid, indicating that the carboxyl group of the amino acid is the site of activation.

From this work and also from that of Berg (1957) the occurrence of an enzyme-bound amino acyl-AMP compound has been postulated as the activated amino acid.

After the amino acids have been activated, it is assumed that there is an enzyme present which will catalyze the union of a series of these building blocks by peptide formation. However, a regulatory mechanism must be applied to ensure the formation of specific completed proteins. Otherwise, as Steinberg and Anfinsen (1952) k- believed, peptide intermediates are possible, Simpson and Velick (195U-) and Askonas et al. (195U) have since shown that the intracellular biosynthesis of peptide intermediates during the formation of proteins is not very likely.

Many formulations for the biosynthesis of protein have been proposed. One mechanism implies that at each step, contact between the appropriate catalyst and the molecule being farmed is limited to relatively small portions of the molecular structure of either. Conse­ quently, each of the many catalysts can have but limited information of what has gone before. This polyenzyme theory QSpiegelman (1957)3 runs into such inherent complications and difficulties that it must be abandoned in favor of a more controllable mechanism. The familiar template theory, first stated by Breinl and Haurowitz

(1930) and generalized by Haurowitz (1950), can account for the exact replication of specific protein molecules, and embodies most of the existing postulated synthesis mechanisms. This theory has as its basis the duplication of protein molecules on the surface of another protein which is fixed in an expanded state by conjugation with a nucleic acid molecule.

Approaching the problem in more specific chemical terms, Bounce (1952) suggested that the activating enzyme, 5 bound to nucleic acid, is the tenplate itself. In his opinion, ATP contributes the necessary energy by means of a phosphotransferase that transfers its terminal phosphate to the phosphate of nucleic acid; the net result is the transfer of a pyrophosphate linkage from

ATP to nucleic acid. The amino groups of amino acids then react to form amino-phosphate compounds on the nucleic acid, and for each amino group so combined the phosphate that came originally from the ATP is displaced and appears as inorganic phosphate. Another enzyme links the free carboxyl group of the adjacent amino acid to the phosphate-bound amino group to form a peptide linkage.

Lipmann (19Sk) postulated that a pyrophosphoryl moiety from ATP is transfer:-ed to the nucleic acid template. It is then displaced by the carboxyl groups of amino acids and upon the influence of an additional enzyme, peptide bond formation between adjacent amino acids takes place, allowing the completed protein to leave the template structure.

By 195b, Borsook had incorporated the newly acquired knowledge concerning amino acid activation into his hypothesis for protein biosynthesis. He feels that after amino acids have been activated, they are attached to the phosphate of nucleic acids by their carboxyl groups. Then 6 another enzyme forms the peptide bonds, thereby removing the amino acids involved from the nucleic acid template.

Recently, Hoagland e_t al. (1957a) and Holley (1957) have postulated the occurrence of anadditional cofactor necessary for protein synthesis. Novelli and DeMoss

(1 9 5 8) have subsequently utilized this information in formulating a hypothesis for the biosynthesis of protein.

They feel that the enzyme-bound activated amino acid is transferred to a factor "X" which can be considered as a transport cofactor. The amino acid moiety of this complex is then donated to a template, and polymerization occurs in the manner described by Borsook (195b).

At the present time, the limiting factor in the elucidation of this mechanism appears to be a lack of information concerning the chemical nature of the template.

Because protein specificity must be passed from generation to generation, there is little doubt that the prime source of this information is in the genes. The actual chemical entity enumerating this information, therefore, could be the protein of the chromosome, the DMA, or the deoxyribonucleoprotein.

That DNA does not play a direct role in all protein synthesis is apparent from the work of Mazia and Prescott

(1955) and others. Thus, in enucleated amoeba, protein 7 synthesis continues for weeks in the apparent absence of

DM. Ultraviolet light and nitrogen mustard, physical agents which destroy DM, do not affect protein synthesis

^Pardee (l95M-)J • ^ can therefore be assumed that D M does not play a direct role in the synthesis of protein.

We must now turn to other macromolecules as chemical formulators in protein metabolism. Novelli and DeMoss

(1 9 5 8) indicate that such an organizer system (l) must contain enough information to regulate the sequence of amino acids in the protein whose synthesis it determines;

(2 ) must be capable of acting catalytically; and (3) must be synthesized under the influence of DNA. Since DNA

itself has been discounted as functioning in this capacity,

only RNA and protein remain as logical candidates for

this organizer system.

The positive aspect of RNA in this situation will be

mentioned shortly. Prescott (1957) has presented un­

equivocal evidence that RNA is synthesized in the nucleus,

but there is no complete evidence to support the idea

that RNA synthesis is exclusively under nuclear or

genetic control. Furthermore,'Gamow (1955) has calculated

the requirements for protein synthesis specificity, and

assuming an RNA structure similar to that of DNA, he

indicates an impossible situation. In order for amino

acids to be polymerized on a template surface they must 8 be closer together than the exclusion diameter of 2,28 A...,

RNA Itself will not specifically arrange amino acids any

closer than 10.2 A., so it appears that RNA alone will

not satisfy the requirements for a template. Also,

though protein molecules contain sufficient information

to play this role, their synthesis is not always genetically

controlled, leaving proteins per se as unlikely candidates

for such duty.

On the basis of this information, Spiegelman (1957)

and Novelli and DeMoss (1958) agree that a ribonucleo-

protein is the most likely raacromolecule to function as

the template. It furnishes the following desirable pro­

perties: (l) it is synthesized in the nucleus; (2) It

can carry more than enough information for the synthesis

of new protein; (3) RNA could act to keep the protein part

of the structure in an extended linear arrangement; and (i^)

either or both of the partners could provide the catalytic

properties of the template.

We are therefore presented with this tenuous informa­

tion on the chemical nature of the template and are forced

to theorize on this basis. New information in this area

Is surely needed to elucidate the biosynthetic mechanism

of protein formation. There remains, however, a rather

pregnant question which bears on the above theory: Is

concomitant protein and ribonucleic acid synthesis obligatory? To answer this question, much research has been con­ ducted in an attempt to separate the biosynthetic activities of the nucleic acids and of protein. Caspersson and Brachet

(191+1) and Brachet (191+7) were the first to draw attention to the possible participation of nucleic acids in protein

synthesis. Since then, many authors have continued this work. Thus, Pardee (1951+) and Pardee and Prestridge

(1956) have demonstrated an absolute uracil requirement for adaptive enzyme formation and a requirement for amino

acids in nucleic acid synthesis in bacteria. Webster and Johnson (1955) report similar requirements for a pea

seedling ribonucleoprotein incorporating system. Non­

specific amino acid incorporation into the protein of these seedling particles is enhanced by the addition of

a mixture of nucleotide triphosphates, whereas Gale and

Folkes (1951+) demonstrated an enhancement of incorporation

of specific amino acids into disrupted Staphyloccocus

aureus cells by differing RNA fragments. Many other such

observations have been reviewed by Borsook (195b) and by

Kamin and Handler (1957).

In contrast, several investigators have reported

other results. Pardee and Prestridge (195b), Yeas and

Brawerraan (1957)> and Neidhardt and Gros (1957) have ex­

tended the work of Wisseman ejb al. (1951+) and Hahn et al.

(1955)» in utilizing chloramphenicol to uncouple this 10 protein-nucleic acid relationship. The important lesson here is that protein and nucleic acids do not have to be synthesized in equal amounts to form a postulated nucleo- protein. During unlimited RNA synthesis in the presence of chloramphenicol, no observable protein is synthesized, but the presence of amino acids is essential. A further development in this problem is presented by Borek and

Ryan (19^8) who report that a methionine requiring mutant of a lysogenic strain of Escherichia coli, K12 accumulates

RNA during periods of methionine, starvation which is not lost upon addition of the required amino acid. It is tempting to postulate after examining the large quantity of anomalous data in this area that there may be more than one method for synthesizing the different types of protein in the cells, e.g., soluble protein, particulate or systemetized protein, deoxyribonucleoprotein, and ribonucleoprotein. It is possible therefore to visualize differing requirements for protein synthesis as opposed to nucleoprotein synthesis and also for soluble RNA in relation to genetically involved ribonucleoproteins.

It should be mentioned that most of the experiments reported in the past few years relevant to the chemical nature of the template have dealt with intact cells.

Many investigators, however, have utilized cell-free preparations In an attempt to examine portions of this 11 biosynthetic process in the absence of reactions which presumably are extraneous to protein synthesis. In such attempts, liver microsomes, bacterial protoplasts and subceilular particles, and plant ribonucleoprotein particles have been used.

It is evident that a ne plus ultra system for protein biosynthesis studies would be one in which a soluble, cell-free enzyme and template mixture catalyzes the incor­ poration of amino acids into a protein which would both contain easily measured enzymatic activity and be easily isolated in a pure form by Immunological techniques.

Therefore, net gain in the amount of protein could be expected, and amino acid sequence studies would indicate the position of the incorporated amino acid.

A cell-free system has been reported by Ullraan and

Straub (195^-) which will synthesize net quantities of amylase. This homogenate of pigeon pancreas required supplementation with a complete amino acid mixture and

ATP at 0.02 M. Also the systems of Webster and Johnson

(1955) and Spiegelman (1957) produce net increases of material, but are not as yet as simplified as could be hoped. Zamecnik and Keller (19514-) succeeded in preparing a microsome fraction from rat liver homogenates which actively incorporated amino acids when supplemented with some component of the supernatant fraction and an ATP 12 generating system. One disturbing aspect of this system is the net loss of protein during amino acid incorpora­ tion.

