A STOW OF OXIDATIVE PHOSPHORYLATION 11 PSEUDOMONAS AERUGINOSA by GEORGE A. STRASDINE B.A., University of Br i t i s h Columbia, 1956. M.Sc. University of British Columbia, 1958. A THESIS. SUBMITTED IN PARTIAL- FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Ph . B . i n the Department of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1961. In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for t financial gain shall not be alloxved without my written permission. Department of »nsi^c The University of British Columbia, Vancouver 8, Canada. I Wc\t PnfajBrsttg of ^rrttsh (Ealumbra FACULTY OF GRADUATE STUDIES PROGRAMME OF T H E F I N A L O R A L E X A M I N A T I O N FOR THE DEGREE OF D O C T O R O F P H I L O S O P H Y of GEORGE A. STRASDBNE B.A., University of British Columbia, 1956 M.A., University of British Columbia, 1958 THURSDAY, SEPTEMBER 28th, 1961 AT 1:30 P.M. IN ROOM 231, BLOCK A, MEDICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: F. H. SOWARD J. J. R. CAMPBELL A. R. P. PATERSON B. A. EAGLES J. J. STOCK G. W. MARQUIS G. M. TENER N. E. NEILSON S. H. ZBARSKY External Examiner: A. F. BRODIE . Harvard Medical School, Boston, Mass. T H E S T U D Y O F O X I D A T I V E P H O S P H O R Y L A T I O N I N PSEUDOMONAS AERUGINOSA ABSTRACT The earlier failure of cell free extracts of Pseudomonas aeruginosa to exhibit substrate-dependent oxidative phosphorylation led to an investigation of the conditions affecting the incorporation of radio-active phosphorus into resting cell suspensions of this organism. In-corporation of radioactive phosphorus was shown to be dependent on the substrate concentration, the presence of magnesium ions, a source of available nitrogen and to be associated with the oxidative enzymes of the cell. The more common methods of cell breakage employed for the preparation of bacterial cell free extracts were considered detrimental to the mechanisms of oxidative phosphorylation and were abandoned in favor of a method involving the osmotic lysis of spheroplasts with versene and lysozyme. These preparations were shown to be easily separated into membranes, cytoplasm and ribosomes by ultracentri-fugation and had the advantage of not having been subjected to severe physical treatments. Previous studies with cell free extracts had demonstrated the formation of ATP 3 2 in the presence of ADP and P 3 2 , presumably through a coupled oxidative phosphorylation process. The formation of ATP 3 2 was shown however to be the result of a coupled enzyme reaction involving polynucleotide phosphorylase and adenylate kinase (equations 1, 2 and 3), and although influenced by a concurrent oxidative phosphorylation process was itself not a measure of oxidative phosphorylation. nADP : — (AMP)n- + nP, (1) (AMP)n + nP •nADP 3 2 (2) 2 A D P 3 2 — , AMP + AMP < v P 3 2 ^ P 3 2 (3) The enzyme mediating the exchange reaction (equation 1) was shown to be polynucleotide phosphorylase and that at least in this organism this enzyme is associated with the ribosomal fraction of the cell. Oxidative phosphorylation was demonstrated in crude cell extracts prepared from succinate grown cultures by the lysozyme-versene treatment. Maximum P:0 ratios of 2.0 with succinate as substrate and 4.3 for^ o -ketoglutarate were obtained thus presenting further evidence for the similarity of this fundamental process in bacterial and animal tissue. GRADUATE STUDIES Field oi Study: Agricultural Microbiology Intermediary Metabolism J. J. R. Campbell G. I. Drummond A. R. P. Paterson S. H. Zbarsky Seminar J. J. R. Campbell S. H. Zbarsky Related Studies: Physical Chemistry R. F. Snider Advanced Organic Chemistry G. S. Dutton R. Stewart Topics in Organic Chemistry D. E. McGreer R. A. Bonnett J. P. Kutney Enzymology ,-. W. J. Polglase PUBLICATIONS 1. Phosphorous incorporation into cell fractions of Pseudomonas aeruginosa. G. A. Strasdine and J. J. R. Campbell, Abstracts of 10th annual meeting, Can. Soc. Microbial, 1960. 2. Substrate-dependent phosphorylation in resting cells of Pseudo-monas aeruginosa. G. A. Strasdine, J. J. R. Campbell, H. P. C. Hogenkamp and J. N. Campbell, Can. J. Microbiol. 7, 19, 1961. 3. A ribosomal polynucleotide phosphorylase in Pseudomonas aeruginosa. G. A. Strasdine, L. A. Hogg, and J. J. R. Campbell, Biochim et Biophys Acta, in press. 4. Localization of enzymes in Pseudomonas aeruginosa. J. J. R. Campbell, L. A. Hogg and G. A. Strasdine, in press. - i i -ABSTRACT The e a r l i e r f a i l u r e to demonstrate substrate-dependent oxidative phosphorylation i n c e i l free extracts of Pseudomonas aeruginosa l e d to an i n v e s t i g a t i o n of. the conditions a f f e c t i n g the incorporation of radio-active phosphorus i n t o r e s t i n g c e l l suspensions of t h i s organism. Incorporation of radioactive phosphorus was shown to be dependent on the substrate concentration, the presence of magnesium; ions, a source of avai l a b l e nitrogen and to be associated, with the oxidative enzymes of the c e l l . The more common, methods of c e l l breakage employed, f o r the preparation of b a c t e r i a l c e l l f r e e extracts were considered detrimental to the mecha-nisms of oxidative phosphorylation and were abandoned i n favor of a method i n v o l v i n g the osmotic l y s i s of spheroplasts with versene and l y s o -zyme. These preparations were shown to be e a s i l y separated i n t o membranes, cytoplasm, and ribosomes' by d i f f e r e n t i a l c e n t r i f u g a t i o n and had the advan-tage of not having been subjected to severe p h y s i c a l treatments. Previous studies with c e l l f r e e extracts had- demonstrated the forma-t i o n of ATP 3 2 i n the presence of ADP' and P 3 2, : presumably through a coupled oxidative phosphorylation process. The formation of ATP 3 2 w a S ! shown however to be the r e s u l t of a coupled enzyme r e a c t i o n i n v o l v i n g polynucleotide phosphorylase and adenylate kinase (equations; 1,. 2 and 3)» and although influenced by a concurrent oxidative phosphorylation process-was i t s e l f not a measure of oxidative phosphorylation. nADP •(AMP) n + nPi (1) (AMP>n + np32 » nADp32 (2) 2ADP 3 2 * AMP • AMP**P32^ P 3 2 (3)) - i i i -The enzyme mediating the exchange r e a c t i o n (equation, 1)) was shown to be polynucleotide phosphorylase and that at l e a s t i n t h i s organism this? enzyme i s associated with, the ribosomal f r a c t i o n of the c e l l . Oxidative phosphorylation was demonstrated i n crude c e l l extracts prepared from succinate-grown cultures by the lysozyme-versene treatment. Maximum P;::0 r a t i o s of 2.0 with succinate as substrate and k*3 f o r -keto-glutarate were obtained thus presenting further evidence f o r the s i m i -l a r i t y of t h i s fundamental process i n b a c t e r i a l and animal t i s s u e . - i v -TABLE OF COMTEN/rS INTRODUCTION HISTORICAL. REVIEW OF THE LITERATURE I. Phosphorus metabolism- - general 1. Permeability to' phosphate 2. I n t r a c e l l u l a r phosphate studies; 3 . The r o l e of metaphosphate h» Polynucleotide; phosphorylase' I I . Oxidative phosphorylation - general HE. Oxidative phosphorylation - b a c t e r i a l 22 MATERIALS AND) METHODS' 32 I . The organism and i t s c u l t i v a t i o n 32. II;. A n a l y t i c a l determinations 32 1. Nucleic acids 32 ' 2 . P r o t e i n 32 3. Ammonia 32 l i . Inorganic phosphate 33 5. E s t e r i f i e d phosphate (P 3 2> 33; I I I . I n i t i a l studies with c e l l f r e e extracts; 3k IV. Studies with r e s t i n g - c e l l suspensions: 36 V. F r a c t i o n a t i o n of whole ce l l s ; 31 yy VI. Column chromatography of hydrolyzed RNA 31 Page 1 2 2 2 ? 9 10-12 - V -V I I . Preparation of c e l l f r e e extracts 38 1. Hughes r press 38 2. Sonic o s c i l l a t i o n . 38 3 . Lysozyme-versene 3& V I I I . Column chromatography of rad i o a c t i v e phosphate esters 39 IX. F r a c t i o n a t i o n of c e l l extracts 111 1. Treatment with protamine sulphate h i (a) Protamine sulphate s o l u t i o n Ul . (b) P r e c i p i t a t i o n of n u c l e i c a c i d Ijl 2. Preparation of pH 5.0j enzyme f r a c t i o n I4I 3 . F r a c t i o n a t i o n with ammonium, sulphate hi k* F r a c t i o n a t i o n by u l t r a c e n t r i f u g a t i o n h2 X. Enzyme assays k2 1. Adenylate kinase (myokinase) 1*2 2. I s o c i t r i c dehydrogenase It2 Xi;. Synthesis and c h a r a c t e r i z a t i o n of polyadenylic a c i d 1*3 1. Synthesis: 1*3 2. Characterization k3 RESULTS AM); DISCUSSION! '' k5 I . I n i t i a l studies with c e l l f r e e extracts h$; I I . Studies with r e s t i n g - c e l l suspensions; 1+6' 1. Reduction of the endogenous r e s p i r a t i o n I4.6 2. Substrate-dependant phosphorylation kit 3 . The e f f e c t of substrate concentration l$< k. P'-^ incorporation as net phosphate uptake: 51* I n t r a c e l l u l a r l o c a t i o n of phosphate $h - v i -6. E f f e c t of metabolic i n h i b i t o r s : 5? 7'V Incorporation with nitrogenous substrates-, 59 I I I . A p p l i c a t i o n of data from whole c e l l studies to c e l l extracts 59 1. E f f e c t of substrate concentration $9 17. The preparation of c e l l f r e e e x t r a c t s 6l 1. Hughes" press 61 2. Sonic o s c i l l a t i o n 62 3 . Osmotic l y s i s of spheroplasts 63 7. Oxygen uptake studies with 1 c e l l fractions- 65 VI. Radioactive phosphate studies with c e l l extracts 66 1. Incorporation i n t o c e l l fractions. 66 2. The e f f e c t of omitting various components 69 3 . E f f e c t of magnesium 70; 111!.. E f f e c t of ADP concentration 70 5. Compounds incorporating F 3 2 ?2 VTr. Studies on the exchange of inorganic and organic phosphate 7k 1. Standard r e a c t i o n conditions Ik 2. P 3 2 i n c o r p o r a t i o n i n t o c e l l f r a c t i o n s 75 3 . ATF or ADP' as acceptor f o r exchange a c t i v i t y ? 7/6 V l i r . The p a r t i c u l a t e nature of polynucleotide phosphorylase77 1. Ammonium sulphate f r a c t i o n a t i o n 77 2. ADP' and inorganic phosphate concentration 78 3 . F r a c t i o n a t i o n by u l t r a c e n t r i f u g a t i o n 79 U. S o l u b l i z a t i o n of the ADP-P 3 2 exchange a c t i v i t y 81 - v i i -5>. Treatment of the particulate fraction with RNA*se and adenylate kinase 81 6 . Exchange activity with various nucleoside diphosphates 82 7 . Synthesis of polyadenylic acid 8.3 V I I l . Effect of the ADP-P32 exchange on oxidative . phosphorylation 85>' 1 . Effect of ATP on the ADP-P32 exchange 8£ 2. Effect of cyanide 86 IX. Studies on oxidative phosphorylation 87 . 1 . Effect of vitamin K x 87 2 . Factors lowering the PsO ratios 8° (a) Fructose-6-phosphatase activity 89 (b) Adenosine triphosphatase: activity 90) GENERAL DISCUSSION! 93 I. Whole c e l l studies 93 I I . Preparation of c e l l extracts; 95 I I I . Radioactive phosphorus incorporation studies 96 17/. Oxidative phosphorylation studies' 98> SUMMARY.. 100 BIBLIOGRAPHY. 102 - v i i i -FIGURES 1. Incorporation of radioactive phosphorus i n t o charcoal adsorb able material of a c e l l extract 2. Phosphorus incorporation i n t o i n t a c t c e l l s as a f u n c t i o n of glucose concentration 3. Phosphorus incorporation i n t o i n t a c t c e l l s with various substrates k* Stimulation of phosphorus in c o r p o r a t i o n by NH^Gl 5. Phosphorus incorporation i n t o r e s t i n g c e l l s (A) Endogenous (B) With glucose 6. Oxygen and phosphorus uptake with aspartate and. asparagine 7 . Oxygen uptake with c e l l fractions^ (A) C e l l f r e e extract (B) Membrane f r a c t i o n (C) Cytoplasm- f r a c t i o n (D) Membranes plus cytoplasm 8'. E f f e c t of the r a t i o of ADP' to inorganic phosphate on the ADP-P;p exchange 9. Orthophosphate l i b e r a t i o n during polynucleotide synthesis from ADP - i x -AGKNCWLEDGEMENT I wish to express my sincere appreciation to Dr. J . J . R . Campbell f o r h i s d i r e c t i o n and c r i t i c i s m throughout t h i s i n v e s t i g a t i o n . INTRODUCTION Iii undertaking this study on the phosphate metabolism of Fseudomonas aeruginosa i t was hoped that a contribution might be made to our knowledge of the mechanism or mechanisms involved in the generation of phosphate bond energy. More specifically the studies were directed towards increas;-ing our understanding of the process of oxidative phosphorylation as; It occurs in microorganisms. The limited information in the literature does not allow generalizations to be drawn and so i t is not possible to conclude whether or not bacteria are similar to animals in respect to the details of this fundamental life process;. The problem has been divided into two partsj; the first is considered prerequisite to the second and is concerned with the conditions affecting incorporation of radioactive phosphorus into whole cells;. It was through this initial study that evidence was obtained in support of the concept that oxidative phosphorylation is an inherent mechanism in this organism. The second part of the study deals with the process per se, including its cytological location in the cell and the various conditions necessary for its demonstration. This part of the work is based on studies performed with cell free extracts and cell fractions. In addition, studies are re-ported which, although not dealing directly with the topic of oxidative phosphorylation, were found necessary to the understanding of the problem of phosphorylation in this organism. These include a modified method of cell breakage, as well as a comprehensive study of enzyme systems which were found to interfere with the demonstration of oxidative phosphorylation. - 2 -HIS TORI GAL WHIM OF THE LITERATURE The historical review of the literature i s designed to follow as closely as possible the general theme of the experimental work. Thus, a brief history of phosphorus metabolismi w i l l be followed by a summary of our present knowledge concerning oxidative phosphorylation. The lat t e r review w i l l of course be predominantly a presentation of data derived from studies using animal tissues since the great majority of the work has; been performed with such tissues. The remainder,, and most intensive part of this section w i l l deal with the current status of our' knowledge of oxi-dative phosphorylation as i t occurs i n bacteria. Phosphorus metabolism - general, 1 . Permeability to" phosphate Phosphate ions have occupied a special position among inorganic ions i n studies; concerning active transport across c e l l membranes because of their extensive participation i n cellular metabolism. In 1 9 U 8 Kamen and Spiegelman (l)i discussed two possible schemes of entry of phosphate into c e l l s and into the metabolic pools of these c e l l s . The f i r s t involved a simple diffusion of