Also, as mentioned previously, Gale and Folkes (1955) have prepared sonically ruptured Staphylococcal cells which incorporate amino acids. This system exemplifies well another problem facing workers in this field. When the disrupted cell preparations, after incorporating radioactive amino acids, are resuspended in a medium containing a mixture of non-radioactive amino acids, the incorporated labeled amino acids are exchanged into the medium. This exchange of amino acids appears to be present when there is unequal labeling of a given amino acid at different loci in a protein molecule or during incorporation of an amino acid into a protein under conditions where synthesis of protein, de novo, from amino acids is excluded. Borsook (195b) envisages this process in the following way:

Protein protein-template-amino acids ; ^ activated amino acids free amino acids.

Some other areas of investigation relevant to the mechanism of protein synthesis are (l) speed of the over­ all reaction; (2 ) inhibition studies on the template;

(3) protein turnover; {Ip) antibody and adaptive enzyme formation; (5) and synthesis regulation mechanisms. It is hoped, however, that the short discussion presented herein is sufficient to gain a general Impression of the problem confronting investigators in this field. With this background in mind, the following studies on some

Intermediate reactions in bacterial protejn synthesis were undertaken. EXPERIMENTAL PROCEDURES

Azotobacter vlnelandll, strain 0, was kindly supplied by Dr* R. H* Burris, University of Wisconsin, and Escherichia coil, strain K-12, was provided by

Dr. James Jay. Bakers yeast was obtained commercially, and animal tissues were supplied either by the Meat

Laboratory, The Ohio State University, or a local meat packing concern,

A. vinelandli was cultured by shaking in the nitrogen-free mineral salt medium of Burk and Lineweaver

(1930) containing 3 percent sucrose. The cells were harvested in the log phase by centrifugation, washed, and suspended in a 0.10 M. phosphate buffer (pH 7.25) containing 0.25 M. sucrose. The E. coli was grown by shaking in a medium containing 8.0 g. of Bacto nutrient broth and l+.O g. of Bacto yeast extract per liter. These cells were harvested and resuspended in a manner identical with that for A. vlnelandll. In some cases, a 0.10 M. tris (hydroxymethyl) amlnomethane buffer (pH 7.25) con­ taining 0.25 M. sucrose was substituted for the phosphate buffer.

In certain experiments concerned with the kinetics of isotope uptake or with the preparation of subcellular Ik 15 fractions, cells were grown in eight liter lots by forced aeration. They were harvested in the same manner and resuapended in about four liters of nutrient medium.

Kinetic experiments were performed in a five liter forced aeration bottle from which one liter samples could be obtained and frozen in thirty to sixty seconds. These and untreated cells were disrupted by exposure for six minutes in a 10 kc. Raytheon sonic oscillator. After

disruption, whole cells were removed by centrifugation

at 8,000 x g. for one hour, and sedimentable fractions were obtained by successive centrifugations at 25,000 x g. for one hour, 80,000 x g. for 25 minutes, and lAjl^.,000 x g.

for three hours. Ultracentrifugal examination of each of

the sedimentable fractions, suspended in the sucrose

phosphate buffer, was performed in a Spinco Model E

Analytical Ultracentrifuge. The fractions were also

examined in a Perkin Elmer Electrophoresis apparatus in

an ethylenediaminetetraacetic acid buffer (pH 8.6).

For the inhibitor studies, a suspension of cells was incubated in a metabolic shaker for one hour at

37° C. in the following medium: 0.25 M. sucrose in

0.10 M. phosphate or tris buffer (pH 7.25)j 0.0U1 M. of

the 1-forms of valine, leucine, isoleucine, serine,

threonine, alanine, phenylalanine, tyrosine, tryptophan,

cystine, methionine, proline, histidine, aspartic acid, 16 glutamic acid, lysine, and arginine with glycine-C^+

(I.5J+ mc/mM.) added to approximately 300,000 cpm/ml.j and the appropriate inhibitor in a total volume of four mis* After incubation, the cells were precipitated by adding 60 percent perchloric acid to a final concentra­ tion of 6 percent, washed twice with 6 percent perchloric acid, once with cold absolute ethanol, and once with hot ethanol. RNA and DNA were extracted from the protein by a modified Schmidt and Thannhauser (191-1-5) technique in which 0.05 M. NaOH at 35° C. for four hours was used for hydrolysis of the RNA. Under these conditions, the ratio

of RNA : DNA : protein was ij. : 1 1 10 in A. vinelandll.

It appears that 0.05 M. NaOH at 35° C. for four hours is sufficient to solubilize most of the RNA in

these cells. When higher concentrations are used, DNA,

as measured by the indole method of Keck (l95h)» appears

in this fraction. It is assumed that the DNA is not

hydrolyzed but merely solubilized to such an extent that precipitation with percholric acid is not complete.

Furthermore, upon hydrolysis with 1.0 M. NaOH, ninhydrin

positive material appears after two hours at 35° C.,

suggesting the breakdown of protein. All of these

observations are true for both A. vinelandii and E. coll. By combining this information with a knowledge of the chemical reactions involved in the Schmidt and Thann- hauser (191+5) and the Ogur and Rosen (1950) techniques, a seemingly unavoidable situation is encountered. Optical density readings, combined with measurements., by the

Indole technique, indicate that a small amount of residual RNA is found in the DNA fraction. Obviously, then, a technique could be used which would produce an

RNA fraction containing a small amount of DNA plus a pure DNA fraction, or one in which pure RNA is extracted before the preparation of the DNA fraction containing small amounts of RNA. It was decided, mainly on the basis of the needs of the chloramphenicol experiments, that the latter assay technique would be more desirable.

It should be kept in mind that all of the data presented as DNA include 10 percent or less RNA.

RNA was determined by its optical density at 260 mu. with an extinction coefficient of 16.2 OD/mg. found by measuring the optical density of RNA prepared from

A. vlnelandll by the method of Crestfield, Smith, and

Allen (1955)• DNA was measured by its optical density at 260 mu. with an extinction coefficient of 20 QD/rag. as determined by the indole method of Keck (1950).

Protein was measured by the optical density values at

260 and 2bO mu. according to Warburg and Christian (l9lpJ. 18

Radioactive assay was performed with a Nuc lear-Chicago model D-47 detector with a micromil window and a standard scaling circuit. Specific activity of the extracts is defined as counts per minute in one mg. of material.

In cell-free amino acid incorporation studies, the particulate fractions isolated by differential centri­ fugation after sonic disruption were used. In addition, a particulate sedimenting between 3*500 x g. and 8,000 x g. for 30 minutes was prepared. A cell-free fraction was also prepared by the osmotic rupture technique of

Robrish and Marr (1957). About 2 mg./ml., dry weight,

of the particles from sonication or the hulls obtained by osmotic rupture was incubated in the following medium:

0.1 M. tris (hydroxymethyl) aminomethane buffer (pH 7»25)

containing 0.25 M. sucrose; the previously mentioned mixture of 18 amino acids at 0,0025 M. including glycine-

at 75*000 cpm/ml,; and 0.5 ml. of a supernatant

solution from cells broken by sonic oscillation and

centrifuged at 30,000 x g. for one hour (about 0.1 mg.

protein) in a total volume of 4 mis.

The transfer of oxygen-18 during amino acid activa­

tion required the synthesis of several amino acids

containing oxygen-18 in the carboxyl group. Tryptophan­

es® was prepared without decomposition by refluxing

tryptophan in H20S® for 2l\. hours in the presence of 19 purified 3*0 N. HC1, and showed an atom percent excess of 0.79* Alanine-0-1-® of approximately the same oxygen-l8 content was prepared in the same manner. A mixture of

0-^-amino acids was prepared by acid hydrolysis of casein in for 40 hours, supplemented with trypto- phan-0-1-® after decolorization with charcoal, and showed an atom percent excess of 0.6U. Complete reaction mixtures contained 0.10 M. tris (hydroxymethyl) amino- methane buffer (pH 7.8); 0.01 M. adenosine triphosphate

(ATP); 0.01 M. MgSO^; 0.01 M. 0l8-amino acid; 1.0 M. salt-free hydroxylamine (pH 7.8); approximately 10 mg. yeast pyrophosphatase; and the enzyme prepared from one pound of pancreas in a total volume of 200 ml. The mixture was shaken for 60 minutes at 38°C. The course of the reaction was stopped with 12 percent trichloracetic acid, and after removal of protein, the pH was adjusted to 8.2. Inorganic phosphate and ATP were precipitated as the barium salts [Lepage, (1949)). ATP was removed with Norite A, and the remaining inorganic phosphate was converted to K^POj^ with at pH 4*5. The adenosine monophosphate (AMP) was precipitated from the reaction mixture with 4 volumes of ethanol, and its phosphate was removed by refluxing in £ N. NaOH for 24 hours. The phosphate removed from the AMP was precipitated as the 20 barium salt, and converted to KH^PO^ with KgSOj^ at pH ^*5*

The two inorganic phosphate samples were pyrolyzed in an

Q evacuated system, and the resulting ^ 0 was equilibrated against CO2 overnight as described by Cohn (1953)* The atom percent excess of the 0^-® in the CO2 was then deter­ mined in a mass spectrometer.

For a control value in these experiments, the oxygen-

18 content in the phosphate groups of ATP was determined*

ATP was refluxed in 5 N. NaOH for 2L\. hours, and the inorganic orthophosphate was isolated and analyzed for atom percent excess in the manner described above. It was found that the average atom percent excess in the phosphate groups of ATP was 0.17^, and this number was used as the background control in all experiments. The values presented as atom percent excess in the results of this work are therefore corrected for the control back­ ground*

For use in these experiments, the tryptophan activat­ ing enzyme was prepared by the method of Davie _et al.

(1 9 5 6), and an approximately fifteen-fold purified alanine activating enzyme from pig liver was kindly provided by Dr. George C. Webster. A mixture of enzymes catalyzing the formation of all of the amino acid hydrox- amates was prepared from A. vinelandli. Ten grams of 21 whole cells were broken by sonic oscillation and centri­ fuged at 30,000 x g. for one hour. The supernatant solution was then adjusted to pH %,0 with 2 N. acetic acid, the precipitated protein separated by centrifuga­ tion, dissolved in tris buffer (pH 7*8) and immediately used in the experiment. Inorganic pyrophosphatase was prepared from bakers yeast by the method of Heppel and

Hilmoe (l9£l).

Experiments were also performed to investigate the nature of the "factor*1 which is the acceptor of the amino acyl group from amino acyl-AMP Intermediates. Whole cells, after centrifugation and resuspension in a 0.10 M* tris (hydroxymethyl) aminomethane buffer (pH 7*25) con­ taining 0.25 M* sucrose, were incubated in the presence of radioactive amino acids. They were then cooled and broken by sonic oscillation for twenty minutes, after which the large cell debris was sedimented at 30>000 x g« for minutes. Perchloric acid was added to a final concentration of 6 percent, and the precipitated material was centrifuged. One gram of Norite A was added per 10 militers of the supernatant solution obtained from the above procedure, and the mixture was warmed to room temperature and allowed to stand for ten minutes. The charcoal was then centrifuged and washed four times with 22

0.05 M. KE^PO^. The sediment from the final washing was

taken up in 2.0 M. salt-free hydroxylamine and after five minutes was filtered. Amino acid hydroxamates, in the

presence of carrier hydroxamates, were separated by paper

chromatography in the solvent system described by Hoagland

et al. (1956). It was found that the hydroxamate assay

of Lipmann and Tuttle (19^-5) was not sensitive enough to measure the amount of amino acid hydroxamate formed as

the procedure outlined above.

Glycine and leucine hydroxamates were prepared by

refluxing equal molar amounts of the methyl esters of

the amino acids with hydroxylamine-HCl for i+S minutes.

After neutralizing with 2.0 M, NaOH, NaCl was precipi­

tated in methanol, and the hydroxamates were isolated

from the filtrate by evaporation.

Another technique involving the supernatant solution

obtained after perchloric acid precipitation of the

sonically ruptured A. vinelandll cells involved dialysis

against distilled water at 3° C. overnight and subsequent

lyophiiization. Dried material was dissolved in distilled

water and separated by paper electrophoresis in U.t»5 M.

veronal buffer (pH 8.6) or in 0.05 M. ammonium formate

buffer (pH 3*8) a potential of 16 volts per centimeter

for 6 hours at 3° C. The paper was then cut Into 27 23

1 .5 cm. sections, and the material on each strip was eluted with water and analyzed by the optical density and radioactive methods previously described. This same procedure was used on freeze-dried material dissolved in

2.0 M. hydroxylamine or in 2.0 M. NaCl.

A dried rumen extract containing cobalt-60 labeled vitamin B^2 was obtained from Dr. Ronald Johnson, Ohio

Agricultural Experiment Station, Wooster, Ohio. When

A. vinelandii cells were grown in the presence of this extract, approximately 2 0 ,0 0 0 cpm were incorporated into one gram of the bacterial cells. Paper electro­ phoretic analysis was conducted on aliquots of the supernatant solution from these cells.

Kinetic studies were performed on the amino acid hydroxamates isolated from charcoal, the major component in the paper electrophoresis assay, and the protein precipitated by perchloric acid after centrifugation at 30,000 x g. In these experiments, a solution of glycine-C^ containing about one million counts per minute was added to 25 mis. of concentrated cells in each of eight different flasks. After times of 5 seconds,

15 seconds, 30 seconds, 2 minutes, 5 minutes, 15 minutes, and one hour, each flask was immersed immediately In a dry Ice - acetone bath until the cells were frozen, thus stopping cellular activity. Control values were obtained by adding the glycine-C^ to a flask of frozen cells, and are designated as 0 second time experiments. After sub­ sequent disihtegratibni of the cells by sonic oscillation for 20 minutes, the cell debris was centrifuged for 45 minutes at 30,000 x g. Perchloric acid was added to the supernatant solution to a final concentration of 6 per­ cent, and the protein in the sedimented material was assayed for specific activity as described before. The supernatant solution obtained by perchloric acid precipi­ tation was then divided into two parts. One-half of the solution was used to assay for amino acid hydroxamates by the charcoal method. Specific activities were crudely calculated here by measuring the optical density of the eluted amino acid hydroxamates at 238 mu. An extinction coefficient of 2.8 OD/mg. hydroxamate was determined by measuring the optical density of glycine hydroxamate at

238 mu. and by using glycine as a blank. The use of this wave length to measure peptide bonds has recently been described by Mitz and Schlueter (1958). The specific activities thus obtained were multiplied by a factor of 10 in order to approximate more closely the

specific activity of the radioactive glycine hydroxamate

in a mixture of all of the amino acid hydroxamates. The second half of the supernatant solution was dialyzed overnight against distilled water, lyophylized, and dissolved in 2.0 M. NaCl. This solution was processed by paper electrophoresis, and the component running

7 .5 cm, toward the negative pole was eluted and the specific activity determined. RESULTS AND DISCUSSION

I. Whole Cell Studies on

Amino Acid Incorporation

It is well known that bacteria, in general, possess the ability to grow rapidly. Some are able to synthesize enough protein and nucleic acid in fifteen minutes to enable them to divide and form a completely new cell.

Because Azotobacter vinelandil and the equipment necessary for culturing large quantities of them were readily accessible in this laboratory, it was of Interest to determine whether these cells might prove useful in studies on protein synthesis. When the rate of uptake of radioactive glycine into the proteins of several different biological systems is determined, an Index to the rate of protein synthesis in living cells is ob­ tained. Table 1 shows that A. vlnelandii Incorporates glycine-C^ into protein at a rate which is I4.0 times as fast as yeast and about 200 times as fast as plant or animal systems. By the use of this microorganism, then,

It appears possible that some insight may be gained con­ cerning the mechanism of amino acid Incorporation into proteins, since this process might be rapid enough to be studied conveniently.

2 6 27

TABLE 1

Incorporation of C^-Glycine into the

Protein of Different Tissues

Specific Activity Tissue cpm/mg. protein**

Beef liver slices 20.75

Pea seedlings 2 7 .0

Bean seedlings 33.75

Yeast 136.5

A. vinelandii 55oo.o

*In an incubation mixture of 5 0 ,0 0 0 cpm/mi.

In order to minimize the number of variables in the incorporation studies that were planned, the following steps were taken: (1) centrifugation of the cells permitting both a resuspension at a greater concentration and also a method of washing the spent medium from them;

(2) resuspension under several experimental conditions; and (3) preparation of aliquots of a common suspension ensuring an equal number of cells in each flask under investigation. However, when cells of A. vinelandli or

I:* CQI1* ^12* are sedimented and resuspended in buffered sucrose containing an amino acid mixture, they no longer exhibit log phase growth. As can be seen from Table 2, during the first hour following resuspension, the levels 28 of RNA, DNA, and protein in E. coli remain constant* In

A. vinelandii, only the protein level remains constant, while the DNA level decreases about 10 percent and the

TABLE 2

Changes in Levels of Protein, RNA, and DNA in

Non-growing Cells of A. vlnelandil and E. coll

Level after 60 minutes as Component a percent of initial level

E. coli A. vlnelandil

Protein 1 0 0 . 1 0 0 .

RNA 1 0 0 . 75.

DNA 1 0 0 . 90.

RNA level decreases approximately 25> percent. Although the cells are definitely not growing during the period of investigation, it is felt that a knowledge of the metabolic reactions concerned with protein and nucleic acid synthesis in this situation is essential to biochemistry today. Therefore, experimentation on amino acid incorporation into these non-growing cells was conducted.

These cells readily incorporate C^-alanine into their protein, P^-orthophosphate into their nucleic acld3, and C^-glycine into both protein and nucleic acid*

In the case of glycine, A. vlnelandil cells incorporate 29

10 to 12 percent of the glycine supplied in one hour,

or approximately 2 ,5 0 0 cpm/mg. into protein, l+,500 cpm/mg.

into RNA and 50,000 cpm/mg, into DNA. Figure 1-A illus­

trates the time course of incorporation of glycine carbon

into the RNA, DNA, and protein of non-growing A. vinelandii

cells. Two phenomena are noteworthy. First, cellular

DNA incorporates glycine considerably more vigorously

than either RNA or protein. This may be due to the

marked formation of new DNA prior to the resumption of

log phase growth by the cells or to a vigorous steady

state exchange between glycine and the purine bases.

Secondly, the incorporaticn of radioactive carbon, after

a rapid uptake during the first 30 seconds of incuba­

tion, exhibits a cessation of incorporation for several minutes before continuing at a steady rate which is

somewhat slower than the initial rate. This interrup­

tion of incorporation is especially striking during the

formation of DNA, but is obviously paralleled by the

incorporation of radioactive carbon into RNA and protein.