inorganic phosphate through the c e l l membrane' followed by esterification within the c e l l . The second involved the esterification of inorganic phosphate i n the membrane. Their evidence was i n favor of the lat t e r scheme, and may be summarized as f o l -lows;: (a) The temperature coefficient was too large to be explained on the basis of simple diffusion, (b) The uptake of inorganic phosphate was inhibitedi by such agents as azide, arsenate and iodoacetate which block phosphate esterification. (c) The rate of exchange increased i n the presence of substrate. The approaches to the e l u c i d a t i o n of t h i s problem, have v a r i e d con-siderably.; The use of radioactive phosphorus has helped to a large extent; however i n t e r p r e t a t i o n of these studies proved, to be very complex since separate phosphate pools are e a s i l y combined during c e l l breakage. The most frequently employed methods are those i n v o l v i n g measurement of the rate of inorganic phosphate uptake from the external medium. For example, i t was shown (2)) with Staphylococcus aureus that net uptake occur-red only with glucose and i n some experiments a l l of the measurable phos-phate was absorbed from, the medium. Thus the movement of inorganic phos-phate was against a concentration gradient. Since gramicidin, i n h i b i t e d t h i s uptake and because t h i s compound was thought to act' on the c e l l , sur-face, i t was suggested that the transport mechanism f o r phosphate was' i n the c e l l membrane. In 19501 Roberts and Roberts (3) explained the exchange of inorganic phosphate between r e s t i n g c e l l s and t h e i r suspending medium by a phenomenon they r e f e r r e d t o as exchange adsorption. The exchange w'as confined to that f r a c t i o n of c e l l u l a r phosphate which was extractable with c o l d t r i c h l o r o -a c e t i c a c i d (TGA). They proposed that t h i s inorganic phosphate was derived not from f r e e inorganic phosphate, but from a l a b i l e adsorption; .complex which remained at a constant concentration i n r e s t i n g c e l l s , because the number of adsorption s i t e s remained constant.. I t was proposed*that each s i t e could r e a d i l y exchange one phosphate f o r another, hence the term, exchange adsorption. These workers b e l i e v e d that the membrane of the organ-ism (Bacterium, c b l i ) was f r e e l y permeable to small molecular weight solutes i n general and that the int e r n a l , "free 1* solutes were held i n the: c e l l as l a b i l e compounds with components of high molecular weight (J+|. . M i t c h e l l and Moyle (£)), also working with E s c h e r i c h i a c o l i , noted a s i m i l a r exchange of inorganic phosphate. They also observed that the r a t e of t h i s exchange was diminished when r e s p i r a t i o n was i n h i b i t e d . Respira-t i o n i n the presence of glucose demonstrated that the inward flow of phos-phate remained the same as during r e s t , but the outward flow f e l l to zero so that phosphate was a c t u a l l y transported inwards. These and other f a c t s l e d to the conclusion that the same transport mechanisms might operate i n the exchange and uptake reactions. E a r l i e r , in. 19k7, Ussing (6) described a one-to-one exchange: of sodium ions across the muscle membrane that could be accounted f o r by an exchange d i f f u s i o n hypothesis according to which the ions: saturate a c a r r i e r on which they pass- through the membrane which would be impermeable to them i n the f r e e s t a t e . The observation i n M i t c h e l l ' s work supported such an exchange d i f f u s i o n system, and, at the same time, contradicted an exchange adsorption hypothesis i n r e s t i n g c e l l suspensions. Some of these points weres (a) Permeability measurements demonstrated the existence of an osmotic b a r r i e r near the surface of the c e l l . (b) Osmotic pressure measurements on i n t a c t c e l l s and protoplasts demon-st r a t e d that the greater part of the small molecular weight solutes, which make up some 20' per cent o f the c e l l ' s dry weight, are: f r e e b d i f f u s e i n the water enclosed by the c e l l membrane and develop the expected osmotic pressure of some 20 to 30 atmospheres. From the known amounts of i n t e r n a l inorganic phosphate and water i t could be computed that the concentration of i n t e r n a l inorganic phosphate i n S'« aureus was about 0.1M. (c) The exchange of inorganic phosphate was susceptible: to the action of inhibitors which also inhibited active transport. It should be mentioned that a considerable portion of the data ob-tained in the studies by Mitchell and Moyle {$) and by Mitchell (?) was obtained with the use of radioactive phosphate. In a recent review, Mitchell ( 8 ) discussed his postulated mechanisms for phosphate exchange. Throughout this review he attempted to treat the: problem of active transport in terms of well known biochemical principles. Very briefly, a molecular group termed the "translocator" becomes covalently linked to the phosphate during translocation. The high specificity of this translocator for phosphate suggests that the translocator region has the properties of a group-transferring enzyme. All of the early work reported by Mitchell and Moyle was carried out with 5". aureus. However the systems which were discussed above were not unique to this organism and these workers have obtained similar data with E. coll (9). Goodman and Rothstein (10) demonstrated, on the other hand, that the resting yeast cell was impermeable to inorganic phosphate. The active transport system was found to be similar to that in S:* aureus, but an ex-change diffusion could not be shown. This was evident from the following observations. (a) A specific substrate, glucose, was required. (b) Inorganic phosphate moved into the cell against a concentration gradient. (c) The inward flow of inorganic phosphate was not accompanied by a -6-release of inorganic phosphate from the c e l l (the s p e c i f i c . a c t i v i t y of the e x t r a c e l l u l a r inorganic phosphate therefore remained constant). (d) The k i n e t i c s of uptake of inorganic phosphate obeyed the Michaelis-Menton equation.. (e) I n h i b i t o r s of phosphorylation reactions such as 2,U-dinitro-phenol (MP), azide and arsenate acted as blocking agents. Considerable data on the e f f e c t s exerted by other ions on phosphate uptake by microorganisms have also been obtained. Thus, potassium has been shown to markedly stimulate, phosphate uptake by yeast (11, 12) , by E. c o l i (3) and by Bacterium, l a c t i s aerogenes (13) • In yeast (10) t h i s a c t i v i t y i s l a r g e l y dependant on pH. The uptake of inorganic phosphate, with or without potassium, i s r e s t r i c t e d at high pU values because of reduced metabolism. However, at intermediate pH values the stimulation by potassium i s considerable although i n d i r e c t since t h i s i o n does not have to be present at the time that the phosphorus i s being taken up. Thus, i f the c e l l s are f i r s t exposed to potassium and glucose, the potas-sium i s r a p i d l y absorbed. The potassium-rich c e l l s w i l l subsequently absorb considerably increased amounts of inorganic phosphate. Goodman et a l . have shown that the problem i s one of t o t a l e l e c t r o l y t e and a c i d -base balance. As H2P0jij~ moves i n t o the c e l l the equilibrium', H + + HP0^= s HgBOfc" . i n the medium, i s s h i f t e d to the r i g h t and the medium' becomes more a l k a l i n e . At the same time the rea c t i o n within, the c e l l , i s s h i f t e d to the l e f t with an increase in 1 a c i d i t y . A potassium:-rich c e l l has however a more a l k a l i n e cytoplasmic p-H. and a large amount of f i x e d base. This c e l l can therefore -7-absorb much more phosphate than a potassium-poor cell without upsetting the acid-base balance^ . 2. Intracellular phosphate studies. The literature cited below will deal principally with studies carried out with resting cell suspensions since i t is with such suspensions that our own experiments were mainly performed. In 191+6 Taylor (lli)) studied the amount and distribution of phosphorus, in the B strain of E. coli and demonstrated that phosphate comprised about 2.72 per cent of the dry weight of organisms from an & hour broth culture. Of this amount 66 per cent was in the form of ribonucleic acid (RNA)^ and 19 per cent as deoxyribonucleic acid (DNA). Of the remaining 1$ per cent, 12 per cent occurred in phospholipid and 3 per cent in unidentified forms;. As a comparison, Roberts et al. (lit) gave a value of 3.2 per cent of the dry weight of E. coli as being phosphorus, 68 per cent of which was nucleic acid phosphate. In 19hk Hotchkiss (l£) reported that inorganic phosphate was; accumu-lated by staphylococcus cells while oxidizing glucose and that the amount taken up was directly related to the amount of oxidation, regardless of the external phosphate concentration. 1 The following abbreviations are used in this presentation: TCA—trichloroacetic acid; DNP'—2,U-dinitrophenolj G-6-P—glucose-6-phosphatej F;-6-P--fructose-6-phosphate; RNA—ribonucleic acid; DNA—desoxyribonucleic acid;, AMP--adenosine monophosphate; ADP--adenosine diphosphate; ATPS—adenosine triphosphate; DPN>—diphosphopyridine nucleotide DPNB--diphosphopyridine nucleotide (reduced form); ATP'ase—adenosine triphosphatase; FAD;—flavin adenine dinucleotide; FMN—flavin mononucleotide; RNase:>—ribonuclease; DNase-#: --desoxyribonuclease; Tris—tris: (hydroxymethyl)aminome thane; TUP—dnosihe diphosphate; UDP!—uridine diphosphate; GDP'—guanosine diphosphate; GDE—cytidine diphosphate;: TPN—triphosphopyridine nucleotide; TPNH—triphosphopyridine nucleotide (reduced form)>$ P^ 2—P 3^ - 8 -Phosphate did not accumulate, however, in the presence of BMP' or gramicidin. Hbtchkiss later ( 1 6 ) exposed resting cell suspensions to radioactive inorganic phosphate under various conditions and analyzed the various cell fractions for labelled phosphate content. He found that the external radioactive phosphate penetrated the cell almost exclusively by metabolic reactions. At 0°G there was very little uptake of label into the cells. At 37°G, in the absence of glucose, there was no net uptake of radioactive phosphorus but rather an equilibrium with the external medium which was complete in 90 minutes. In contrast, in the presence of glucose at 37°C, a net uptake and labelling of all the cell phosphate fractions occurred. The inorganic phosphate fraction acquired a high percentage of labelling with or without glucose, but in the presence of glucose the label passed to a much greater extent to organic fractions of the cell. The most pronounced effect was'}into the nueleoprotein fraction which increased and. received one-fifth of the radioactive phosphorus taken up. These findings were extended ( 1 6 ) by studying the effect of amino acids on radioactive phosphate incorporation. Amino acid utilization was accompanied by a marked decrease in phosphate uptake, the greater difference again occurring in the nueleoprotein fraction. The greater the complement of amino acids added, the greater the reduction in incorporation. The experiments sugges-ted that for every two amino nitrogen atoms combining in peptide linkage there was either one less phosphorus atoms esterified or one ester phosphate returned to inorganic form. A study of metabolic poisons revealed the close dependent nature of both amino acid and phosphorus incorporation on the energy made available by oxidation.. Most interesting, was: DEE which stimu-lated oxygen uptake to 1 3 1 per cent of normal, while inhibiting amino acid uptake by 90 per cent and completely stopping the incorporation of radio-- 9 -active phosphate. Am explanation f o r the above r e s u l t s was proposed on the basis of the amino acid-catalyzed exchange between pyrophosphate and adenosine t r i p h o s -phate (ATP)). The b a c t e r i a l systems that have been shown to possess t h i s a c t i v i t y e x h i b i t an increased a c t i v i t y when any one of a v a r i e t y of amino acids i s present (17). By t h i s mechanism amino acids would be.acylated to form adenyl-amino acids at. the expense of ATP. This lowering of the concen-t r a t i o n of ATP would consequently lower the accumulation of the acidi-insol-uble phosphate to the extent that the amino acids are coupled. M a l l i n and Kaplan (18) studied the incorporation! of radioactive phos-phorus i n t o r e s t i n g c e l l suspensions of the anaerobic organism, Clostridium perfringens. Although f r a c t i o n a t i o n of the. c e l l components was not under-taken, i t was shown that under aerobic conditions these organisms incorpor-ated radioactive phosphorus only when an oxidizable substrate,"ethanol, was present and, that t h i s substrate^dependent phosphorylation was i n h i b i t e d by gramicidin and by DNE. Oxygen uptake was not affected by these i n h i b i t o r s , when ethanol was the substrate. The importance of the substrate being oxidized was demonstrated when i t was observed that with glucose, under aerobic conditions, DNP enhanced phosphate inc o r p o r a t i o n and gramicidin . abolished i t . S i m i l a r r e s u l t s were obtained with another anaerobe; Clostridium k l u y v e r i . ' The e f f e c t s observed with DNP and gramicidin were also exhibited under anaerobic conditions. The authors state that i t i s p o s s i b l e that the r e s u l t s obtained with oxidation of ethanol may be l i n k e d to the oxidation of reduced diphosphopyridine nucleotide (DPNH). 3. The r o l e of metaphosphate The a b i l i t y of a number of microorganisms to store phosphorus i n the form of polymetaphosphates, and the i m p l i c a t i o n t h a t these compounds might -ID-serve as energy sources, has l e d a number of workers to f o l l o w the formation and h y d r o l y s i s of these compounds i n whole c e l l s and c e l l f r e e e x t r a c t s . Kornberg et a l . (19) i n 1956 prepared an enzyme from E. c o l i which could synthesize polymetaphosphate from. ATPj an u n i d e n t i f i e d primer was required to i n i t i a t e the r e a c t i o n * Later Kornberg demonstrated (20) t h a t the enzyme also catalyzed the reverse reaction, i . e . the synthesis of ATE from polymetaphosphate and adenosine diphosphate (ABE'). In the same year a s i m i l a r enzyme was found i n Corynebacteriumi dlphtheriae (20)). The enzymes responsible f o r the formation and hydrolysis of the poly-metaphosphates have been found to vary considerably i n t h e i r mechanism of act i o n . Thus, Kornberg"s enzyme (19)) may be described as a transferase i n t r a n s f e r r i n g phosphate from polymetaphosphate to ADP and form; ATE., An enzyme from Gorynebacterium- xerosis was i s o l a t e d by Muhammed et a l . (21) which apparently attacks only long-chain metaphosphates. The s i g n i f i c a n c e of these enzymes i s s t i l l rather vague and two b a s i c views e x i s t at the present time. A Russian group, Belozersky et a l . (22), regard the metaphosphates as energy stores which are capable of phosphory-l a t i n g other compounds without the i n t e r v e n t i o n of ATE or other nucleoside: triphosphates. ; The alternate suggestion (and the one most widely, accepted by workers i n t h i s f i e l d ) i s that these compounds act simply as a source of phosphate. This l a t t e r view also f i t s rather w e l l with the experimental f i n d i n g s that polymetaphosphates accumulate when c e l l s are given ample phosphate and energy supplies but the accumulation i s retarded by nitrogen s t a r v a t i o n (23). k» Folynucleotide phosphorylase. A discussion of the enzyme, polynucleotide phosphorylase, may seem -11-unwarranted i n a survey of systems which may be implicated in. b a c t e r i a l oxidative phosphorylation. The enzyme has however played a considerable r o l e i n the i n t e r p r e t a t i o n of data i n our own studies and may i n f a c t e x p lain some of the anomalous data already appearing i n the l i t e r a t u r e . For t h i s reason the enzyme i t s e l f and a number of i t s c h a r a c t e r i s t i c s w i l l be b r i e f l y reviewed. Polynucleotide phosphorylase, was discovered by Grurtberg-Manago and Ochoa i n extracts: of Azotobacter a g i l e (2h, 2 5 ) . The r e a c t i o n catalyzed by t h i s enzyme may be formulated as f o l l o w s : n(nucleoside-pp) *^ (nucIeoside-p) n + n(p) where nucleoside-pp and p represent nucleoside-^ 1 1-diphosphate and inorganic phosphate r e s p e c t i v e l y . The polymers that are formed i n t h i s reaction (nucleoside-p) n have a l l the s t r u c t u r a l features of i s o l a t e d RNA and are attacked i n a s i m i l a r way by h y d r o l y t i c enzymes. The r e a c t i o n i t s e l f may be followed i n various ways, thus one can measure the release of inorganic phosphate from ADP (26))j the formation of acid-insoluble polymer i n the forward d i r e c t i o n or, i n the reverse d i r e c t i o n , one can assay the rate of phosphorolysis of such polymers as RNA or adenylate; polynucleotide (2?) . T h i r d l y , and of more importance to oxidative phosphory-l a t i o n studies, one can measure the rate of exchange between r a d i o a c t i v e phosphorus and ADP or other nucleoside diphosphates (25). Q u a n t i t a t i v e l y however the three assays, under optimum; conditions,, do not y i e l d the same results,. Thus, as shown i n a recent a r t i c l e by Heppel et a l . (28), the micromoles (/umoles) per milligram; (mg) of protein; per hour f o r the three assays were as follows:: inorganic phosphorus from; ADP, 1000; phosphorolysis of polyadenylic a c i d , k$i', and ADP-P 3 2 exchange,, 100!. -12-Brummondl et a l . ( 2 9 ) confirmed, the wide d i s t r i b u t i o n of t h i s enzyme by i t s i s o l a t i o n from, numerous b a c t e r i a l extracts (P'» aeruginosa was not among those listed.)). The widespread occurrence of the: enzyme has warranted the assumption, that i t may be i n v o l v e d i n the biosynthesis of RNA (30). ' In. l i n e with t h i s view are the. recent studies with ribonucleoside: diphosphates containing analogues of the; n a t u r a l l y occurring bases ( 3 1 ) ) j . that polynucleotide phos-phorylase: can br i n g about the synthesis of an, ,RNA of the same nucleotide composition and molecular weight as that I s o l a t e d f romi. Azotobacter ( 3 0 ) , and f i n a l l y that the a c t i v i t y of the enzyme: i s highest i n young1 growing c e l l s ( 2 9 ) . However, there i s no evidence that the enzyme i s able to r e p l i c a t e the primer molecules as in. the case: of Kornberg ,'s DNA polymerase! ( 3 2 ) . Very l i t t l e evidence about the. i n t r a c e l l u l a r l o c a t i o n of t h i s enzyme: i s a v a i l a b l e . The: method of i s o l a t i o n normally employed involves c e l l rupture by sonic o s c i l l a t i o n or grinding followed by s e l e c t i v e p r e c i p i t a -t i o n and elution. with ammoniumi sulphate and calclurai. phosphate gels r e s p e c t i v e l y . I I , . Oxidative phosphorylation - general Oxidative phosphorylation, i n b i o l o g i c a l , systems may occur by two sepa-rate mechanisms v i z . substrate-linked phosphorylation and respiratory-chain phosphorylation. I n the former process the exergondic dehydrogenation. (oxidation) of a metabolite i s coupled to the endergonic phosphorylation of ADP' to form' ATP'. This method is: best exemplified I n anaerobic g l y c o l y s i s by the. formation of 3-phosphoglyceric acid, from 3-P'hosphoglyceraldehyde with the concurrent phosphorylation of ADP: to form ATP. The o v e r a l l reaction may be wr i t t e n as follows ( 3 3 ) : -13-3-phosphoglyceraldehyde DPNH + H 1 , 3-diphosphoglyceric a c i d j ADP 3-phosphoglyceric acid ATP Since no molecular oxygen i s involved i n these phosphorylations the reac-t i o n s are termed, anaerobic phosphorylations- at the substrate l e v e l . The mechanisms involved are now w e l l established and w i l l not be discussed here. Respiratory chain phosphorylation,, or what i s commonly r e f e r r e d to as; oxidative phosphorylation, accounts f o r the greater part of the energy f o r -mation i n aerobic a l l y r e s p i r i n g c e l l s . . The r e a c t i o n involves the enzymatic t r a n s f e r of electrons along the r e s p i r a t o r y chain to molecular oxygen with the coupled phosphorylation of ADP' to f o r ATP. The mechanisms involved. l e v e l . Ih recent years there has been a concentrated e f f o r t i n many labor-atories on the problem of e l u c i d a t i n g these mechanisms and, although a con-siderable amount of l i t e r a t u r e has been published and our knowledge has g r e a t l y increased i n p a r t i c u l a r areas, the o v e r a l l mechanism i s not y e t f u l l y understood. ' ' S l a t e r (3U) demonstrated that ADP was a s p e c i f i c acceptor i n oxidative phosphorylation and much attention has since been devoted to the s t o i c h i o -metric r e l a t i o n , between the equivalents of ADP phosphorylated and the atoms of oxygen taken up (or electrons t r a n s f e r r e d to molecular oxygen). This; r e l a t i o n s h i p i s generally termed the P':0 r a t i o since i t denotes the number of atoms of inorganic phosphate incorporated i n t o organic phosphate per atom of oxygen consumed. i n t h i s process are considerably more complex than those at the substrate -1U-The determination of the P:.0 r a t i o i s complicated by the presence of enzymes which hydrolyze. ATPto ADP' and inorganic phosphate. To reduce t h i s l o s s , f l u o r i d e i s commonly added to i n h i b i t these enzymes; (35))« Another method employed t o reduce t h i s l o s s i s to add an e f f i c i e n t "trapping" system such as the hexokinase-catalyzed transphosphorylating system used by Kornberg (36). Here, the ATP' generated is; r a p i d l y t r a n s f e r r e d to glucose or fructose t o form glucose-6-phosphate (Gi-6-P) or fructose-6-phosphate (F-6-B') r e s p e c t i v e l y . ^ Whereas substrate-linked phosphorylation occurs i n homogeneous s o l u t i o n , the mechanisms responsible f o r r e s p i r a t o r y - c h a i n phosphorylations, i n animal t i s s u e at l e a s t , are associated with p a r t i c u l a t e f r a c t i o n s of the c e l l . Consequently the majority of studies are performed on whole mitochondria or mitochondrial fragments. Oxidative phosphorylation was f i r s t described i n 19,39 by Kalckar (37)) when he showed that phosphorylation occurred during the oxidation of such substrates as fumarate, malate, and c i t r a t e by kidney homogenates. S h o r t l y afterwards B e l i t z e r (38) and Ochoa (39) demonstrated that t h i s was:at l e a s t i n p a r t a d i f f e r e n t mechanism to that occurring at: the substrate l e v e l , since here, the P:0 r a t i o exceeded u n i t y . Both groups suggested that phos-phor y l a t i o n must be occurring not only when the substrate was dehydrogenated but also during the f u r t h e r oxidation of reduced DPS by molecular oxygen. In 19U3 Ochoa (35) attempted to a r r i v e at an accurate F:.0 r a t i o by taking i n t o account losses caused by adenosine triphosphatase-type reactions. He obtained a quantitative p i c t u r e of such losses by examining\ under iden-t i c a l conditions, the y i e l d of phosphorylation from reactions known to produce a s i n g l e phosphorylation (substrate-linked)). He concluded from t h i s work that the corrected P:0 r a t i o f o r pyruvate oxidation would be 3.0:1.0, -IS-that i s , three phosphorylations with each of the f i v e o xidative steps i n the metabolism of pyruvate v i a the c i t r i c a c i d c y c l e . He could not demon-stra t e a substrate-dependent phosphorylation with BPNM as substrate. Ten years l a t e r F r i e d k i n and Lehninger (1*0) showed d i r e c t l y that phos-phor y l a t i o n accompanied the oxidation of DPNH i n l i v e r mitochondria. Sub-sequently, with cleaner preparations, and the use of a hexokinase trapping system,, Lehninger (1*1) obtained P:G r a t i o s of 2.6 f o r t h i s oxidation. The f i n d i n g of a P:0 r a t i o greater than two has been i n t e r p r e t a t e d to mean that the actual value i s threej the lower experimental value being the r e s u l t of ATP1 h y d r o l y s i s . The majority of the work up to t h i s time had been performed on i n t a c t mitochondrial preparations;, attempts to obtain extracts: or fragments being f o r the most p a r t unsuccessful. In 1956 Lehninger, Cooper and.'Devlin (1*2, kky 1*3) prepared d i g i t o n i n extracts of r a t l i v e r mitochondria. These pre-parations were active with&-hydroxybutyrate and succinate as substrates, and exhibited a s p e c i f i c requirement f o r adenosine-^ "-phosphate (AMP)) as phosphate acceptor. ADP i s now considered to be the primary acceptor i n oxidative phosphorylation (1*£,1*6), the involvement of AMP being due t o the presence of adenylate kinase i n idle preparations. The s i t e s of phosphorylations i n the e l e c t r o n chain began to be estab-l i s h e d i n 1951 when Lehninger (1*1) demonstrated a requirement f o r cytochrome c. Using C^-hydroxybutyrate, as substrate, DPN as coenzyme and f e r r i c y t o -chrome c as el e c t r o n acceptor, Bergstrom et a l . (1*7) demonstrated that the substrate oxidation per se produced phosphorylation. By 1955 i t had been demonstrated (1*8) that the oxidation of ferrocytochrome c caused coupled phosphorylation, the PrO r a t i o being about 1.0. This would i n d i c a t e t h a t the remaining two s i t e s of phosphorylation occurred somewhere between DPNH -16-and ferrocytochrome c. Bergstrom et a l . found that when the cytochrome oxidase was blocked by cyanide and ferricytochrome c served as e l e c t r o n acceptor, P :0 r a t i o s approaching two were achieved f o r the oxidation of Q -hydroxybutyrate (hi). A tent a t i v e s i t e f o r one of these two phosphory-l a t i o n s i s between DPNH and the antimycin A-sensitive step (k9)a second between cytochrome b and ferrocytochrome c (50). Thus the aerobic oxida-t i o n of succinate by mitochondria i s knoxm to give P:0 r a t i o s between one and two, suggesting only two s i t e s of phosphorylation. In that the dehy-drogenation of succinate does not involve a pyridine-nucleotide system, i t may be i n f e r r e d that a phosphorylation site; near the DPNH to flavoprotein. step of the chain has been by-passed. A s i m p l i f i e d scheme of the r e s p i r a t o r y chain showing the t e n t a t i v e phosphorylating s i t e s i s given below, where A and AH2 designate o x i d i z e d and reduced substrate r e s p e c t i v e l y (33)• Possible s i t e s of phosphorylation of ADP + 5 § I «••+ AH 2 DPN v * . FH 2 \ / 2Cyt b, Fe . * 2Cyt c Fe , 2Cyt a Fe HgO A A A *v V +«y\ DPNH F ' 2Cyt b Fe ' 2Gyt c Fe 2Cyt a Fe ' N | 0 2 H + » 2H+ T : i succinate ascorbate Important information concerning the s i t e s of phosphorylation has come from the spectroscopic studies of Chance and Williams- (51) on the steady-state l e v e l s of the e l e c t r o n c a r r i e r systems i n l i v e r mitochondria. I t had been shown e a r l i e r (52) that the rate of ele c t r o n transport from DPNH to oxygen was c o n t r o l l e d by the concentration of ADP and inorganic phosphate. Addition of ADP caused a very large increase i n oxygen uptake which was -17-accompanied by d i s t i n c t changes i n the steady-state r a t i o s of the o x i d i z e d to reduced forms of the EFHi, f l a v i n s , cytochrome h and cytochrome c systems, a l l of which became more oxidized. Chance and Williams i n f e r (5"!)