Furthermore, it has been observed, as is illustrated

in Figure 1-B, that preincubation of the cells for 10

minutes in a glycine-free amino acid mixture results

subsequently in a greater initial incorporation of

glycine carbon and also a marked increase in the length

of the pause. . vinelandii. A. i, . — 1. Pig, Specific Activity 11,000 3,000- 1,000 500 Glycineincorporation DNA Protein RNA 30 0 30 ie n inutes M in Time into RNA, DNA, and protein of protein and DNA, RNA, into RNA Protein . J _LJ 15O DNA, Protein

31

There appears to be no available explanation for this observation, but one possibility might be suggested.

Dalgliesh (1957) has pictured the biosynthesis of a single protein molecule to occur as a series of peptide bond formations along a template. As soon as this bond is formed, that portion of the protein molecule is set free from the template and the molecule then appears to peel off. In this case, as in all other such postula­ tions, the absence of a single amino acid would inhibit the whole process. If, however, the delinquent amino acid were added after all of the others had been positioned on the template, the sudden formation of one protein molecule per template could be expected. A finite time would then be required to orient all of the building blocks necessary for the biosynthesis of subsequent protein molecules. Dalgliesh also feels that several partially formed proteins may be peeling from the template at one time. This author’s explanation would be equally valid In this light if a glycine molecule were needed near both ends of the protein, making it impossible to even start a second polymer.

With this information as a background, experiments were conducted to determine whether the synthesis of protein could be divorced completely from the synthesis 32 of RNA and DNA by the use of differential inhibitors.

The first inhibitor used was chloramphenicol. Figure. 1-C illustrates the effect of chloramphenicol on the incor­ poration of C^-glycine into RNA, DNA, and protein of

A. vine land il. In contrast to previously reported results

[Gale and Folkes (1953)J Pardee and Prestridge (195b);

Wisseman et al. (195^)» and Hahn e_t al. (1955)3 » chlor­ amphenicol inhibits the incorporation of C^-glycine not only into protein, but into RNA and DNA as well. Chlor­ amphenicol also eliminates the interruption of incorpora­ tion noted in untreated cells.

Figure 2 shows the effect of varying chloramphenicol concentrations on C^-glycine incorporation into the RNA,

DNA, and protein of non-growing A. vinelandii and E. coll cells. In both bacteria, incorporation into DNA and protein are inhibited in a similar manner over the entire range of concentrations, but glycine incorporation into

RNA is less inhibited with decreasing concentrations of chloramphenicol. With both kinds of cells, chloram­ phenicol has no significant effect on the levels of

RNA, DNA, or protein — - only on the amount of isotope

Incorporated.

As chloramphenicol produces a differential inhibition of RNA from DNA and protein, a series of other inhibitors init . ieadi n . coli. E. and vinelandii A. into tion Pig. 2. — The effect of chloramphenicol on glycine-C^ incorpora­ glycine-C^ on chloramphenicol of effect The — 2. Pig.

Percent of Normal Growth 100 80 60 40 20 02 03 04 05 06 07 0'8 I0 10'7 I0~6 10"5 10'4 I0'3 I0"2 ______i ______RNA ocnrto o Clrmphenicol Chloram of Concentration i ______DNA, I ______Protein l ______i ------1— 34 of either protein or nucleic acid synthesis was tested in this respect. Table 3 indicates the absence of effect of RNAase and DNAase on whole A. vinelandli cells. It is assumed that these two enzymes are not able to get through the cell wall.

TABLE 3

Effect of RNAase and DNAase on the

Incorporation of Radioactive Amino Acids

Radioactivity in cpm. Treatment i frr* 2 hrs.

Control 110 548

RNAase 163 628

DNAase 164 61j2

Levy et al. (1949) have reported that cobalt ions inhibit growth (including protein synthesis) in Proteus vulgaris without inhibiting nucleic acid synthesis. In contrast, McQuillen (1955) observed that uranyl ions inhibit nucleic acid synthesis in protoplasts of Bacillus megaterium without inhibiting protein synthesis. The effects of these two ions have been tested in addition to penicillin, purine, 8-azaxanthine, rubidium ions,

B-2-thienylaIanine, and allylglycine. Figure 3 indicates that only three of the above mentioned inhibitors, Co++, . vinelandii. A.

Fig. Fig. Percent Inhibition 3 — — . 0- o' w 10'6 w '4 io -2 ,0 ob t Ions Co bo It The inhibition inhibition The

f o netain f Inhibitor of oncentration C I0 N, N, n rti ytei in synthesis protein and DNA, RNA, 2 ' I0 rnl Ions Uronvl "4 ICT 6 10 llylqlycine A 2 ’ I0 '4

VJT 36

UOg+t, and allylglycine, have any effect on the incor- i )i poration of glycine-C into A. vinelandii cells and that

the inhibition elicited by them is not differential. It

can therefore be suggested that Co++ and U 02++ do not have

the same effect on all cells or that the biosynthetic metabolism of vlnelandi1 differs during different

periods of its life-cycle.

Chloramphenicol is the only inhibitor that has been

found which inhibits incorporation into RNA, DNA, and

protein to different extents. The smaller chloramphenicol

inhibition of C^-glycine incorporation into RNA could be

due either to a lesser sensitivity of RNA metabolism to

chloramphenicol or to the formation of a particular

polyribonucleotide In response to the presence of chloram­

phenicol. Information on these possibilities has been

sought by an examination of the effect of this inhibitor

on C^-glycine Incorporation into various centrifugal

fractions of A. vinelandii cells. If chloramphenicol

elicits the formation of a particular new polynucleotide,

then glycine incorporation might be inhibited to an extent

comparable with DNA and protein in most fractions, but

actually increased In one of them. Alternatively, if

RNA metabolism is simply less sensitive to chloramphenicol

than DNA or protein metabolism, then the inhibitory effect 37 of chloramphenicol on incorporation into RNA should parallel, but be less than, its effect on DNA or protein in all fractions. Table 4 shows that chloramphenicol

inhibits the incorporation of glycine into the RNA, DNA

and protein of all cellular fractions. Although glycine

TABLE 4

Effect of Chloramphenicol on C-^-glycine Incorporation

into the Protein, RNA and DNA of Various Centrifugal

Fractions of A. vinelandii

Incorporation as a percent of glycine incorporation in the Centrifugal Fraction______absence of chloramphenicol Protein DNA RNA

Sediment from 25,000 x g. for 60 minutes 33 45 85

Sediment from 80,000 x g. for 25 minutes 28 51 73

Sediment from 1144,000 x g. for 3 hours 39 47 72

Supernatant solution from H 44,UUO x g. for 3 hours 19 26 77

incorporation Into RNA of all fractions is less inhibited

than into DNA or protein, the difference is especially

striking in the fraction not sedimented in 3 hours at

1144,000 x g. The importance of this finding is intensified

when a time course of incorporation Is plotted against 38 the percentage of label contained In each of the above fractions. As can be seen In Figure ij., the non-sedi­ mentable fraction appears to be the site of early

incorporation into the cells. This observation is

similar to those of Hoagland (1955) in liver cells

and DeMoss and Novelli (1955) in extracts of A. vine­

landii cells. It would necessarily follow that inhibi­

tion resulting from chloramphenicol at the site of

initial incorporation would affect the subsequently

labeled fractions, whereas the reverse would not be

true.

It has been shown that amino acid activation by

extracts of pea seedlings is not inhibited by chloram­

phenicol [Webster (1957)3 • Therefore, this differential

inhibitor must act at some biosynthetic site subsequent

to amino acid activation but previous to the biosynthesis

of the protein itself, involving nucleic acid metabolism.

In a series of papers, the latest being Hahn et al.

(1957), Drs. Hahn, Hopps, Wisseman and co-workers have

investigated the mode of action of chloramphenicol

extensively. From all of this work, they state [Hahn ejb al.

(1957)] that chloramphenicol causes an imbalanced synthesis

of normal nucleic acid rather than the formation and

elimination of abnormal polynucleotides. Protein RNA DNA * B+C 4 0 4 0 4 0

•; 30 30 3 0 B + C

° 20 20 20

vO

-A _L 3 0 10 20 3 0 20 3 0

Time in Minutes

Pig. 4. — Jhe distribution of glycine-C1^- in the RNA, DNA, and protein of centrifugal fractions. Although it has been attempted [Hahn et_ al. (1957)3 » no clear evidence has been presented that chloramphenicol

has no effect on the intracellular levels of ATP and ADP.

It is well known that protein and DNA synthesis requires

ATP tHoagland (1955) and Kornberg et al. (1956)1 » where­

as RNA synthesis uses ADP and other diphosphates as

substrates [Grunberg-Manago et al. (1956)) . Furthermore,

Gale and Paine (1951) have shown that aureomycin and

chloramphenicol at very high concentrations will inhibit

oxidative phosphorylation, although they seem to have no

such effect at concentrations which Inhibit protein

synthesis. It is therefore felt that the effect of

chloramphenicol might be explained on the basis of an

inhibitory action on the rephosphorylation of adenine

nucleotides in the biosynthetic "factories" of the cell.

If chloramphenicol, at a certain concentration, would have

the ability to increase the nucleoside diphosphate/nucleo­

side triphosphate ratio, then the observed effects would

be expected e.g., an increased synthesis of RNA resulting

from an increase in the concentration of Its normal sub­

strates. Furthermore, an effect on phosphorylation in

general would explain the Inhibitory action of 3 x 10”3

chloramphenicol on RNA, in addition to DNA and protein.

Perhaps in non-growing A. vinelandii cells, there Is

a different over-all biosynthetic metabolism being 0

ui performed than in log phase cells. The protein-nucleic

acid interrelationship here appears to be rather involved

and leads to the impression that the cells are making

nucleoprotein to a great extent. It would therefore be

difficult to divorce one biosynthetic activity from the

other in intact cells.