! that ADP exerts i t s e f f e c t on e l e c t r o n t r a n s f e r at the three oxidation-reduction reactions i n d i c a t e d i n the above diagram and that these reactions may repre-sent the three s i t e s of oxidative phosphorylation i n l i v e r mitochondria. The present evidence on the nature of the intermediates i n oxidative' phosphorylation should be mentioned since the recent data.on t h i s subject m ay explain c e r t a i n fragments of experimental data to be discussed l a t e r . The various theories that have been proposed are constantly undergoing modifications as new evidence i s obtained. Although a number of speculations concerning the mechanism have been, proposed, one finds; that many of these are i n f a c t quite s i m i l a r . This i t s e l f i s somewhat encouraging since i n most cases these t h e o r i e s have been a r r i v e d at through d i f f e r e n t experimental approaches and with a v a r i e t y of preparations. I t has been proposed by a number of i n v e s t i g a t o r s that at each of the three phosphorylating s i t e s i n the r e s p i r a t o r y chain coupled e l e c t r o n trans-f e r r e s u l t s i n the formation of a "high-energy" intermediate of an electron c a r r i e r , e i t h e r with phosphate or with some other compound.. This intermedi-ate may thus be viewed as; the chemical form i n which the energy l i b e r a t e d during e l e c t r o n t r a n s f e r has been conserved. I t i s then proposed th a t t h i s intermediate can donate i t s high-energy phosphate group to ADP t o form ATP, e i t h e r d i r e c t l y or through intervening group-transfer r e a c t i o n s . The theories proposed by S l a t e r (53,5ii:,55)) and Lehninger (56,57)) d i f f e r i n one p r i n c i p l e respect from that advocated by Chance (58). That i s , t h e i r theory suggests that two c a r r i e r s , A and B, adjacent to each other i n the chain undergo oxidation-reduction i n the presence of a t h i r d substance, I . -18-The nature of I. i s not known but may be an enzyme, thus:-AH 2 +• I ^ * AH 2-I Cl) During oxidation and reduction the bond between AH 2 and I." i s : converted, to one of high-energy. AH 2-I +• B A ' v l 4- BHg (2) The next step i s a r e a c t i o n between the high-energy intermediate, A~I., and a compound, X, to form another high-energy intermediate, X ~ l and regenerate the c a r r i e r , A. A ^ I . +• X ^ ^ X ^ I +• A (3> The involvement of the intermediate, X arose from the "kinetic data of Chance (51) when he demonstrated that the c a r r i e r s do not act d i r e c t l y with ADP or inorganic phosphate. The f i n a l step i s then the r e a c t i o n between X-v I, inorganic phosphate and ADP to form ATP. X~ I *- ADP + P j L — - ATP + I. + X ' (It)) Lehninger prefers a r e v e r s i b l e phosphorylation of A'v JL p r i o r to phos-phor y l a t i o n of ADP thus forming an energy-rich compound between. A and -inorganic phosphate (P^ ), i . e . A ~ I + P ± ^ P ^ I + A (5) P^/1. + ADP ^ = ± : ATP' + I ( 6 ) The mechanism proposed by Chance (51) d i f f e r s frdm that' given above i n t h a t the high-energy form of the c a r r i e r i s i n the reduced form ( B H ^ I ) . The evidence given f o r t h i s i s that the c a r r i e r s e x i s t i n an i n h i b i t e d form i n mitochondria lacking phosphate acceptors (59). Probably the best evidence f o r the existence of an oxidized c a r r i e r -high- energy intermediate has been forwarded by Purvis (60') who demonstrated that a f t e r a l l the i n i t i a l DPN.H ipresent i n the l i v e r mitochondria had been oxidized, the amount of DPN; appearing g r e a t l y exceeded the amount of DPNH -19-oxidized. DNP could replace ADP i n t h i s system whereas ATP was i n e f f e c t i v e , thus s t r o n g l y suggesting that the precursor i s connected to oxidative-phos-p h o r y l a t i o n . I t should be stated however that the proposed DPN*JE has not as yet been i s o l a t e d and i t s existence i s i n f e r r e d from the increased (DPN + DPNH) which i s found, a f t e r incubation w i t h ADP and Inorganic phosphate. Uncoupling agents of oxidative phosphorylation have been postulated to act by accelerating the hydrolysis of X ^ i ; i n the reactions given above (55"): DNP X/v/I + H£0 X + I (7) or, by combining with X - y l to l i b e r a t e X (51)• E i t h e r of these p o s s i b i l i t i e s w i l l d i s s o c i a t e the phosphorylation process from e l e c t r o n t r a n s f e r . Experimentally DNP stimulates the l i b e r a t i o n of inorganic phosphate from ATP', that i s , i t appears to accelerate adenosine triphosphatase (ATP 1-ase) a c t i v i t y (6l). S l a t e r explains t h i s s t i m u l a t i o n by a r e v e r s a l of r e a c t i o n (1*.) coupled with r e a c t i o n (7) thus: ATP + I. + X ^ = ± X v l . + ADP + Pv (UR) X ~ I + H 20 7 — X • + I (7)) ATP 4- H 20 v ADP + p i sum In t h i s connection, Hulsmann and S l a t e r (55) have observed f o u r d i f f e r e n t enzyme systems i n heart mitochondria capable of bringing about the hydrolysis of ATP'. They suggest that three of these (DNP'-stimulated) are r e l a t e d to the three DNP-sensitive steps i n the r e s p i r a t o r y chain phosphorylation. The concept that the ATPl,!ase a c t i v i t y of mitochondria was f u n c t i o n a l l y r e l a t e d to the mechanism by which phosphorylation of ADP is; coupled to electron transport was i n f e r r e d e a r l i e r by Cooper and Lehninger (62). -20-D i r e c t evidence has been given f o r this; concept by Penefsky et al 7. i n a recent report (63). P r i o r to t h i s these workers (6I4;) managed to resolve beef-heart mitochondria i n t o p a r t i c u l a t e and soluble p r o t e i n components j the soluble component being required f o r the coupling of phosphate e s t e r i -f i c a t i o n to r e s p i r a t i o n catalyzed by the p a r t i c l e s . The soluble component also catalyzed a DBP-stimulated h y d r o l y s i s of ATP. The evidence obtained may be summarized as f o l l o w s : (a) By addition of the h i g h l y p u r i f i e d ATP*ase i t was p o s s i b l e to r e c o n s t i t u t e oxidative phosphorylation and adenosine triphosphate-radioactive inorganic phosphate (P^ 2-ATF) exchange. (b) A v a r i e t y of p h y s i c a l treatments affected both a c t i v i t i e s . i n the same manner. (c) Exposure ..to elevated temperatures' activated the two a c t i v i t i e s to the same extent. The adenosine triphosphate-adenosine diphosphate (ATP-ADP) exchange reported by Cooper and Lehninger (57) has also been implicated as being a manifestation of the terminal steps of oxidative phosphorylation i n d i g i t o n i n fragments of mitochondria where ADP reacts with an unknown high-energy intermediate generated during e l e c t r o n transport to form ATP'. These conclusions have been;.based on the s p e c i f i c i t y of ADP and ATP and the i n -h i b i t i o n of t h i s exchange by the uncoupling agents;, DNP, dicumarol and gramicidin* Azide, an uncoupling agent of oxidative phosphorylation and an i n h i b i t o r of the DNP-stimulated ATP'rase, prevented the i n h i b i t i o n of the ATP-ADP exchange, although by i t s e l f i t exerted no e f f e c t on the rate of the reaction (65). The aging of mitochondrial p a r t i c l e s r e s u l t e d i n a - Z L -l o s s , at s i m i l a r rates, of the DNP-sensitlvity of the ATP-ADP' exchange, of oxidative phosphorylation, and of the DNPi-stimulated. ATP'rase a c t i v i t y . However there was no s i g n i f i c a n t l o s s of ATP-ADP exchange a c t i v i t y . Wadkins ( 6 6 ) observed i d e n t i c a l r e s u l t s with whole mitochondria and was l e d to the conclusion that DHP does not act d i r e c t l y on the ATP-ADP exchange reaction but at some previous step i n the coupling reactions by depleting the system of high-energy intermediates which are i n e q u i l i b r i u m with the ATP-ADP' exchange r e a c t i o n . The exchange reactions discussed above may be r e a d i l y explained by employing equations f o r the mechanism of oxidative phosphorylation, formu-l a t e d e a r l i e r . C a r r i e r + X ,^ C a r r i e r A/X ( 8 ) Carrier'*'X • P^ ^ C a r r i e r + F ~ X (9) P^X + ADP ^ ATP •» X (10)1 32 The ATP'-P exchange i s represented by equation (9); followed by equation (10) j ATP^ase a c t i v i t y by a r e v e r s a l of equations; (10)/ and (9); followed by hydro l y s i s of carriier/"X; and f i n a l l y the ATP-ADP5 exchange by equation (10)'. In agreement with the mechanisms o u t l i n e d by S l a t e r , Wadkins and Lehninger p r e f e r to think t h a t the intermediate, c a r r i e r - v X , exists; i n an oxidized form. Their evidence f o r t h i s conclusion i s based on the obser-v a t i o n that the ATP-P^ 2 exchange and the ATP"ase a c t i v i t y are maximum'when the c a r r i e r s are f u l l y oxidized and nea r l y completely i n h i b i t e d when r e -duced. F i n a l l y , the group of Seikevitz,, Low, Ernster and Lindberg have also published considerably on t h i s problem (67;, 6 8 , 6 9 , 7'0))» Their approach i s -22-s i m i l a r to that of Wadkins and Lehninger, i . e . studying the terminal, reactions of oxidative phosphorylation* The evidence given however sup-ports the view that the ATE-P 3 2 exchange reflects- one of the phosphory-l a t i o n s that occurs i n the diaphorase-flavin region of the r e s p i r a t o r y chain and i n d i c a t e s a d i r e c t phosphorylation of r e s p i r a t o r y pigments. Most of t h i s work i s based e x c l u s i v e l y on i n h i b i t o r studies. I t does however o f f e r evidence against the view held by other workers that phosphate does not p a r t i c i p a t e i n the formation of the primary high-energy'bond, (cf Brodie to f o l l o w ) . Support f o r t h e i r conclusions has come from the thermochemical data of Grabe et a l . (71)• III... Oxidative phosphorylation - b a c t e r i a l . U n t i l recently..studies on the transformation of inorganic phosphate i n t o high-energy phosphate during r e s p i r a t i o n of b a c t e r i a l systems lagged f a r behind s i m i l a r studies with animal c e l l s . During the past decade how-ever data concerning oxidative phosphorylation i n b a c t e r i a l extracts has appeared at an increasing rate and,, although controversy s t i l l e x i s t s as to the mechanisms involved and t h e i r s i m i l a r i t y to animal systems, our knowledge has v a s t l y increased. I t w i l l be seen i n the f o l l o w i n g review of the l i t e r a t u r e that as new information i s obtained more and more p a r a l l e l i s m s may be drawn between b a c t e r i a l and animal oxidative phosphorylations. At present however (and t h i s may be l a r g e l y a matter of technique) t h i s seems to be a function of the p a r t i c u l a r bacterium, employed. The f i r s t evidence f o r the oxidative formation of energy-rich phos-phate bonds i n b a c t e r i a l metabolism was. obtained by Lipmann i n 1939 (72) when he demonstrated that the oxidation of pyruvic a c i d by Lac t o b a c i l l u s d e l b r u c k i i required inorganic phosphate, and that the products of the oxidation were a c e t y l phosphate and carbon dioxide. Active c e l l f r e e extracts were obtained from Clostridium: butylicum by Koepsell and Johnson i n 191*2 (7/3)' and from: E. c o l i by K a l n i t s k y and Werkman ini 19k3 (Ik)* Vogler i n 191*2 (75) demonstrated, with the autotrophic organism,, T h i o b a c i l l u s thiboxidans that the oxidation, of sulphur was coupled to the t r a n s f e r of inorganic phosphate from the medium to the c e l l s . I n 1951, Hersey et a l . (76) demonstrated a requirement f o r inorganic phosphate during the single-step oxidation of succinate to fumarate i n c e l l - f r e e extracts of E. c o l i . ; Although the nature of the a c i d - l a b i l e phosphate formed was not determined, P'tO r a t i o s of 0 . 5 to 0 . 6 were obtained. The f i r s t d i r e c t evidence that at l e a s t c e r t a i n b a c t e r i a l species behaved i n a manner s i m i l a r to animal systems with respect to oxidative phosphorylation was given by Brodie and Gray i n 195>5 (77,78)) when they reported the existence of a system which y i e l d e d P'::0 r a t i o s greater than one i n crude extracts of Mycobacterium', pfalei. Moreover phosphorylation, was uncoupled by EH3F and by gramicidin. In a more d e t a i l e d report of this: work (79) i t was shown that sonic extracts of t h i s organism with succinate as substrate y i e l d e d P::0 r a t i o s of 1 . 7 8 . This value approached very c l o s e l y the t h e o r e t i c a l r a t i o of 2.0 i n d i c a t e d f o r animal c e l l s . The authors stated however that with the exception of Corynebacterium. creatinovorans s i m i l a r systems could not be demonstrated i n a number of other organisms studied. The advantages o f f e r e d by p a r t i a l l y p u r i f i e d systems f o r the study of the mechanism of oxidative phosphorylation, are, of course, obvious. That f r a c t i o n a t i o n i n t o p a r t i c u l a t e and soluble components was possible w i t h the ( b a c t e r i a l system was shown by Brodie and Gray In 1 9 5 > 7 ; (80). C e n t r i f ugation o f the crude extract at 11*0,000 x g y i e l d e d a p a r t i c u l a t e f r a c t i o n which,, although capable of some coupled phosphorylation per se, was' stimulated four to seven f o l d by the addition of the 1U0,000 x g supernatant.. Again, c e r t a i n s i m i l a r i t i e s to animal systems were obvious, v i z . the detrimental e f f e c t of f r e e z i n g on P::0 r a t i o s j the osmotic c h a r a c t e r i s t i c s of the p a r t i c l e s j the greater l a b i l i t y of the phosphorylation a c t i v i t y of p a r t i c l e s compared to t h e i r a b i l i t y to transport electrons to oxygen; and. the necessity f o r maintaining h i g h l y organized p a r t i c l e s at the molecular l e v e l f o r oxidative phosphorylation a c t i v i t y . Differences exhibited between the b a c t e r i a l system and animal oxidative phosphorylation were however also evident. These included the greater s t a -b i l i t y of the p a r t i c l e s and the soluble nature of f a c t o r s necessary f o r coupled a c t i v i t y . Of i n t e r e s t t o our own work wit h P. aeruginosa was the i n i t i a l f a i l u r e of Hartman,. Brodie and Gray ( 8 1 ) to obtain s a t i s f a c t o r y r e s u l t s with the azotobacter system when they applied the methods used i n the study of M. p h l e l . Upon r e i n v e s t i g a t i o n of the Azotobacter ag i l e system' ( . 8 1 ) they found th a t the i n i t i a l f a i l u r e s could be traced t o the extreme l a b i l i t y of oxidative phosphorylation i n the crude ex t r a c t . The presence of magnesium; ions and. ATP during a l l stages of preparation of the extract' s t a b i l i z e d t h i s a c t i v i t y . . Nbn-dialyzed extracts demonstrated very l i t t l e substrate-dependant phosphorylation. Dinitrophenol. only p a r t i a l l y uncoupled phos-phor y l a t i o n from, oxidation. EtO r a t i o s i n excess of one were not obtained; with c i t r i c acidl cycle Intermediates; other than.o< - k e t o g l u t a r i c acid.. In 1959 and i960 a d e t a i l e d study on the r o l e of soluble f a c t o r s in. -25-the mycobacterium. system was reported by Brodie (82) and by Brodie and B a l l a n t i n e (83,81*). The f i r s t of these reports demonstrated t h a t the soluble f r a c t i o n (ll* 0 , 0 0 0 x g supernatant) contained h e a t - l a b i l e and heat-stable f a c t o r s necessary f o r coupled phosphorylation. Treatment of t h i s supernatant with ammonium, sulphate demonstrated the requirement i n the system f o r both f l a v i n adenine dinucleotide (FAD) and vitamin K^. Also w i t h the use of t h i s f r a c t i o n a t e d system, ADP' was found to be the s p e c i f i c acceptor of inorganic phosphate. I r r a d i a t i o n of the extract with l i g h t at 360 millimicrons, (mp.) wavelength r e s u l t e d i n the destruction of a bound "vitamin-K-like" compound and a concurrent l o s s i n the a b i l i t y to conduct oxidative phosphorylation ( 8 2 ) . This a b i l i t y could be restored by the addition of vitamin or a n a t u r a l b i o l o g i c a l l y - a c t i v e napthoquinone which had been i s o l a t e d p r e v i o u s l y from extracts of M. p h l e i ( 8 5 ) . The addition of FAD; alone r e a c t i v a t e d oxidation only, i n d i c a t i n g the presence of a non-phosphorylative pathway of e l e c t r o n transport. The s p e c i f i c i t y of the b a c t e r i a l system f o r vitamin has been s i n c e studied with the l i g h t - t r e a t e d preparation ( 8 3 ) . Although oxidation could be, restored with numerous napthoquinones, the requirements f o r phosphory-l a t i o n were more s p e c i f i c , thus, compounds: containing a methyl group i n the carbon-two p o s i t i o n and an unsaturated side chain of at l e a s t f i v e carbon atoms i n the carbon-three p o s i t i o n of the napthoquinone r i n g restored both oxidation and phosphorylation. A proposed mechanism f o r the action of the vitamin K-type compound has been o u t l i n e d by Brodie (81*). I t involves the d i r e c t phosphorylation of the reduced napthoquinone. A high-energy bonded phosphate then arises when electrons are t r a n s f e r r e d to the cytochrome chain. The unstable enol-- 2 6 -phosphate d e r i v a t i v e of the napthohydroquinone i s then postulated to enter i n t o r e a c t i o n with ADP r e s u l t i n g i n the t r a n s f e r of the energy-rich phos-phate to this; compound forming ATP and regenerating the napthoquinone. The IhOyOOOxg supernatant i n the M. p h l e i system has been shown to contain the f l a v i n s (FMHI and. FAD)) i n greater concentration than the UiO.OOOxg p e l l e t . I t i s thought (86) that the r e l a t i v e l y 'high concentra-t i o n of FMN i n t h i s supernatant f r a c t i o n , explains the observed non-phos-phorylative FMEft-linked pathway of oxidation i n t h i s f r a c t i o n . On the other hand, the napthoquinone was located predominantly in.the p a r t i c u l a t e f r a c -t i o n as was cytochrome b. Since the vitamin. K]_ was necessary f o r the oxidation of reduced DPN! and f o r the reduction ofthe terminal r e s p i r a t o r y pigments i t was concluded that the vitamin K, acts between 'these two c a r r i e r s ( 8 0 ) . , Quinones have also been i s o l a t e d from microorganisms other than M. p h l e i . Lester and Crane i n 1959 (87) reported the presence of both a benzoquinone and a napthoquinone i n E. c o l i . The benzoquinone was found only i n c e l l s grown under aerobic c o n d i t i o n s . In 1 9 6 l Kashket and Brodie (88) working with E. c o l i obtained c e l l f r a c t i o n s which on recombination, coupled phosphorylation t o o x i d a t i o n . D i f f e r e n t i a l c e n t r i f u g a t i o n y i e l d e d a p a r t i c u l a t e preparation, sedimenting at lOUjOOOxg, which contained a napthoquinone. I r r a d i a t i o n of the p a r t i -culate f r a c t i o n r e s u l t e d i n decreased oxidative phosphorylation which could be restored on a d d i t i o n of quinones. Rose and Ochoa i n 1956 (89) obtained coupled oxidative phosphorylation w i t h Azotobacter v i n e l a n d i i p a r t i c l e s . I t should, be mentioned however, that " p a r t i c l e s " as reported i n t h i s work were obtained by c e n t r i f u g i n g a c e l l f r e e extract (prepared by grind i n g with alumina powder) at lU,000xg. I t i s therefore d i f f i c u l t to equate such f r a c t i o n s with those p a r t i c u l a t e f r a c t i o n s obtained by Brodie et a l . Among the substrates; demonstrated by Rose and Ochoa to be oxidized with coupled phosphorylation were molecular hydrogen, DPNH and TPNH. PtO r a t i o s however at no time exceeded onej the highest reported being 0.52. Radioactive phosphorus was employed i n t h i s work to f o l l o w e s t e r i f i c a t i o n of inorganic phosphate and the existence of an ADP-ATP' exchange r e a c t i o n was; noted. Whether or not t h i s exchange was re l a t e d t o oxidative phosphorylation could not be ascertained. The presence of a polynucleotide phosphorylase-catalyzed exchange of ADP and ra d i o a c t i v e inorganic phosphate was not discussed. Phosphorylation was only very s l i g h t -l y uncoupled by DNP. In that the oxidation of substrate was independant of the presence or absence o f phosphate acceptor ( i n contrast t o f r e s h l y p r e -pared mitochondria) the question was r a i s e d as t o whether the b a c t e r i a l system was l a r g e l y uncoupled. This point could also be used to explain the r e l a t i v e i n s e n s i t i v i t y to DNP. 4 ' '' In 19-56 Nbssal et a l . (90) prepared, p a r t i c u l a t e , m i c r o s c o p i c a l l y v i s i b l e f r a c t i o n s from bakers yeast, Proteus v u l g a r i s and Aerobacter aero-genes; by high-speed shaking methods. Conditions which allowed oxidative phosphorylation by the yeast and aerobacter p a r t i c u l a t e f r a c t i o n s f a i l e d with P. vulgaris:. Since the P. v u l g a r i s p a r t i c l e s ; allowed good oxidative a b i l i t y and since no loss; of inorganic phosphate from ATP1 or hexose mono-phosphate could be shown, i t was assumed that complete uncoupling of the system had occurred* Phosphorylative a c t i v i t y with aerobacter was i n t e r e s t -i n g i n that good S'tO ratios; were obtained, with endogenous systems; i n both the crude c e l l f r e e extract and supernatant systems, but on the addition of -28-substrate these r a t i o s were lowered 50 per cent. ¥ith the p a r t i c u l a t e f r a c t i o n a small, but nevertheless substrate-dependant, phosphorylation was observed. T i s s i e r e s e t a l . i n 1957 (91) f r a c t i o n a t e d c e l l f r e e e x t racts of Azotobacter v i n e l a n d i i by c e n t r i f u g a t i o n , y i e l d i n g a l a r g e - p a r t i c l e f r a c t i o n , sedimenting at 22,000xgj a small p a r t i c l e fraction,, sedimenting at lU$,.OO0xg, and a supernatant f r a c t i o n from the l a t t e r c e n t r i f u g a t i o n . The highest P:0 r a t i o s were obtained w i t h the s m a l l - p a r t i c l e f r a c t i o n although at no time d i d they exceed one. Sonic treatment of the l a r g e - p a r t i c l e f r a c t i o n increased t h e . s p e c i f i c a c t i v i t y of DPHEt-oxidase and succinic-oxidase systems t o values s i m i l a r to those obtained f o r the s m a l l - p a r t i c l e f r a c t i o n * The r e s p i r a t o r y chain i t s e l f was located i n the s m a l l - p a r t i c l e f r a c t i o n . . I t was suggested by the authors that these p a r t i c l e s may represent the granules evident i n the cytoplasm of whole c e l l s by e l e c t r o n microscopy. However, the p o s s i -b i l i t y that these p a r t i c l e s were derived from the c e l l membrane could not be excluded* DNP' again d i d not uncouple phosphorylation from oxidation. In a l a t e r p u b l i c a t i o n Hovenkamp (92) studied the e f f e c t of various, suspending s o l u t i o n s on the oxidative phosphorylation a c t i v i t y ofthe small-p a r t i c l e f r a c t i o n and found that t h i s a c t i v i t y was i n a c t i v a t e d by d i l u t e s a l t s o l u t i o n s . Increasing the s a l t concentration brought about a p a r t i a l r e v e r s a l of this^ i n h i b i t i o n . Contrary to data obtained with mitochondrial suspensions however, phosphorylative a c t i v i t y i n the azotobacter system was l o s t i n sucrose s o l u t i o n s . Serum albumin, as i n the case w i t h mitochondrial suspensions, protected t h i s a c t i v i t y . Regardless of the suspending medium P:0 r a t i o s with DPNH as substrate did: not exceed 0.50. Vitamin K]_ had no e f f e c t on phosphorylative a c t i v i t y , whereas menadione (vitamin Kyj< uncoupled -29-i t . uncoupling by t h i s l a t t e r agent has also been reported f o r Alcaligenes f.aecalis by Pinchot (93) and by Martius and Mt z - I i t z o w (9k) i n mitochon-d r i a . In 1951 Pinchot and Racker (95) demonstrated the e s t e r i f i c a t i o n of i n -organic phosphate during the oxidation of ethanol. by sonic extracts of E. coli... Two phosphorylating reactions were shown both of which were dependant upon the ad d i t i o n of DPN. One of- these (formation of a c e t y l phos-phate) occurred at the substrate l e v e l , the second was thought to be due to the f u r t h e r oxidation of DPNH;- However since phosphorylation with DPNH as substrate could not be shown, exclusion of g l y c o l y t i c substrate-linked phosphorylation could not be excluded* In 1953 Pinchot (96), working w i t h sonic extracts of'Alcaligenes f a e c a l i s , demonstrated phosphorylation during the DPN-linked oxidation of ethanol to acetaldehyde. Since acetaldehyde was metab o l i c a l l y i n a c t i v e the phosphorylation was assumed to occur during the flrther o x i d a t i o n of DPNH by the r e s p i r a t o r y chain.. This was confirmed by demonstrating an increase i n the formation of G-6-P w i t h DPNH as substrate. With the crude extract there was no i n h i b i t i o n of phosphorus uptake with DNP. The crude A. f a e c a l i s : extract was: f r a c t i o n a t e d by ammonium, sulphate treatment i n t o three parts., a l l of which were necessary f o r the demonstration of coupled oxidative phos-phorylation w i t h DPN®. The three components consisted of a soluble heat-l a b i l e f r a c t i o n , a p a r t i c u l a t e DPNH oxidase (obtained by eent r i f u g a t i o n at 100,000xg) and a polynucleotide of the RNA type (97).. The function of the polynucleotide, i t was stated, was to bind the soluble h e a t - l a b i l e f a c t o r t o the p a r t i c l e s * In a l a t e r communication (98) a high-energy intermediate of oxid a t i v e phosphorylation was proposed on the basis of d i s s o c i a t i o n of p a r t i c u l a t e and soluble components: by preincubation of the f r a c t i o n s with DPNH. A f t e r e l u t i o n the soluble f r a c t i o n (supposedly now containing the high-energy Intermediate) would then be capable of producing net ATP' synthesis front ADP and inorganic phosphate. To measure phosphorylation both radioactive phos-phorus incorporation i n t o organic phosphate and. ATP' formation were followed. The p o s s i b i l i t y that the eluted supernatant contained a soluble phosphory-l a t i n g system was r u l e d out on the b a s i s of i n h i b i t o r studies. Again, the p o s s i b i l i t y that incorporation of r a d i o a c t i v e phosphorus was due to an ATP-p32 e x change was eliminated on the b a s i s o f ADP" being a b e t t e r acceptor f o r phosphate than ATP. F i n a l l y , the p o s s i b i l i t y that an ADP-P^2 exchange catalyzed by polynucleotide phosphorylase accounted f o r radioactive phos-phorus: incorporation was thought to be improbable since addition of poly-nucleotide i n h i b i t e d the incorporation of radioactive phosphorus. However, no explanation i s given as to why a marked i n h i b i t i o n should occur on the a d d i t i o n of a polynucleotide. Miidd. et a l . (99) reported the presence i n Saccharomyces- cerevisae of cytoplasmic granules with s t a i n i n g properties resembling those of mitochon-d r i a . In 1958 Utter, Keech and Nossal (100) studied the a b i l i t y of these p a r t i c l e s t o perform oxidative phosphorylation.- Particles: were obtained by grinding yeast c e l l s with alumina and c o l l e c t i n g the m a t e r i a l sedimenting between 13,000xg and 25>,000xg. In that these p a r t i c l e s were capable of e x h i b i t i n g oxidative phosphorylation without the addition "of soluble com*-ponents and since BMP' completely uncoupled t h i s a c t i v i t y , the yeast p a r t i c l e s were thought to be more c l o s e l y a l l i e d to mitochondria than p a r t i c l e s obtained from b a c t e r i a l systems. Unlike animal systems however P::0 r a t i o s -31-rarely exceeded one arid in some cases were lowered by the presence of an oxidizable substrate. MATERIALS AND; METHODS I* The organism and i t s c u l t i v a t i o n The organism used throughout these studies was Pseudbmonas aeruginosa ATCC 9027. The c e l l s were cultured i n the ammonium phosphate-glucose medium of N o r r i s and Campbell i n Roux f l a s k s at 3Q»°C (101). Each f l a s k contained 100! ml of media.- In the studies conducted with i n t a c t c e l l s the mediumi also contained yeast extract at a f i n a l concentration of 0.1 per cent. In l a t e r studies on oxidative phosphorylation, succinate (Q;.3 per cent) was s u b s t i -tuted f o r glucose as the carbon source. To d i s t i n g u i s h between c e l l s grown on a succinate medium from those grown on glucose, the former w i l l be r e -f e r r e d to as, succinate-grown. The incubation time unless otherwise stated was 18 hours. I I . A n a l y t i c a l determinations 1. Nucleic acids RNA was determined by the method o f Stuy (12l±) and DNA by the method of Burton (1 25), using the e x t r a c t i o n procedure of Stuy. 2. P r o t e i n P r o t e i n concentration was determined by the Biuret method (126) and by the method of Sutherland et a l . (102). The l a t t e r allowed a bet t e r c o r r e l a t i o n between samples and p r o t e i n concentrations given i n the text were determined by t h i s method. 3. Ammonia determinations; Ammonia was determined by the Conway microdiffusion, technique (103)). -33-U. Inorganic phosphate Inorganic and t o t a l phosphate were determined by the method of King (ioH). 5. E s t e r i f i e d phosphate E s t e r i f i e d P 3 2 was determined by the isobutanol-benzene e x t r a c t i o n procedure of Ernster et a l . (10$) as modified by Nielsen and Lehninger ( I 4 . 