II. Cell-free

Incorporating Systems

It became increasingly apparent from studies on

the incorporation of radioactive precursors into the RNA,

DNA and protein of whole cells that mechanistic investi­

gations into the chemical nature of the biosynthesis of

protein were not very feasable in such a complicated

system. It was felt that by utilizing an incorporating

system which did not have (l) a membrane transport

problem; (2 ) unrelated physiological obstacles, such as

temperature shock; or (3) a great number of other metabolic

pathways, more information could be obtained concerning

the biosynthetic metabolism of protein and its relation

to nucleic acids. Attempts were undertaken, therefore,

to isolate from whole A. vlnelandil cells a subcellular

particulate fraction which would catalyze the incorpora­

tion of amino acids into protein. 42

Marr and Cota-Robles (1957) have presented evidence that sonic disruption of A. vinelandii does not cause inactivation of hydrogenase or glucose-6-phosphate dehydrogenase in the broken preparations. Alexander

(1 950 ) has also examined enzymatic activity after sonic disruption of this microorganism, and Schachman et al.

(1 952 ) have shown the presence of submicroscopic particles in bacteria. It was no surprise, therefore, that when a heavy suspension of A. vlnelandil cells is exposed to sonic vibration, active particulate material can be isolated by differential centrifugation. After whole cells were removed at 3*500 x g., sedimentable fractions were obtained at 8,000 x g. for 30 minutes (Fraction A);

2 5 ,0 0 0 x g. for one hour (B); 8 0 ,0 0 0 x g. for 25 minutes

(C); and 1144*000 x g. for 3 hours (D). Examination of

Fraction A by phase microscopy indicated a fairly homo­ geneous suspension. The spherical particles were about one-sixth the size of whole A. vinelandii cells, measuring

600 - 700 mu;* in diameter. Upon chemical analysis, an over-all 260/280 mu. ratio of about 1 .7 was obtained, and their RNA : DNA : protein ratio was 10 : 1 : 100. On the other hand, the resuspended sediment from the

25*000 x g. centrifugation contained a whole spectrum of particles under the phase microscope. Because of lj-3 the size of the particles, the sediments from the final two centrifugations and the supernatant solution from the last one were examined for homogeneity by analytical ultracentrifugation and flow electrophoresis. Figure £ indicates that there is a total of at least 15 components in these three fractions.

When the particulate fractions, which are designated

B, C, D, and the supernatant solution of the D fraction, were tested for the ability to incorporate radioactive glycine into their protein, no activity was found. In fruitless attempts to obtain incorporation into these fractions, either individually or in combination, the pH was varied from 6.0 to 8.5* molarity and particle concentration changed, nucleoside mono-, di-, and tri­ phosphates and mono- and divalent ions were added, and energy sources such as ATP, hexose diphosphate (HD?), phosphoenolpyruvic acid (PEP), and phosphoglyceric acid

(PGA) were supplied. After this work was completed, it was decided to investigate fraction A which previously had been discarded. Surprisingly, it was found that fraction A did contain incorporating activity. This activity was enhanced upon the addition of a small amount of the supernatant solution from a 3 0 ,0 0 0 x g. centrifugation for one hour. Table 5 indicates the specific activity hk

ELECTROPHORESIS ULTRACENTRIFUGE I I

1200 &. to 600 X.

. o 600 A. to 200 1.

Below 200 %.

Pig. 5?. — Electrophoretic and ultracentrifugal analysis of the particulate fractions C and D and the supernatant solution of D. 1*5 of the protein Isolated from this particle after two hours of incubation. Burma and Burris (1957) have reported a similar incorporating system.

TABLE 5

Amino Acid Incorporation into Particulate

Protein of A. vinelandii in Vitro

Specific Activity of Protein System Fraction A Hulls

Complete 68 358

Complete minus Sup. 16 1*1*5

Complete plus ATP-Mg++ both 1 0 “5 M. 92 292

Complete plus ATP-Mg++ both lO”^ M. 58 220

Complete plus HDP 10“^ M. - . 507

Complete plus HDP 10"^- M. - 305

Complete plus PEP 10“^ M. - 0

Complete plus PEP 1 0 “^- M. - 2i|6

Also listed in Table 5 are the characteristics of

another incorporating system. When cells which have been

suspended in 1.5 M. glycerol for two hours are poured

into four volumes of cold distilled water, they are ruptured by osmotic shock. The broken cells, or hulls, can then be

isolated by centrifugation and used for amino acid incor­

poration studies. From the table, it can be seen that a ¥> supernatant solution is not necessary for enhancement of amino acid incorporation, and that ATP appears to be inhibitory, Indicating two definite differences in these fractions.

The hull fraction seems to be the more stable of the two incorporating systems. After two hours, incorpora­ tion of gLycine-C^ into this preparation was still linear, and the label appeared in RNA and DNA in addition to the protein. Under optimal conditions, the specific activity of the RNA and DNA was much greater than that of the protein, e.g., - 15»^00 cpm/mg. into RNA and

38,000 cpm/mg. into DNA.

These hulls appeared as flattened cells under the phase microscope in which I4. to 6 dense areas could be discerned, being approximately the same size as the particles in fraction A. The ratio of RNA : DNA : protein in the hulls was $ : 1 : 100.

It is felt that the utilization of these particulate cell-free incorporating systems may prove fruitful in further Investigations on the problems of protein bio­ synthesis and of protein-nucleic acid interrelationships. H7

III. Amino Acid Activation.

Since Hoagland (1955) first published his observa­ tions on an amino acid dependent exchange between inorganic pyrophosphate and ATP, catalyzed by a soluble prepara­ tion from liver, many other similar reports have appeared

in the literature. In addition to the 22 references given by Kamin and Handler (1957)» encompassing prepara­

tions from yeast, liver, beef pancreas, a number of micro­

organisms, and several plant materials, Scarano and

Maggio (1957) reported this activity in unfertilized sea urchin eggs. Specific activating enzymes have been reported by Davie et al. (1956) for tryptophan, Schweet

et al. (1957) for tyrosine, Holley (1957) for alanine,

and Berg (1956a) for methionine. The evidence to date

indicates that amino acid activation may be formulated

as follows:

(a) E + ATP + amino acid -«---- E-AMP-amino acid + PPi

(b) E-AMP-amino acid + acceptor ^ - 1 E + amino acyl- acceptor + AMP.

In all of the studies cited in which the second reaction

was observed, hydroxylamine has been used as the acceptor.

In this function hydroxylamine becomes a trapping agent

and the second reaction is not reversible. Recently both

Holley (1957) and Webster (1957) have shown the presence 10 of an exchange between AMP and ATP in their activating systems, and this exchange was inhibited by ribonuclease.

All of the data mentioned thus far are very suggestive concerning the exact mechanism of amino acid activation.

Berg (1957) has gone one step further in suggesting that an amino acyl-AMP is the chemical intermediate in the formation of the activated amino acid. In his system the addition of chemically synthesized amino acyl-AMP and inorganic pyrophosphate to the enzyme preparation caused the formation of ATP in the backward reaction of equation (a). This reaction is analogous to the acetate activation whfch is reviewed by Berg (1956b)•

Because the amino acyl-AMP intermediate has never been isolated in a free state, it is thought to be enzyme- bound. Therefore, definite and complete proof of its existence has not been demonstrated, and it was felt that this was necessary before other studies on activation could be performed. In view of this, the following investigation was undertaken.

When tryptophan, labeled with oxygen-l8 in its carboxyl group, Is activated by a purified activating enzyme [Davie et al. (1956)3 , it should form a phosomono- ester linkage with AMP if the existing theories are correct. In the presence of hydroxylamine this bond U-9 would be cleaved and the remain on the phosphate moiety of the AMP. Alternatively, if an enzyme-bound amino acyl-pyrophosphate intermediate is formed, oxygen-

18 from the carboxyl group of the amino acid would be found in the pyrophosphate upon hydroxamate formation.

By isolation of the phosphate from AMP and from pyro­ phosphate and subsequent analysis for oxygen-l8 content, definite proof for the formation of the intermediate in amino acid activation would be found.

Table 6 shows the results of these experiments.

0xygen-l8 labeled tryptophan does transfer its carboxyl

TABLE 6

Oxygen Transfer During Amino Acid Activation

Atom Percent Excess Phosphate Phosphate from from Pyrophos- AMP Enzyme Preparation Substrate phates

Trypt ophan-ac t ivat- Tryptoghan- ing enzyme Olo 0 .0 0 2 0 .0 3 0

Alanine-activating Alanine- enzyme 0l8 0 .0 0 4 0 .0 3 2

Extract from A. Mixture of vinelandii 0-Lo labeled amino acids 0.006 0.016 oxygen to the phosphate group of AMP during the formation

of tryptophan hydroxamate. Within experimental error, the amount of oxygen-l8 found in the AMP corresponds to 5o that expected for the transfer of one oxygen from the tryptophan carboxyl. In contrast, essentially no oxygen-l8 from tryptophan appears in the phosphate groups derived from the liberated pyrophosphate. These results are consistent with the formation of a tryptophan-AMP intermediate during tryptophan activation.

In order to learn whether these results might apply to amino acid activation in general, the same experiment was performed with alanine-0 -1-® and an alanine activating enzyme purified from pig liver some fifteen-fold by

Dr. George C. Webster. When this enzyme catalyzes the conversion of alanine-0 -1-® to alanine hydroxamate, oxygen-

18 is again found in the liberated AMP (Table 6 ).

A pH 5*0 amino acid activating preparation ^Hoagland

(1955)3 which seems to catalyze the activation of most of the commonly occurring amino acids was prepared from a supernatant solution of sonically disrupted A. vinelandll cells. This crude enzymatic mixture, when incubated with an Ol®-labeled casein hydrolysate and ATP, also catalyzes the transfer of carboxyl-0^® to the phosphate moiety of AMP during the formation of a mixture of amino acid hydroxamates. As is shown In Table 6, however, the total oxygen transferred is considerably less than is elicited by the purified enzymes. This Is probably due to a marked phosphatase activity of the preparation 51 which produces considerable unlabeled AMP* Furthermore, the reaction results in the transfer of fair quantities of oxygen-l8 to inorganic phosphate. It must be remembered that inorganic pyrophosatase is added to the incubation medium to allow a more complete reaction, thus making it

impossible to determine whether or not the O^-labeled

Inorganic phosphate came from pyrophosphate. This

significant quantity of transfer, however, could be due

to the activation of some amino acids through the inter­ mediate formation of amino acid phosphates or to the

operation of glutamine synthetase, which Boyer, et_ a l ♦

(1956) and Kowalsky et^ al. (1956) have shown to transfer

glutamate carboxyl oxygen to inorganic phosphate. A

decision between these two possibilities must await the

purification of other amino acid activating enzymes.

After this work was completed, Hoagland at al. (1957b)

published results on similar work. By using only the

tryptophan-activating enzyme and carboxyl labeled \ fl tryptophan- 0 , they were able to show the transfer of the

oxygen-l8 to the phosphate group of AMP, thus corrobor­

ating the findings reported here.

It is felt, therefore, that direct proof has been

found for the formation of a phosphomonoester linkage

between amino acids and AMP during amino acid activation. 52

On this basis, the presence of an enzyme-bound amino acyl-AMP is assumed, and work was undertaken to examine reaction (b) with the hope of gaining more insight into the transfer of the amino acyl moiety to an undetermined acceptor.

IV. Intermediates in the Biosynthesis

of Protein

When the equations which have been formulated for the activation of amino acids are examined,

(a) amino acid + ATP + E - ^ 1 - > E-AA-AMP + PPi

(b) E-AA-AMP + X AA-X + AMP + E, it can be seen that the techniques used to measure amino acid activation are completely useless in determining the chemical nature of the acceptor in reaction (b).

Both the exchange between inorganic pyrophosphate and

ATP and the formation of amino acid hydroxamates are independent of the physiological acceptor, X. However, the exchange between AMP and ATP does require the presence of X. Recently, Holley (1957) and Webster (1957) have reported some results from the use of this technique.

In addition, they find that ribonuclease will inhibit this exchange, suggesting that either X or some other necessary cofactor may be a polyribonucleotide. 53 By using a different approach to the problem, Hoagland et_al. (1957a) have discovered the presence of a low molecular weight ribonucleic acid (S-RNA) in their pH 5 amino acid activating preparation. When this activating preparation is incubated with ATP and carboxyl labeled leucine, at pH 7.5* the S-RNA subsequently isolated is found to be labeled. The leucine labeled

S-RNA so obtained is non-dialysable and is charcoal and

Dowex-1 adsorbable. The C”-^-leucine-RNA bond is acid

stable and alkali labile, and when the material is

incubated with hydroxylamine, paper chromatography

indicates that all of the radioactivity is found in the

spot corresponding to leucine hydroxamate. Hoagland,

therefore, feels that S-RNA is the actual physiological

acceptor of the activated amino acid during the biosynthesis

of proteins. Ogata and Nohara (1957) have published

similar results. Also applicable in the present context

is the work of Konningsberger et al. (1957). Paper

electrophoresis of a cell extract produces a component

showing nucleotide characteristics. Upon addition of

hydroxylamine to this component, a ferric-hydroxamate

spot can be developed, and after acid hydrolysis,

ninhydrin positive material is found.

To this information concerning an RNA-amino acid

intermediate during protein biosynthesis can be added the 514- work of Rabinovitz and Olson (195b). They presented evidence for a ribonucleoprotein Intermediate In the synthesis of globin by reticulocytes. However, Jencks

(1957) has shown that amino acyl-adenylate Intermediates are too labile to be considered as likely physiological intermediates. Thus, as can be further postulated, a carboxyl-phosphomonester linkage between amino acids and a soluble RNA could meet with the same chemical stability difficulties making it necessary to revise this theoretical approach. As the matter now stands, no good chemical proof exists for the occurrence of an S-RNA cofactor in protein biosynthesis. However, some stabilized active amino acid complex does exist, and investigations were undertaken in an attempt to further classify it.

If A. vinelandii cells are incubated in the presence of C”1^-glycine or C'^-leucine, an intracellular soluble radioactive complex is formed. After 3onic rupture and subsequent precipitation of all protein material with perchloric acid, the solution is cleared by centrifuga­ tion. When charcoal is added to the supernatant solution, radioactivity, which is not due to free amino acids, is adsorbed. That the adsorbed material is actually the carboxyl activated complex is suggested when the charcoal

is washed and subsequently incubated in salt-free hydroxy­

lamine. After filtering, paper chromatography indicates 55

the formation of both glycine and leucine hydroxamates•

Table 7 shows the Rf values obtained by paper chromato­

graphy of known hydroxamates.

TABLE 7

Paper Chromatography of Glycine, Leucine

and Their Hydroxamates

Component Rf

Glycine .11

Glycine hydroxamate .2 2

Leucine .65

Leucine hydroxamate .57

Figure 6 graphically illustrates a typical paper

chromatogram from these experiments. After drying the

paper, on which a band of the hydroxylamine eluate has

been chromatographed, eight sections are cut out. These

strips correspond to Rf values from 0 to .12, .12 to .25*

.25 to .37, and so forth. It can be seen that most of

the radioactivity appears in the strips having Rf’s

corresponding to the hydroxamates of the amino acids and

not to the amino acids themselves. This conclusion

was further justified by finding that all of the activity leucine hydroxamates. leucine

Pig. Pig. Rf on Paper Chromatogram 0 0 . 1 .87 .75 .62 .25 .50 .12 .37 .00 6 — Ppr hoaorpy f lcn and glycine of chromatography Paper . — 40 oa Counts Total 80 56 120 Leucine-NHOH Glycine- NHOH Glycine- 6 200 160 moves on the paper with carrier hydroxamates. The spot can then be assayed by the ferric-hydroxamate test of

Lipraann and Tattle (1914-5 )•

In order to show that the amino acid-X is actually a soluble intermediate, ruptured cells were prepared by the osmotic method of Robrish and Marr (1957)* Fifteen minutes before dilating the cells, C“^-glycine was added, and after breaking the cells, the hulls were sedimented and exposed to sonic oscillation for twenty minutes. Both the supernatant from the sedimented hulls and the sonically broken hulls themselves were assayed for AA-X content. Table 8 shows that almost all of the

AA-X was in the supernatant solution obtained by osmotic rupture.

TABLE 8

Distribution of AA-X in Ruptured Cells

Cell Fraction cpm In AA-NHOH formed

Hulls 10.

Soluble fraction after rupture 275.

An assay was thus established for the proposed amino

acid-X intermediate. This allowed the Investigation of

the conditions necessary for the in vitro formation of

this complex as well as some of its chemical properties. £>8

It wa3 found that no measureable AA-X was formed by any of the cell-free Incorporating systems utilized previously.

Even supernatant solution containing the amino acid activating enzymes would not catalyze the biosynthesis of this factor, so attempts along this line were dropped.

The effect of neutral pH on the stability of AA-X was determined. In 5 percent perchloric acid, AA-X remains intact overnight at 3° C. However, by adjust­ ing the pH of the perchloric acid supernatant to 8.0 with 1.0 N. KOH, about 75 percent of the charcoal adsorbable radioactivity is lost after one hour. Table

9 shows that in addition to H* ion concentration, dialysis also causes the breakdown of this intermediate*

TABhE 9

Sensitivity of AA-X to pH and Dialysis

cpm in amino acid Treatment hydrbxamate formed

Control 528 pH 8 for one hour 139

Dialysis at pH 1 for three hours 88

Dialysis at pH 7 for three hours 61

During dialysis It Is possible that AA-X Is not decomposed

but actually passes through the dialysis tubing. That this 59 is not true has been shown by analyzing the solution outside of the tubing. In several attempts, no AA-X could be measured In this solution.

While the dialysis experiments were being conducted, it became evident that there was a great deal of radio­ active material remaining inside of the dialysis tubing.

In the Beckman DU this dialysate shows a very high

260/280 mu. ratio, and as can be seen in Figure 7> contains much absorbing material at wave lengths below

260 mu. Also the figure indicates the spectra obtained by incubating this dialyzed solution in 2 M. NH2OH or

2 M. NaCl. The spectrum of the dialysate in NaCl is

of great interest because upon aging at 3° C. for 2

days, the dialysate itself produces an identical curve.

To investigate further the nature of this nondialyz-

able material containing bound glycine-C^, it was

concentrated by lyophilization and subjected to paper

electrophoresis. At pH 8 .6 in veronal buffer, the

radioactivity moved in a manner similar to free glycine—

about 1 to 2 cm toward the positive pole— indicating a

cleavage of the bound glycine-C^ at pH 8.6. On the

other hand, in sodium formate buffer, pH 3*8, radio­

active material was found over the entire paper.