8 ) . S l i g h t modifications i n the method were required t o adapt the procedure to our own s i t u a t i o n . Acid molybdate reagent: Dissolve gm of ammonium, molybdate i n I4.O ml of 10 N HgSOj^ and make to 100 ml with d i s t i l l e d water. One ml of the r e a c t i o n supernatant (containing TCA) was added to 1.0 ml of anhydrous acetone i n a glass-stoppered tube. Two ml of water (satur-ated with isobutanol) and 7.0 ml of a water-saturated mixture of equal volumes of benzene and isobutanol were added and the mixture thoroughly shaken. A f t e r separation of the two phases, 1.0 ml of acid-molybdate reagent was added to the aqueous phase, gently mixed with i t , and the tube allowed to stand $ minutes. The tube was then shaken vigorously f o r 30 seconds to extract phosphomolybdic acid i n t o the Isobutanol-benzene l a y e r . The aqueous l a y e r was then removed with a Pasteur pipette (quan-t i t a t i v e removal unnecessary) and f i l t e r e d through Whatman No. $0' paper. The f i l t r a t e was c o l l e c t e d i n a second glass-stoppered tube. ( I t was found that when the p r o t e i n was previously removed by ce n t r i f u g a t i o n , or when very small quantities of p r o t e i n were present i n the reac t i o n mixture the f i l t r a t i o n step was not required and the aqueous layer could be placed d i r e c t l y i n t o the second tube). A f t e r adding one drop of 0.02 M KEgPO^ -3k-( c a r r i e r ) and 3.5 ml of water-saturated isobutanol-benzene, the contents were vigorously shaken f o r 30 seconds;. Following these two extractions with isobutanol-benzene, the aqueous l a y e r was withdrawn by means of a Pasteur p i p e t t e and exactly 1.0 ml counted (dried) i n an aluminumi planv chet. From the s p e c i f i c a c t i v i t y (A) of the orthophosphate present i n the r e a c t i o n medium (cpm per mumole of orthophosphate), the r a d i o a c t i v i t y of the 1.0 ml a l i q u o t of e s t e r i f i e d p 3 2 separated as; above (B) (cpm), and appropriate f a c t o r s f o r a l i q u o t s i z e , dilution,, e t c . , the amount of ortho-phosphate uptake per 1.0 ml of the o r i g i n a l r e a c t i o n medium, can be c a l -culated -A P i = (B) x U.00 mumoles w The f a c t o r U.00 i s the t o t a l volume of the aqueous phase (1.0, super-natant; 2.0, water; 1.0' reagent) i n the e x t r a c t i o n ; the acetone added to the aqueous phase i n the f i r s t step i s q u a n t i t a t i v e l y extracted by the isobutanol-benzene phase. In l a t e r experiments the exchange reactions were performed d i r e c t l y i n the glass-stoppered tubes, stopped by the addition of TCA and e s t e r l f i e d P 3 2 determined as above. The s e n s i t i v i t y of t h i s method f o r e s t e r l f i e d P^2 w a s determined i n the presence of 1 to.8 yumoles of c a r r i e r phosphate. Erirploying the same volume of components as given above;, 99.80 per cent of the inorganic phos-phate was removed over t h i s range of c a r r i e r phosphate. I I I . I n i t i a l studies with c e l l f r e e extracts I n i t i a l studies were performed on c e l l extracts prepared by means of a Hughes' Press. -35-C e l l s were harvested by c e n t r i f u g a t i o n , washed once i n d i s t i l l e d water, once i n 0.2 per cent glutathione and the wet c e l l paste packed i n t o glass tubes of an i n s i d e diameter of about 1 mm less than, the diameter of the hole i n the Hughes11 Press.. The cy l i n d e r s were corked with rubber stoppers and immersed i n an ethanol-dry-ice bath f o r 20 minutes. C e l l s were f r e s h l y harvested, frozen, and crushed f o r each experiment. The Hughes' Press; i t s e l f was assembled and held at l e a s t 2k hours at -15°C. The frozen c e l l paste was crushed by applying 10 to 12 thousand pounds per square inch o f pressure with a Carver hydraulic press. The Hughes' press was q u i c k l y opened and the frozen c e l l extract placed i n a di l u e n t to give a f i n a l concentration of 200 mg per ml (wet weight)-. DNA',;-ase (0 .2 ml of a s o l u t i o n containing 1 mg per ml) was added t o decrease the v i s c o s i t y of the preparation and the mixture homogenized by means of a manually-operated Potter homogenizer. The composition ofthe diluent was: 0.05 M g l y c l g l y c i n e , 0.25 M sucrose and 500 mg per cent egg albumin (106). Phosphate incorporation studies w i t h the c e l l e x t r a c t s contained the foll o w i n g concentrations of components:; g l y c y l g l y c i n e (50 /jmoles/0.3 ml) plus orthophosphate (2 /umoles/0.3 ml) 0..5 ml;, MgCl6*6i£0 (100)/imoles/ml)) 0.5 ml; ADP; (to ^moles/ml) 0.5 ml; cytochrome c (0.05 /moles/ml) 0.2 ml; P 3 2 (2QjucurfLes/ml) 0.2 ml; c e l l extract 3-0' ml; substrate (100 /moles/ml) 1.0 ml; sucrose (0.05 M) to 10 ml. Reactions were st a r t e d by the addition of e xtract. Aliquots were removed at various time intervals,., deproteinized with kO per cent TGA ( f i n a l concentration of 10! per cent) and the pH qu i c k l y adjusted to U.0! by the addition of 1 M sodium; acetate to a f i n a l concentra-t i o n of 2I4. per cent... The suspension was then centrifuged and 1.0 ml of supernatant mixed with 80;/ig of acid-washed N o r i t . The charcoal was c o l --36-l e c t e d by ce n t r i f u g a t i o n , washed 3 times i n d i s t i l l e d water, resuspended i n 95 per cent ethanol,- poured i n t o planchets, d r i e d and counted. A l l r a d i o a c t i v i t y measurements were made on a Nuclear Chicago Scaler model 181 A equipped w i t h a gas-flow detector. IV1.. Studies x^ith r e s t i n g c e l l suspensions; 1. Reaction mixtures. C e l l s were harvested by centrifugation,. washed i n cold d i s t i l l e d water and suspended i n 0.1 M tris(hydroxymethyl)aminomethane (Tris.) buffer, pH 7,.k at a f i n a l concentration of 50; mg per ml (wet weight). Experiments t o determine ]?32 uptake and oxygen consumption were con-ducted w i t h a Warburg respirometer, i n double-side-arm f l a s k s . V^Z as; orthophosphate was tip p e d i n from one side arm with substrate and c a r r i e r -phosphate. The t o t a l r e a c t i o n mixture contained approximately 0.1 ^ic of 32 P"^ . Twenty per cent KOHI'solutiomi was added t o the center-well of the re a c t i o n v e s s e l s . TCA was added from the second side-arm at' the required time to stop the re a c t i o n , and p r e c i p i t a t e the p r o t e i n * Duplicate one ml samples were removed, centrifuged and the p e l l e t s ( c e l l s ) washed twice in; cold d i s t i l l e d water, resuspended i n ethanol, poured i n t o planchets, d r i e d and counted. A t y p i c a l r e a c t i o n mixture i s represented as fo l l o w s : K^HPOjj, (0.1 M) 0.15 mlj P 3 2 (1.0 /uc/ml) 0.1 ml; substrate. (100/amoles/ml) O.05 ml; c e l l suspension, 1.0 ml; T r i s b u f f e r , pH 7+k (0.1 M) 1.0 mil; TCA (60$) 0.3 ml; KOH (20$). 0.15 ml; water to 3*l5 ml. Concentrations of other components, where added, w i l l be given i n the t e x t . -37-V/. F r a c t i o n a t i o n of whole c e l l s The d i s t r i b u t i o n of F 3 2- among the- various c e l l constituents was deter-mined by employing the f r a c t i o n a t i o n procedure of Roberts et a l . (U)• Removal and estimation of the charcoal-adsorbable material of the c o l d TCA soluble f r a c t i o n was c a r r i e d out by adding 20' mg of acid-washed Norit (found by experiment to remove 93 per cent of 260 mui absorbing material present) to 1.0 ml of the cold. TGA soluble f r a c t i o n . Further f r a c t i o n a t i o n of the cold TCA soluble material"with bariumtwas accomplished according t o the method of Umforeit e t a l . (107). VJ. Column chromatography of hydrolyzed RNA The separation of the nucleotide: components of hydrolyzed RNA was achieved with a Dowex-1 iom-exchange column1.. The method employed was that given by Hurlbert et a l . (108), i n which the r e s i n i s used i n the formate form and eluted with solutions of formic acid and ammonium' formate. Sam-ple s were c o l l e c t e d i n 5 ml amounts and assayed f o r 260> mn absorbing material and r a d i o a c t i v i t y . Tubes corresponding to a s i n g l e peak of a c t i v i t y were pooled and. concentrated i n vacuo. The nucleotides were converted to the hydrogen-form by passing the concentrated solutions-through Dowex-5>0 (H +) columns. I d e n t i f i c a t i o n of nucleotides was achieved by an analysis of t h e i r c h a r a c t e r i s t i c absorption, spectra at pH 1.0 and 11.0 and paper chromato-graphy. A solvent system, composed, of i s o b u t y r i c acid-lM NHj4.OH-Q.lM sodium versenate (100:60:1.6) f o r paper chromatography gave good separation i n 16 hours on ffo. 3 rami f i l t e r paper (109). R a d i o a c t i v i t y determinations were made: d i r e c t l y on the paper with a Nuclear Chicago Actigraph II. equipped with an - 3 8 -automatic recorder and a gas-flow detector. VIE. Preparation of c e l l f r e e extracts: 1. Preparation of extracts by means, of a Highes' Press:. The method has been p r e v i o u s l y o u t l i n e d ( I I ) . 2. Preparation of extracts by means of sonic o s c i l l a t i o n . C e l l s were harvested and washed once i n cold distilled water and r e -suspended i n e i t h e r 0.2 per cent s o l u t i o n of KC1 or Qi.05 M. T r i s b u f f e r pH I'.li t o a f i n a l concentration of l+OO; mg per ml, wet weight. The suspen-sion was placed i n the % chamber of a IO Kc sonic o s c i l l a t o r p r e v i o u s l y cooled toU°C by means of a c i r c u l a t i n g pump and ice-water. Before sonicating, nitrogen gas was bubbled through the c e l l suspension f o r two minutes. When samples were to be removed at various times,for the determination of O.D., enzyme a c t i v i t y e t c . the volume of the remaining suspen sion: was kept con-stant by the addition of c o l d d i l u e n t . Viable counts on the extracts were made by standard method plate-counts. When extracts were employed f o r oxid a t i v e phosphorylation studies, the sonic-time was U minutes:. Extracts were centrifuged at 500Qxg f o r 10) minutes at i±°C to remove unbroken c e l l s and c e l l u l a r d e b r i s . 3 . Preparation of extracts; by means of lysozyme-versene s o l u t i o n . C e l l s were harvested, washed and suspended i n 0.0)3 M.' T r i s b u f f e r p i 8.0; to a f i n a l concentration of 8 0 0 mg per ml,, wet weight. To obtain o p t i -mum, breakage the complete system required the following concentration of components per ml of c e l l suspensions T r i s (Q.12!?M pH 8 . 0 ) O.6I4 ml; Versene ( 3 2 mg/ml pH 8.0) 1.28 ml; lysozyme (It mg/ml) 0*6It ml. The complete mixture minus c e l l suspension was brought to room temperature and the c e l l s added -39-slowly, while being s t i r r e d by means of a magnetic s t i r r i n g apparatus. Lys i s was complete i n 30'minutes at room temperature. The preparation at t h i s time was extremely viscous. To decrease the v i s c o s i t y , 0.2 ml of 1 M. MgCl2 and 0.03 ml of Ulase; ' (1 mg/ml) were added per ml of c e l l suspension and the s t i r r i n g continued an a d d i t i o n a l 5 minutes. The preparation was centrifuged twice at 5000xg; f o r 15 minutes at l*0© to remove whole c e l l s and the supernatant ( c e l l f r e e extract) removed with a Pasteur p i p e t t e . Viable counts on the c e l l extract i n d i c a t e there are approximately 8: x 10^ c e l l s per ml. The p r o t e i n concentration of the extra c t from glucose-grown c e l l s averages about 16 mg per ml. Centrifugation of the crude c e l l extract at 26,000xg; f o r 30: minutes at l4°C y i e l d s a red p e l l e t designated,, membrane! fraction.- The supernatant (cytoplasm) i s c a r e f u l l y decanted and. centrifuged 2 hours at litO,000'xg i n a Splnco preparative u l t r a c e n t r i f u g e . The p e l l e t from t h i s l a t t e r c e n t r i f u -gation i s termed the ribosome f r a c t i o n , the supernatant i s the soluble cytoplasm. Both membranes and ribosomes are washed and suspended i n 0).l M T r i s b u f f e r pH 7.-U containing 0.01 M. MgCl^ u s u a l l y to 2 times the o r i g i n a l concentration. Modifications of the above f r a c t i o n a t i o n procedure were made; occasion-a l l y and w i l l be given i n the t e x t . V I I I . Column chromatography of rad i o a c t i v e phosphate es t e r s The TGA used t o stop the re a c t i o n and p r e c i p i t a t e the p r o t e i n was extracted with e t h y l ether (3 to 5 times). The phosphate esters were then converted to t h e i r hydrogen-form by hatch-*jise treatment with Bbwex-50 (M*) ion^-exchange resin.'„ The preparation was d i l u t e d f i v e f o l d , and run on t o a Dbwex-1 anion-exchange column i n the c h l o r i d e form (200-liiOO' mesh, 10%, cross-linked!). The column was eluted i n a step-wise manner using the f o l -lowing sequence and volumes of eliutants (110.) • (a) 0.001 M NH^OH, 100 ml (b) 0.O25: M NH^Gl 0.01 M K ^ O y , 1000 ml (c) 0.025 M NH^Cl 0.0025 M NH^OH 0.001 M KgBj^Oy, 100O ml (di) 0.025 M NHhCl 0.0025 M N;%0H 0.00001 M K 2B^0 7 ;, 1000 ml (e) 0.03 M; NB^Cl,, 500 ml. (£) 0.005 M HCl, 1000 ml (g) 0.01 M HCl, 1500 ml (h) 0.02 M HCl 0.02 M KC1, 5&0' ml (i) 0.02 M HCl 0.20 M KCl, 1000 ml A pressure of 2 lbs; per square i n c h was applied to the head of the columni w i t h nitrogen gas. Volumes: were c o l l e c t e d from the columns i n 1Q> ml amounts with an automatic f r a c t i o n c o l l e c t o r . The r a d i o a c t i v i t y and the o p t i c a l density at 260 mp. were determined on every f o u r t h tube except i n the region of r a d i o a c t i v e or 2601 mu absorbing peak, In which case each consecutive tube was examined. AMP', ABP and ATP' were i d e n t i f i e d by paper chromatography as ou t l i n e d in, p a rt V of Methods, and by paper electrophoresis with known solutions of these compounds. For paper electrophoresis of nucleotides, a O.OU M c i t r i c a c i d b u f f e r pH 3*8 was employed (79). Electrophoresis; f o r 3 hours at 750 v o l t s gave good separation. R a d i o a c t i v i t y and. 260; mu absorption were determined as before. Inorganic phosphate was. determined by the method of KIng.; Stcgar phosphates were i d e n t i f i e d e l e c t r o p h o r e t i c a l l y (111). Concentrations of adenine nucleotides were determined by the s p e c i f i c a c t i v i t y of t h e i r E 3 2 content, and also by t h e i r respective extinction! c o e f f i c i e n t s at,259 mu (l$.k x 10 3) (112). IX. F r a c t i o n a t i o n of c e l l e x t r a cts. 1. Treatment with protamine sulphate. (a) Ereparation of protamine sulphate s o l u t i o n . One gram of protamine sulphate was added to 50; ml of water and heated, u n t i l d i s s o l v e d . A f t e r cooling the pffiwas adjusted to ?.0< with KOH (1.2 ml of a 1 N. s o l u t i o n ) . The t u r b i d s o l u t i o n was d i l u t e d to 100 ml with water, cooled 20 minutes i n an ice-bath, centrifuged at 10,000 x g f o r 10) minutes and the p e l l e t discarded. (b) E r e c i p i t a t i o n of the nu c l e i c a c i d . The protamine s o l u t i o n was slowly added to the enzyme preparation i n an ice-bath with s t i r r i n g . Samples were removed, centrifuged and t h e i r O.D. at 260 mu and 280 1191. determined. Frotamine s o l u t i o n was added u n t i l a 280): 260 r a t i o of at l e a s t 0.65 was obtained. When t h i s was achieved the enzyme preparation was centrifuged at 10,000 x g at k°G f o r 15 minutes, and the p e l l e t discarded. 2. Ereparation of the pM 5>»0> enzyme f r a c t i o n . This i s the enzyme f r a c t i o n obtained by adjusting the pH of the 100,000' x g supernatant to pM 5.0 with I M acetic a c i d and c o l l e c t i n g the p r e c i p i t a t e by c e n t r i f u g a t i o n at 10,000 x g.. The p r e c i p i t a t e dissolves r e a d i l y i n 0.1 M. T r i s b u f f e r pH l.Ij.. 3. F r a c t i o n a t i o n with ammonium, sulphate. C r y s t a l l i n e (M^)2 ^ ' % w a s added slowly to the enzyme preparation i n an -1*2-ice-bath with s t i r r i n g . When, the desired l e v e l of s-aturation was obtained, the extract was centrifuged at 10,00© x g; f o r 15 minutes at U°G, the p e l l e t resuspended i n 0.1 M T r i s b u f f e r pHi 7.1* and dialyzed against 0.01 M T r i s b u f f e r pH ?.l* at 1*;°C with s t i r r i n g . 1*. F r a c t i o n a t i o n by u l t r a c e n t r i f u g a t i o n . This method has been p r e v i o u s l y o u t l i n e d 3 . ) . X. Enzyme assays. 1. Determination of adenylate kinase (myokinase) a c t i v i t y . The method.involves assaying f o r the disappearance of a c i d - l a b i l e phos-phate with ADP, glucose and hexokinase (113)• Reaction mixtures contained the following concentration of components: T r i s (0.1 M pffi 7.U) 1.00 ml; ADP (25 umoles/ml) 0.20) ml; MgClz (100 pmoles/ml) 0.10 ml; glucose (200 yumoles/ml) 0.20 ml;; hexokinase (10 mg/ml) Q).20i ml; TCA (60$) 0).3O ml; enzyme f r a c t i o n , 1* mg p r o t e i n ; water to 3.0 ml. Reactions were stopped a f t e r 15 minutes incubation at 30°C by the addition of TCA from a second side-arm of the Warburg v e s s e l , the f l a s k s r i n s e d with one ml of 5< per cent TCA and the combined suspension centrifuged at 10,000 x g; f o r 10; minutes., at 1*°C. One ml of 2.0 Ni HCl was added to 1.0 ml of supernatant and the s o l u t i o n heated 7 minutes at I J O O P C . Inorganic phosphate was determined by the method of King. S u f f i c i e n t controls were conducted to account f o r any release of inorganic phosphate that might occur by the assay method per se. A c t i v i t y i s expressed as /moles of a c i d - l a b i l e phosphate converted t o : inorganic phos-phate per mg of p r o t e i n during the 15 minute incubation, period. 2. I s o c i t r i c dehydrogenase assay. I s o c i t r i c dehydrogenase a c t i v i t y was determined spectrophotometrically -%3-by following the reduction of TPNi at 3k0] mu (Ul i i ) . The enzyme has been found to be associated with the soluble cytoplasm f r a c t i o n of the c e l l (115)}. XI. Synthesis and c h a r a c t e r i z a t i o n of polyadenylic acid., 1. Synthesis. The r e a c t i o n mixture contained the following components t Tris- (1.0 M pH S.0>) 0.50' ml; MgCl 2 (1.0 M) 6..0I ml; ADP (50> mg/ml) 0.50 ml;' ribosomal enzyme f r a c t i o n , 2 mg p r o t e i n , i n a f i n a l volume of 1.0- ml. The mixture was incubated at 30°G and. the progress of the reaction followed by deter-mining the formation of inorganic phosphate from; ADP'. P u r i f i c a t i o n of the polyadenylic acid was according to the method of Grunberg-Manago et al. (25), and involved the p r e c i p i t a t i o n of the polymer with i c e - c o l d ethanol, d i a l y s i s , r e p r e c i p i t a t i o n and drying from the frozen s t a t e . The y i e l d of the l y o p h i l i z e d m a t e r i a l was lU mg. 2. Characterization of the polyadenylic a c i d . Two 5 mg samples were removed and dissolv e d i n 1 ml of d i s t i l l e d water gi v i n g a very viscous s o l u t i o n . One ml of 2 N NaOl was added to one sample and the s o l u t i o n heated at 100°G to hydrolyze the polymer. A f t e r c o o l i n g , the s o l u t i o n was passed through a small column (Pasteur p i p e t t e ) of Dowex 50' lonvexchange r e s i n i n the ammonium form to remove sodium Ions. Samples from each s o l u t i o n were spotted on No. 3 mm f i l t e r paper along with c o n t r o l spots of AMP and ADP. Electrophoresis was c a r r i e d out on the hydrolyzed and non-hydrolyzed samples i n 0.0lt M c i t r a t e b u f f e r , p>M 3»8 f o r 2 hours at 750' v o l t s . The non-hydrolyzed s o l u t i o n gave one u l t r a v i o l e t absorbing' spot at the o r i g i n (polyadenylic acid)) and a trace spot corresponding to ADP'. The hydrolyzed. sample produced two spots, one corresponding to AMP, and. a f a i n t spot corresponding to ADP'. The presence of AMP' i n the hydrolyzed sample c o r r e l a t e d with an increase i n inorganic phosphate concentration concurrent with a l o s s i n o p t i c a l density at 260 mn^therefore^characterizes the product of the r e a c t i o n as polyadenylic a c i d . RESULTS AND DISCUSSION I.. I n i t i a l studies with c e l l f r e e extracts Previous work i n t h i s laboratory (106,ll6)> f a i l e d to demonstrate a substrate-dependant phosphorylation i n c e l l extracts of P. aeruginosa* A number of experiments were therefore undertaken to confirm these observa-t i o n s . The experiments' demonstrated that c e l l extracts;, prepared by means of a Hughes;* Press, were capable of o x i d i z i n g glucose, gluconic a c i d and suc-c i n i c a c i d , and that incubation i n the presence of radioactive inorganic phosphate (P 3 2)> r e s u l t e d i n a t r a n s f e r of phosphorus from the medium to a charcoal-adsorbable form (concurrent with a loss of P 3 2 from the external medium). In the presence of an oxidizable substrate however there was always l e s s P 3 2 incorporated. The r e s u l t s of a t y p i c a l experiment, wi t h glucose as substrate, are shown i n Figure 1. (Reaction conditions and determination of P 3 2 incorporation are given i n Methods.) That the i n h i b i t i o n of inc o r p o r a t i o n by substrate was accompanied by l e s s ATP formation was shown by i s o l a t i o n of ATP and by spectrophotometry assay ( U 6 ) . Attempts to demonstrate e s t e r i f i c a t i o n of orthophosphate by following the l o s s of inorganic phosphate from the system e i t h e r f a i l e d or were i n -conclusive (106). Repeated attempts to obtain p o s i t i v e data f o r the presence of oxidative phosphorylation i n the organism were unsuccessful and l e f t the impression that e i t h e r the process d i d not e x i s t or i t was completely disrupted, (un-coupled) . Hoyenkarap (92), studying oxidative phosphorylation i n c e l l extracts of A. v i n e l a n d i i , demonstrated that when sucrose was employed as a sus-pending di l u e n t f o r the c e l l e xtract, P:0 r a t i o s were considerably reduced. Since a l l previous work i n t h i s laboratory was performed with sucrose i t was thought worthwhile t o determine the e f f e c t of sucrose i n our system. Employing i d e n t i c a l r e a c t i o n mixtures as above, the same r e s u l t s were: obtained with or without sucrose, i . e . i n h i b i t i o n of P 3^ incorporation on addition of an oxidizable substrate. To help e x p l a i n the anomolous: r e s u l t s obtained with the c e l l extracts, a search of the l i t e r a t u r e was made pe r t a i n i n g to phosphorus metabolism i n aeruginosa. I t was soon evident however that very l i t t l e such informa-t i o n was a v a i l a b l e . A study of phosphate metabolism i n i n t a c t c e l l s there-fore seemed to be the most l o g i c a l place t o s t a r t our i n v e s t i g a t i o n s . The object of t h i s p art of the work was then to obtain Information concerning the conditions which a f f e c t the i n c o r p o r a t i o n of phosphorus i n t o r e s t i n g c e l l suspensions, the dependance of t h i s i n corporation on substrate and f i n a l l y , knowledge as to the i n t r a c e l l u l a r l o c a t i o n and turnover of the phosphorus. I I . Studies with r e s t i n g c e l l suspensions 1. Reduction of the endogenous r e s p i r a t i o n The high endogenous r e s p i r a t i o n exhibited by t h i s organism i n t e r f e r e d with the i n t e r p r e t a t i o n of data from studies on i n t a c t c e l l s and c e l l ex-tracts.- For t h i s reason experiments- were, undertaken to reduce or remove t h i s a c t i v i t y . C e l l s were harvested i n the usual way and resuspended i n a complete medium minus a source of nitrogen: (A)! or a source of carbon (B), or both (G-). The preparations were then aerated at 30°G f o r varying lengths of time, the c e l l s c o l l e c t e d and t h e i r endogenous a c t i v i t y determined mano-metrie a l l y . (Table 1). Table 1. Reduction of endogenous r e s p i r a t i o n by aeration i n various media. Aeration Per cent reduction of rate: of oxygen uptake time (min): ; Mediumt A Medium: B Mediumi G 30 hi 2 $h 60 51 it 55 120 60; 8: 59 Although i t was po s s i b l e to reduce the endogenous a c t i v i t y about 6b per cent by these methods, the ex t r a manipulations required were not con-sidered s u f f i c i e n t l y rewarding and a l e s s troublesome method was sought. The omission of yeast extract from the cu l t u r e mediumi was the method, of choice,. The y i e l d of c e l l s on t h i s medium, was 20 per cent lower than with media containing yeast extract, however t h i s was compensated f o r by a 65 per cent reduction i n endogenous a c t i v i t y . 2. Substrate-dependant phosphorylation R e s t i n g - c e l l suspensions were prepared as: o u t l i n e d i n Materials and Methods and incubated 90 minutes at 30°C' with glucose, gluconate or 2-keto-gluconate as substrate, the reactions stopped by the addition of TCA and the r a d i o a c t i v i t y of the c e l l s determined. The amount of phosphorus i n -corporated was shown to be a f u n c t i o n of the amount of substrate oxidized (Table 2). Table 2. Substrate dependant incorporation of orthophosphate. jumoles phosphorus incorporated Substrate glucose gluconic 2-keto concentration gluconic O.Oyumoles. .037 .037 .027 0>.5 " .0U6 .OUU .039 1.0 » .065 .051 .01*8: 2.5 " .090 .081 .068 5.0: » .113 .102 .095^ I t was also evident that equivalent amounts of the more oxidized substrates gluconate and 2-ketogluconate gave lower in c o r p o r a t i o n of radioactive, phos-phate. Since there i s no evidence t o suggest that glucose oxidation v i a gluconate and 2-ketogluconate generates energy at the substrate l e v e l i n t h i s organism, the observed differences i n available energy from the three substrates suggests that energy i s gained by the passage of electrons to oxygen by the e l e c t r o n transport system of t h i s organism. C a l c u l a t i o n of P:0 r a t i o s from such data i s not p o s s i b l e since i t has been shown (117) that there i s considerable incorporation of ammonia on the addition of sub-s t r a t e under the conditions employed and presumably t h i s r e a c t i o n would involve the expenditure of considerable amounts of energy. 3. The e f f e c t of substrate concentration A study of conditions a f f e c t i n g i n c o r p o r a t i o n of ' F 3 2 i n t o r e s t i n g c e l l s was i n i t i a t e d with experiments designed to examine the e f f e c t of substrate concentration on phosphate in c o r p o r a t i o n . Oxygen uptake was also followed. -k9-Oxygen consumption was shown to increase p r o p o r t i o n a t e l y with inh-ere asing substrate concentrations over the range of glucose concentrations: employed. F ^ 2 incorporation, on the other hand, increased with increasing substrate concentration over a r e l a t i v e l y l i m i t e d range. (Figure 2). In-creasing the concentration of glucose beyond 10/imoles/vessel lowered, incorporation to a value which nei t h e r increased nor decreased on f u r t h e r addition of glucose. S i m i l a r r e s u l t s were obtained w i t h gluconate and 2-ketogluconate (Figure 3). Again, with the l a t t e r substrates i t may be seen that concentrations, higher than those required with glucose were needed to allow equivalent amounts of P^ 2 Incorporation. From the shape of the curves obtained above, one might expect that some f a c t o r i n the system other than substrate became l i m i t i n g . That the r e s u l t s could p a r t l y be explained by such a view was shown to be correct when the e f f e c t of adding ammonium ions (Nflj^ ) on P^ 2 incorporation was studied. The choice of ammonium' chloride was not completely a matter of conjecture since, as mentioned above, during glucose oxidatidn by r e s t i n g c e l l s there i s a l o s s of ammonia from the suspending medium* The r e s u l t s obtained with added NH^Cl are shown i n Figure 1*. To say t h a t F V incorporation stops when NHj^ becomes l i m i t i n g i s only p a r t l y true. Thus, i f one analyzes the r e a c t i o n v e s s e l contents from a mixture containing i n i t i a l l y 3© /umoles of glucose arid 25 /umoles of NH^Cl, one f i n d s that at the end of the r e a c t i o n (maximum; phosphorus incorporation); 12 /umoles of glucose and 1? yumoles of N % remain. Yet, the addition of more NHj^ * to the r e a c t i o n mixture w i l l , produce a f u r t h e r stimulation of p32 i n c o r p o r a t i o n . A complete explanation f o r t h i s l a t t e r observation and also why one should observe an i n h i b i t i o n of P^ 2 i n c o r p o r a t i o n a f t e r a given • endogenous O glucose -o ! ! I 1 I 1 D 10 20 30 T I M E (minutes) F I G . I I n c o r p o r o t i o n of radioactive, phosphorus into charcoal adsorbable material of a cell extract. /JMOLES PHOSPHORUS INCORPORATED -52-0.30 r Q LU < O Q. DC O U Z C f t z> o s V) O a. co ID d 0.20 L 0-10 I-O glucose 3 gluconate © 2-ketogluconate 10 20 30 /JMOLES SUBSTRATE 40 FIG- 3 Phosphorus incorporation into intact* cells with various substrates. -53--5U-concentration of.substrate, i s not as yet a v a i l a b l e . I t would seem probable that the r e s u l t s might r e f l e c t a c o n t r o l mechanism i n the c e l l which i s involved i n maintaining a carbon-nitrogen, balance. J|. E Incorporation as net phosphate uptake. The p o s s i b i l i t y that the observed incorporation of radioactive phos-phate i n t o whole c e l l s was an exchange of phosphate and riot a net increase was examined by following the change i n t o t a l phosphate concentration of the c e l l s as a f u n c t i o n of time. Inorganic phosphate was determined by the method of King (10U). The net increase i n i n t r a c e l l u l a r phosphate during the incubation period (Figure £) confirms the suggestion that the r e s u l t s with E ^ 2 were i n d i c a t i v e of net phosphate i n c o r p o r a t i o n . i: $>. I n t r a c e l l u l a r l o c a t i o n of phosphate. The high rate of the endogenous r e s p i r a t i o n i n t h i s organism r e s u l t e d i n the incorporation of appreciable quantities of E-^ 2. However, as may be' seen in. Figure !