Thereafter, all electrophoretic studies were performed 20

-k

1.6 X-k -[- = Dialysate H; • = Dialysate + NH2OH \ O = Dialysate + NaCl or Age -■g 12 v-k c CD o 6 O' o o u 9 s-k Q- O \

'0— 0— 0- 0 2 4 0 260 280 300 Wave Length in mu.

Pig. 7. — The spectra of the dialyzed and treated supernatant solution. 61 at pH 3.8. Figure 8 (+-+-+) shows the pattern of label produced in a typical run. Specific activity here is designated as cpm/OD unit at 2b0 mu., and it can be seen that a considerable amount of the bound radioactivity has moved more than 20 cm. toward the negative pole.

The 260/280 mu. ratio of eluted material remains about

1.3 to 1.5 throughout the length of the paper. If, however, the freeze-fried dialsate is dissolved in 2 M.

NH2OH or 2 M. NaCl, an entirely different pattern develops. Figure 8 (-0-0-0) shows that one peak with a very high specific activity is found near the origin.

By comparing this with Figure 9, which indicates only the total activity of radioactive material in the same experiment, two observations are noteworthy.

First, it appears that the NHgOH or NaCl has caused the splitting of some bound glycine found in the dialyzed material. This radioactivity appears where both glycine and glycine hydroxamate usually run in this system. At the same time, radioactivity is lost from that portion of the paper 25 cm. or more toward the negative pole.

The second observation concerns the material which moves about 7 .5 cm. and contains a great majority of the radio­ activity. On Figure 8, this same component has a low

specific activity, indicating that there is a large

amount of it. Furthermore, although the 260/280 mu. electrophoresis.

Pig. Pig. Specific A ctivity 0 0 5 2 1500 0 0 5 8 . — The specific activity of components separated by paper by separated components of activity specific The — . Pole + ------°^o^o-o-° ° °-0-°^0 N-^, ^ - N 0 ^ ° - 0 - ° ° ° - o - o ^ o ^ ° . 0 - 0 rgn i Origin o S ------itne n cm. in Distance Dialysate = + = = O 20 ------ilst + Dialysate . % * V V ^°°3°0O ° ° ^ NHgOH or Pole — NaCl 0 0 0 0 0 0 *40 electrophoresis• Pig, Pig,

Total Activity 1000 1500 00 50 9 , — ‘i'he total activity of components separated by paper by separated components ‘i'he of activity , — total itne n m. cm in Distance o T ^

6k ratio of the solutions eluted from the paper is 1 .3 to l«k for all other components, the ratio for this fraction is about 0 .?, and reacts positively with nin- hydrin.

While this work was in progress, Wagle at al. (1957) published experiments concerned with the role of vitamin b 12 *-n protein synthesis. They showed that the majority of the Co-60 incorporated as vitamin B]_2 by rat liver, in vivo, was found in the microsome fraction and in a supernatant solution. In addition, upon preparing the pH 5 activating enzymes from the supernatant solution,

80 percent of the label remained with this enzymatic fraction. They also investigated the in vitro activities of these preparations and compared them with similar

ones isolated from vitamin B-^ deficient rats. The amino acid incorporating activity of the vitamin B^2

deficient system was three times less than that of the microsome and supernatant preparation containing Co-60 labeled vitamin B-j^* Prom these results, they postulated that vitamin Bi2-enzyme acts as an "activator carrier" for amino acid to the template.

In order to determine if this vitamin is involved

in any of the phenomena which are being examined In

this study, A. vlnelandll cells were grown in the presence

of Co-60 vitamin B1 2. by utilizing all of the techniques 65 outlined above, no Co-60 activity could be found. It was not bound to the charcoal during analysis for AA-X, nor did it appear in the dialysate of the supernatant solution. Therefore, two explanations are possible.

Either the conclusions of Wagle and co-workers are questionable or the factor being studied here has no relationship to the postulated carrier for the activated axnino acids.

The most interesting explanation for the observa­ tions that have been presented concerning AA-X and the major component (called peptide material for the purpose of terminology) in the electrophoretic studies is also the most obvious. When amino acids enter a cell, they are immediately activated by the activating enzymes at the expense of the energy of ATP. The amino acyl group of the bound amino acyl-AMP is transferred to X, and this soluble intermediate is then free to donate the amino acid to a template. Assuming the presence of only a catalytic amount of X, it is possible that this co­ factor could be saturated with amino acid almost as soon as the amino acids enter the cell. After the desired number of different amino acids have been delivered to the template, stepwise polymerization occurs and a completed protein is synthesized. If, however, this whole procedure is stopped by freezing, it is possible 66 to visualize the presence of a saturated amount of

AA-X, partially completed protein molecules bound to templates, and soluble free proteins which have just been synthesized. By sonically breaking the cells,

a homogenate would be formed containing AA-X, broken template with bound peptide material, and soluble proteins.

In order to determine whether the peptide material

isolated by paper electrophoresis may have originated from uncompleted protein during synthesis, as is postu­

lated in the above visualization, a kinetic experiment

has been performed. At differing times after the

addition of glyclne-C1^, A. vinelandii cells were frozen,

sonically ruptured, and analyzed for (a) AA-X, (b) the

paper electrophoresis component, and (c) the protein

precipitated following centrifugation of 3 0 ,0 0 0 x g.

for one hour. The results of this experiment are shown

in Table 10. The results are not conclusive, but it

can be seen that both of the suggested intermediate

components become labeled much faster than the soluble

protein. Furthermore, both AA-X and the peptide appear

to be a plateau in specific activity as early as five

seconds.

Rather than attempting to survey all of the possible

explanations for the data in Table 10, only one observa­ 67 tion will be made. The results do not conflict with the premises upon which the experiment was undertaken, but more investigation will have to be applied on this problem before any positive statements can be made.

TABLE 10

Kinetic Uptake of Glycine-C-^

R Specific activity cpm/mg. of material Time AA-X Peptide Protein

0 seconds 120 58 0.

5 seconds 325 1+07 0.

15 seconds 398 1*01+ 0.

30 seconds 1+25 1*10 9.

2 minutes 1*38 1+38 27.5

5 minutes 513 622 117.

15 minutes 625 975 311.

60 minutes 606 1250 3300.

It is hoped that the results and ideas presented in this study may prove helpful in further experiments on the mechanism of the biosynthesis of protein. SUMMARY

The problem of the elucidation of the mechanism of protein biosynthesis has recently evolved from investi­ gations concerning the total process to experimentation on each of the chemical steps. This study deals with attempts to uncover the relationship between nucleic

acids and protein synthesis and with observations on the intermediate reactions in this biosynthesis.

By utilizing sedimented cells from log-phase

Azotobacter vlnelandil cultures, the characteristics of the incorporation of radioactive glycine into the ribo­ nucleic acid, deoxyribonucleic acid and protein of these

cells were investigated. It was found that the deoxy­ ribonucleic acid, which comprises about 7 percent of the weight of the cell, becomes the most radioactive on a

specific activity basis. In addition, the specific

activities of all three macromolecules appeared to

remain constant for about three minutes after the

introduction of glycine-C1^. This cessation of incor­

poration was not observed when chloramphenicol was present

in the incubation medium. A second effect of chloram­

phenicol on the metabolism of these cells concerns its

ability to inhibit differentially the biosynthesis of

proteins and nucleic acids. Other inhibitors of one

68 69 of these processes, such as penicillin, purine, 8-azaxan- thin§, B-thlenylalanine, allylglycine, or rubidium, cobalt and uranyl ions, do not produce this differential

inhibition. Prom the experiments performed, it appears that chloramphenicol does not cause the synthesis of

abnormal ribonucleic acid, but that some initial bio­

synthetic process is inhibited.

A. vinelandil cells were fragmented by sonic

oscillation and centrifugal fractions obtained. When

the specific activities of ribonucleic acid, deoxyribo­

nucleic acid, and protein in these sedimentable fractions

and in the supernatant solution from the high speed

centrifugation were determined on a time basis, it was

found that the initial incorporation of label appears

in the supernatant solution. This solution contains a

majority of the amino acid activating activity, and in

studies on cell-free incorporating systems it was found

that a heavy particulate fraction obtained after sonic

oscillation required the presence of this solution.

In contrast, a cell-free amino acid incorporating system,

which was inhibited by the supernatant solution, was

prepared by the osmotic rupture of these bacterial cells.

Incorporation of glycine-C1^ into this preparation was

enhanced by hexose diphosphate or by an adenosine tri-

phosphate-magnesium mixture. Both of these particulate 70 systems catalyze the Incorporation or glycine-C^ into ribonucleic and deoxyribonucleic acids*

Because definite proof for the occurrence of an

amino acid-adenosine monophosphate intermediate in

amino acid activation had not been reported, the enzymatic mechanism of this reaction was studied. When tryptophan,

labeled in its carboxyl group with oxygen-l8, was incu­

bated in the presence of adenosine triphosphate, a

purified tryptophan activating enzyme and hydroxylamine,

it was found that the oxygen-18 was transferred to the

terminal phosphate of adenosine monophosphate. In order

to determine whether thi s mechanism is true for amino

acid activaticn in general, similar experiments were

performed with alanine-01® and an alanine activating

enzyme, and with an oxygen-18 labeled, casein hydrolysate

and the activating enzymes from the A. vlnelandii super­

natant solution. In both cases, carboxyl oxygen-i8 was

transferred to adenosine monophosphate, indicating the

formation of an amino acid-adenosine monophosphate

intermediate during the activation on all amino acids.

Investigation was also Initiated in an attempt to

characterize the soluble cofactor involved in protein

synthesis. This cofactor appears to be the acceptor

for activated amino acids, since hydroxylamine, which

attacks high energy carboxyl bonds, when incubated in the presence of a solution containing the amino acid- factor, causes the formation of amino acid hydroxamates.

A series of experiments was performed to characterize the chemical and physical properties of this amino acid- factor complex. BIBLIOGRAPHY

1. Alexander, M., Bact. Revs. 20, 67 (1956).

2. Askonas, B. A., P. N* Campbell and T. S. Work, Biochem. J. 58, 326 (1951+).

3. Berg. P., J. Biol. Chera. 222, 1025 (1956a).

Berg. P., J. Biol. Chem. 222, 991 (1958b).

5. Berg. P., Fed. Proc. 16, 152 (1957). 6. Block, K., J. Biol. Chem. 179, 121+5 (191+9). 7. Borek, E., and A. Ryan, J. Bact. 75, 72 (1958). 8. Borsook, H., J. Cell, and Comp, Physiol. 1+7 Suppl., 35 (1956).

9. Boyer, P. D., 0. J. Koeppe and W. W. Luchsinger, J. Am. Chem. Soc. 78, 356 (1956). 1 0 . Brachet, J., Cold Spring Harbor Symposia of Quant. Biol. 12, l8 (191+7). n. Brachet, J., Biochim, at Biophys. Acta 18, 21+7 (1955). 12. Breinl, F., and F. Haurowitz, Hoppe-Seyler1s Zeit, Physiol. Chem. 192, 1+5 (1930).

13. Burk, E., and H. Lineweaver, J. Bact. 19, 389 (1930). 111-. Burma, D. P., and R. H. Burris, J. Biol. Chem. 225, 723 (1957).

15. Caspersson, T., and K. Brandt, Protoplasma 35, 507 (I9t+1).

16. Cohn, M., J. Biol. Chem. 201, 735 (1953).

17. Crestfield, A. M., K. C. Smith and F. W. Allen J. Biol. Chem. 216, 185 (1955). 72 73

18. Dalgliesh, C. E., Science 125, 271 (1957).

19. Davie, E. W., V. V. Konningsberger and F. LIpmann, Arch. Biochem. Biophys. 65» 21 (1956).

20. DeMoss, J. A., and 0. D. Novell!, Biochiru* et Biophys. Acta 18, 592 (1955)*

21. Dounce, A., Enzymologia 15, 25l (1952).

22. Gale, E. F., and T. F. Paine, Biochem. J. l*-8, 298 (1951).

33. Gale, E. F., and J. P. Folkes, Biochem. J. 53, 1*93 (1953).

21*.. Gale, E. F. and J. P. Folkea, Nature 173,1223 (1951*-). 25. Gamow, G., Kgl. Dansk, Urdenskab. Selokab., Biol. Medd. 22, No. 8 (1955).

26. Grunberg-Manago, M., P. J. Ortiz and S. Ochoa, Biochim. et Biophys. Acta 20, 269 (1956).

27. Haurowitz, F., The Chemistry and Biology of Proteins. Academic Press, Inc., New York, (1950).

28. Hahn, F. E., C. L. Wisseman and H. E. Hopps, J. Bact. 69, 215 (1955).

29. Hahn, F. E., M. Schaechter, W. S. Ceglowski, H. E. Hopps and J. Ciak, Blochim, et Biophys. Acta 26, 1*69 (1957).

30. Heppel, L. A., and R. J. Hilmoe, J. Biol. Chem. 192, 87 (195D.

31. Hoagland, M. B., Biochim. et Biophys. Acta 16, 288 (1955).

32. Hoagland, M. B., E. B. Keller and P. C. Zamecnik, J. Biol. Chem. 218, 3kS (1956).

33. Hoagland, M. B., P. C. Zamecnik and M. L. Stephen­ son, Biochim, et Biophys. Acta 21*., 2l5 (1957a). 714.

34* Hoagland, M. B., P. C. Zamecnik, N. Sharon, F. Lipmann, M. B. Stulberg and P. D. Boyer, Biochim, et Biophys. Acta 26, 215 (1957b).

35. Holley, R. W., J. Am. Chem. Soc. 79, 658 (1957).

36. James, T. ¥., Biochim, et Biophys. Acta 15, 367 (1954).

37. Jencks, W. P., Biochim. et Biophys. Acta 24, 227 (195 7).

38. Kamin, H., and P. Handler, Ann. Rev. of Biochem. 26, 464 (1957).

39# Keck, K., Arch. Biochem. Biophys. 63, 446 (1956).

40. Konningsberger, V. V,, C. 0. Van der Grinten and J. T. G. Overbeek, Biochim. et Biophys. Acta 26, 483 (1957).

4JL. Kornberg, A., J. R. Lehman, M. J. Bessman and E. S. Simms, Biochim. et Biophys. Acta 21. 197 (1956).

42. Kowalsky, A., C. Wyttenbach, L. Langer and D. E. Koshland, Jr., J. Biol. Chem. 219, 719 (1956).

43. Lepage, G. A., in "Manometric Techniques and Tissue Metabolism”, (Umbreit, W., R. H. Burris and J. Stauffer, Eds.) p. 1 8 5, Burgess Publishing Co., Minneapolis, (1949).

44* Levy, H. B., E. T. Skutch and A. L. Schade, Arch. Biochem. 2 4, 189 (1949).

45 * Lipmann, F., and L. C. Tuttle, J. Biol, Chem. 159, 21 (1945a).

46. Lipmann, P., J. Biol. Chem. 160, 173 (19456).

47. Lipmann, P., in "The Mechanism of Enzyme Action", (McElroy, N. D., and H. B. Glass, Eds.) p. 599, Johns Hopkins Press, Baltimore, (1954).

48. Marr, A, G., and E. H. Cota-Robles, J. Bact. 74, 79 (1957).

49. Mazia, D., and D. M. Prescott, Biochim, et Biophys. Acta 17, 23 (1955). 75

50. McQuillen, K., Biochim. et Biophys. Acta 17* 362 (1955).

51. Mitz, M. A., and R. J. Schuter, Biochim. et Biophys. Acta 27* 168 (1958).

52. Neidhardt, P. C., and F. Gros, Biochim. et Biophys. Acta 25* 513 (1957).

53* Novell!, G. D., and J. A. DeMoss, J. Cell. Comp. Physiol, in press (1958).

5^4-* Ogata, K., and H. Nohara, Biochim. et Biophys. Acta 25, 659 (1957).

55* Ogur, M., and G. Rosen, Arch. Biochem. 25* 262 (1950).

56. Pardee, A. B., Proc. Natl. Acad. Sci., U. S. I4O, 263 (1954-).

1|7. Pardee, A. B., and L. S. Prestidge, J. Bact. 27, 677 (1956).

5 8. Prescott, D. M., Exptl. Cell Res. 12, 196 (1957).

59. Rabinowitz, M., and M. E. Olson, Exptl. Cell Res. 10, 7147 (1956).

60. Robrish, S. A., and A. G. Marr, Bact. Proc. 10, 130 (1957 ).

61. Scarano, E., and R. Maggio, Exptl. Cell Res. 12, 1403 (1957).

62. Schachman, H. K*, A. B. Pardee and R. Y. Stanier, Arch* Biochem. Biophys. 3 8, 2I4.5 (1952).

63. Schmidt, G., and S. J. Thannhauser, J. Biol. Chem. 1 6 1, 83 (191+5) -

6I4.. Schweet, R. S., R. W. Holley and E. H. Allen, Arch. Biochem. Biophys. 71* 311 (1957).

65* Simpson, M. V., and S. F. Velick, J. Biol. Chem, 2 0 8, 61 (19514).

6 6. Spiegelman, S., in "The Chemical Basis of Heredity", (McElroy, W. D., and H. B. Glass, Eds.) p. 232, Johns Hopkins Press, Baltimore, (1957). 76

67* Steinberg, D., and C. B, Anfinsen, J. Biol. Chem. 199, 25 (1952).

68. Ullman, A., and F. B. Straub, Acta Physiol. Acad. Scl., Hung., 6, 377 (1954).

69. Wagle, S. R., R. Mehta and B. C. Johnson, Arch. Biochem. Biophys. 72, 241 (1957).

70. Warburg, 0., and W. Christian, Biochem. Zelt* 310, 3 8 4 (194L).

71. Webster, G. C., and M. P. Johnson, J. Biol. Chem. 2 1 7 , 6ipL (1955).

72. Webster, G. C., J. Biol. Chem. 2 2 9 , 535 (1957).

73. Wisseman, C. L., J. E. Smadel, F. E. Hahn and H. E. Hopps, J. Bact. 67, 662 (1954).

74* Yeas, M,, and G. Brawerman, Arch. Biochem. Biophys. 68, 118 (1957).

75* Zamecnik, P. C., and E. B, Keller, J. Biol. Chem. 2 0 9 , 337 (1954). AUTOBIOGRAPHY

I, Robert William Bernlohr, was born in Bexley,

Ohio, April 20, 1933. I received my secondary school education in the public schools of Bexley, Ohio, and my undergraduate training at Capital University, which

granted me the Bachelor of Science degree in 1955* In

June, 1955* I enrolled in the Graduate School of The

Ohio State University, where I specialized in the

Department of Agricultural Biochemistry. While there,

I was a teaching assistant until 195h, a Charles F.

Kettering Fellow during 1956** 1957# and a Public Health

Service Fellow from 1957 to 1958* During the final

two years, I was completing the requirements for the

degree Doctor of Philosophy.

77