>, t h i s uptake was increased almost three f o l d by the pre-sence of substrate. F r a c t i o n a t i o n of the c e l l contents i n t o inorganic, nu c l e i c acid, sugar, charcoal adsorbable and ether, ether-alcohol soluble phosphorus, demonstrated, tha t most of t h i s increase In E ^ 2 could be. 32 accounted f o r by an Increased E^ content of the n u c l e i c a c i d f r a c t i o n * T h i s agrees very c l o s e l y with the data of Hotchkiss f o r S. aureus (16)). To determine i f the £ ^ incorporated i n t o the n u c l e i c a c i d f r a c t i o n was concentrated i n DMA,. RNA or both, the residue remaining a f t e r treatment with ether-alcohol mixture (see Methods) was extracted with. 1 N NaOH! at 100°C f o r 30 minutes. This treatment s o l u b l i z e s the RNA. A f t e r cooling and adding TGA to p r e c i p i t a t e the DNA, the s.upernatant was; again examined. o UJ I-< or o a or o o z CO or o x a. co o x a. w yj _j o z 0 . 4 0 0 . 3 0 0 . 2 0 0.10 ® o whole c e l l s • n u c l e i c a c i d s © s u g a r P, inorganic P. m, c h a r c o a l adsorbab le R 9 e t h e r ft e t h e r - a l e . s o l . P 4 0 8 0 i UT. I 4 0 8 0 TIME FIG. 5 120 ( m i n u t e s ) P h o s p h o r u s i n c o r p o r a t i o n into r e s t i n g c e l l s . ( A ) E n d o g e n o u s . I B ) W i t h g l u c o s e . 120 f o r r a d i o a c t i v i t y * The r e s u l t s are tabulated i n Table 3 Table 3 . F r a c t i o n a t i o n of nu c l e i c acids. P 3 2 incorporated (cpm) Fr a c t i o n Endogenous Glucose Whole c e l l s 5lU0 ' 9500 T o t a l n u c l e i c a c i d 267T3 5300 (hot TGA) Tota l n u c l e i c a c i d 2661 1*796 (hot NaOH)-RNA only 2650 1*630* % RNA-P'32 of t o t a l 99.6 .,, 97-1+ As would be expected, almost a l l of the p 3 2 Incorporated i n t o n u c l e i c acid.could be accounted f o r as RNA phosphate. To confirm that the inc o r p o r a t i o n of F 3 2 i n t o RNA represented a tu r n -over or synthesis of t h i s m a t e r i a l and not a p r e f e r e n t i a l l a b e l l i n g of one nuc l e i c acid, residue, the mat e r i a l obtained by a l k a l i n e hydrolysis was; fr a c t i o n a t e d on a Dowex-1 ion-exchange column. RNA nucleotides were iden-t i f i e d by t h e i r c h a r a c t e r i s t i c absorption spectra and by paper chromato-graphy with standard nucleotides. R a d i o a c t i v i t y was determined d i r e c t l y on the chromatograms. I t was possible to show that a l l f o u r bases of the RNA v i z . GMP, CMP, UMP* and AMP' contained radioactive phosphate i n s u f f i c i e n t quantity to account f o r the r a d i o a c t i v i t y observed i n the undegraded RNA. 6. E f f e c t of metabolic i n h i b i t o r s . The e f f e c t of a number of metabolic i n h i b i t o r s on phosphate incorpora-t i o n i n t o whole c e l l s was examined (Table U). The substrate-dependant i n -corporation of P^ 2 was not appreciably changed with e i t h e r DNP' or sodium, f l o r i d e . I t would appear however that the endogenous incorporation of P^ 2 was markedly decreased by f l u o r i d e and s l i g h t l y increased by DIP. By the use of the i n u l i n space method, i t has been found that DNP! f r e e l y penetrates the c e l l membrane o f t h i s organism. (11&). At concentrations of chloram-phenicol shown to prevent induced enzyme formation i n . t h i s organism an almost two-fold increase i n the rate of P^ 2 Incorporation occurred.. The observation again i s i n agreement wit h the concept that p r o t e i n synthesis causes a decrease i n net phosphate uptake.. The experiment w i t h cyanide: serves to emphasize that the incorporation, of P 3 2 i s dependant on the oxidative enzymes of the organism. The concentration of pyocyanin employed was s u f f i c i e n t to stop oxidation of glucose at the 2-ketogluconate stage and under these conditions no substrate-dependant incorporation of P 3 2 occurred. The addition of magnesium reversed the i n h i b i t i o n , of both oxygen uptake and P-' incorporation, thus suggesting that the a b i l i t y of pyocyanin to uncouple oxidative phosphorylation i s due to it's a b i l i t y to bind mag-nesium. (119). The oxygen and P^2 values obtained with whole: c e l l s plus pyocyanin resemble the data obtained e a r l i e r with c e l l extracts and no added pyocya-n i n . In each instance the oxidation of glucose stops when two atoms of oxygen have been taken up and P^ 2 Incorporation I n the presence of added substrate i s . below that of the endogenous. -58-Table h* E f f e c t of i n h i b i t o r s on phosphate incorporation of re s t i n g c e l l s of B. aeruginosa. . Control DNP NaF CMP KCN Pyocyanine (1X1Q~J) (1X10~^) (20) ug/ml) (IXlor3)) (1.5X10^) Phosphate uptake (uM/ml) _ — (min) E S' E S E S E S E S' E S 15 .08; .13 .08 .11 ,o5: .08 .o? .11 .ok .ok 30 .09 .15 .10 .19 .07 .18 .07 .25 .05 .05 -Ok .03 60 .11 .36 .17 .33 .08 .21+ .09 .1+2 .05 .05 .05 .Ok 90s .1$ .39 .20 .kk .09 .30 .12 .1*8 .05- .0% -x- Endogenous -:H;- + substrate •5HHS- Chloramphenicol 7. Incorporation with nitrogenous substrates. The importance of available nitrogen to the metabolism of resting c e l l suspensions of this organism was again, evident from the results obtained with nitrogenous substrates. Preliminary experiments using numerous nitro-gen-containing substrates, indicated that compounds differing to the extent of only one amino group could produce marked effects on P^2 and oxygen uptake. In Figure 6 oxygen uptake and. B^ 2 incorporation are compared with equimolar concentrations of aspartate and asparagine as substrates. Resting; cells took up twice as much oxygen with asparagine as substrate as they did with aspartate* This was accompanied by an additional Q-.Ol*>umole of phos-phorus incorporation* Assuming there are no permeability differences between the two substrates, the additional amide group on asparagine must be respon-sible for this increased activity. III. Application of data from whole c e l l studies to studies" with c e l l extracts The studies below were performed with c e l l free extracts prepared by means of a Hughes1 Press as outlined In Methods and Materials. 1. The effect of substrate concentration* At high concentrations of glucose, i t was observed that there was an inhibition of P ^ Incorporation into whole c e l l s . Reduction of p32 incor-poration i n c e l l extracts on addition of substrate might then occur by a similar mechanism but at a lower concentration of substrate. To test the v a l i d i t y of this hypothesis, c e l l extracts were incubated with glucose concentrations ranging from; zero to l+Oyumoles per ml of reaction mixture. P'32 Incorporation into charcoal-adsorbable material was followed as an index of phosphorylation, activity. It may be seen i n Table £: that the inhibition by -60w 1 T I M E (minutes) F IG . 6 Oxygen and phosphorus uptake with a s p a r t a t e and a s p a r a g i n e . -61-substrate was complete at a glucose concentration of O.OOUyuraoles per ml, Table g. Influence of glucose concentration on the in c o r p o r a t i o n of phosphate by c e l l extracts Glucose concentration Phosphate uptake ^umoles/ml) (jumoles/ml) 0.000 0.02k 0.00k 0.01? o.oUo 0.017 o.Uoo 0.017 U.000 0.017 Uo.ooo 0.017 I t should be mentioned that values f o r phosphate incorporation such as those l i s t e d i n Table 5> are cal c u l a t e d on the basis of s p e c i f i c a c t i v i t y and r e f l e c t large differences i n counts per minute of radioactive phosphate. The stimulation of incorporation observed w i t h whole c e l l s on the addition of NHj^Cl was not apparent with c e l l e x t r a c t s . The use of pyruvate, succinate and g l u t am ate as substrates also f a i l e d to support a substrate-dependant phosphorylation i n the c e l l f r e e e x t r a c t s . IV. The preparation of c e l l f r e e extracts 1. Preparation of extracts by means of a Hughes' Press. The method has been given i n d e t a i l i n Materials and Methods and involves at le a s t one marked disadvantage namely, the f r e e z i n g and subsequent -62-thawing process. The detrimental e f f e c t of f r e e z i n g on oxidative phosphory-l a t i o n systems has: been observed i n both animal and b a c t e r i a l preparations (120). The: p o s s i b i l i t y t h a t the method of. preparing our extracts might account f o r t h e i r p e c u l i a r behaviour l e d us to i n v e s t i g a t e other methods of c e l l breakage. 2. Sonic o s c i l l a t i o n . Preparations of c e l l f r e e extracts by t h i s method i s given i n Materials: and Methods. When these extracts were incubated with P 3 2 under the same conditions as the Hughes* Press: extracts, the r e s u l t s were i d e n t i c a l i . e . i n h i b i t i o n of incorporation by substrate. The s e v e r i t y of t h i s method, on. p a r t i c u l a t e c e l l f r a c t i o n s was examined (Table 6). Table 6. The e f f e c t of sonicing time on o p t i c a l density and. v i a b l e count. Sonicing time- % of i n i t i a l (min.) O.D:. Viable count 0 100 100 1 71 7/6 2 55 51 k 39 33 6 2k 21 8 19 1% 12 . 1 2 6 16 - 8 3 -63-The degree of solublization of the cell suspension as a function of sonicing-time is readily observed by following the decrease in optical density (O.D.) at 6^ 0 mu. The requirement for particulate systems in oxidative phosphory-lation observed with animal mitochondria as well as the extreme lability of these preparations, led us to abandon this method for the routine prepara-tion of cell extracts. When used, a sonic time of four minutes was em-ployed. - 3. Preparation of cell extracts by osmotic lysis of spheroplasts. Bacterial protoplasts have been shown to perform identically to com--plete cells with respect to their general metabolism. When the protoplasts are suspended in, hypotonic solution* rupture of the membrane occurs and a cell free extract is obtained. The advantages of cell extracts prepared in such a manner are obvious: the cell has not been subjected to vigorous rupturing processes or severe changes in temperature; solublization of particulate components would be expected to be at a minimum; and finally, breakage by such a method should allow easy separation of cytological fractions (cell membrane, ribosomes; etc.). Relatively few bacterial species (all. Gram positive) are capable of undergoing protoplast formation by the usual method of treatment of a bacterial suspension with lysozyme. Recently however, numerous reports-have appeared referring to the preparation of "protoplasts'* of Gram negative organisms. The criterion for protoplast formation being a conversion of a rod-shaped organism to a spherical form in a medium, of suitable composition (5 to 20. per cent sucrose) and osmotic sensitivity of these structures. Since these structures may not be true protoplasts, in terms of those ob--6U-.tained w i t h Gram p o s i t i v e c e l l s , and remnants of c e l l wall, m aterial might e a s i l y remain attached to the s p h e r i c a l structures, the term protoplast has been modified to p r o t o p l a s t - l i k e or "protoplast" 1. When the c e l l w a l l structure has been modified by such treatment as growth .in p e n i c i l l i n t o produce globular forms, the r e s u l t i n g structures are u s u a l l y termed sphere— p l a s t s (121) . Repaske (122) found that i n a lysozyme-versene-tris system, c e l l s . b f E. c o l i , A. v i n e l a n d i i and P. aeruginosa were susceptible to lysozyme treatment. ; Mahler and Eraser (123) went on to show that i n the case of E. c o l l l y s i s could be prevented and the action arrested at a stage of spher-i c a l , o smotically shockable, "protoplasts" i f sucrose (0 .5 M) was present. A d e t a i l e d study of the method reported by Repaske was made to determine the optimum conditions f o r r a p i d i t y and completeness of c e l l breakage by the lysozyme-versene method wit h P. aeruginosa. C e l l s were; harvested, washed and suspended i n 0*03 M T r i s b u f f e r pH 8 .0 to a f i n a l concentration of 800' mg per ml wet weight. : The degree of c e l l breakage was determined under various conditions by following the decrease In 0 *D;<. of the c e l l suspension at 65Q mu i n a Model DU spectrophotometer. To obtain optimum- bre.akage the complete systems required the following concentrations of components per ml of c e l l suspension: T r i s (0.125 M pH 8 .0) 0»6h ml; Versene (32 mg per ml pM 8.©) 1*28) ml; lysozyme (1*. mg per ml) 0 .6U ml. The omission of Versene from the mixture reduced breakage by 53 per cent* Without added lysozyme a 2l* per cent reduction in. O.D.. occurred. Thus, versene alone i s active i n producing c e l l l y s i s * The p H of the mixture was shown to have a marked influence on a c t i v i t y ; -65-the more a l k a l i n e mixtures producing more complete l y s i s . Varying the pS from 6.5 to 8.5 increased l y s i s from 29 to 7/6 per cent. A pH of 8.5 was considered too high however f o r the retention of active e x t r a c t s and a l l subsequent preparations were made at pH 8'.0. Increasing the molar i t y of the T r i s b u f f e r to 0.03 M increased! both the rate and the t o t a l amount of l y s i s . No f u r t h e r increase i n l y s i s was noted with m o l a r i t i e s above 0.03 M. The concentrations of versene and lysozyme given above were optimum, incr e a s i n g the concentration of e i t h e r component d i d not a l t e r the a c t i v i t y . When sucrose was included i n the re a c t i o n mixture at a f i n a l concen-t r a t i o n of 0.6 M a conversion of the rod-shaped c e l l s to s p h e r i c a l forms could be observed by phase contrast microscopy. Unless otherwise stated, c e l l f r e e extracts employed i n the experiments to follow were prepared by the lysozyme-versene method as o u t l i n e d i n Methods. A separate study has been undertaken i n t h i s laboratory (115) to deter-mine the I n t r a c e l l u l a r l o c a t i o n of numerous enzyme systems of P. aeruginosa w i t h c e l l f r a c t i o n s obtained by d i f f e r e n t i a l c e n t r i f u g a t i o n of the lysozyme-versene c e l l e xtract as o u t l i n e d i n Methods. Thus i t has been possible to characterize enzyme a c t i v i t y as: being associated with the ribosomes, the membrane or the soluble cytoplasm of the c e l l . Some of these a c t i v i t i e s i . e . adenylate kinase, ATP'ase and polynucleotide phosphorylase, were deter-mined i n the studies given below because of t h e i r e f f e c t s on oxidative phosphorylation or exchange a c t i v i t y . V. Oxygen uptake studies with c e l l f r a c t i o n s A measure of the a c t i v i t y of the c e l l f r a c t i o n s was achieved by deter-mining t h e i r a b i l i t y to oxidize various substrates. C e l l f r e e e x t r a c t s -66-prepared by methods; other than the lysozyme-versene technique were shown e a r l i e r to take up two atoms of oxygen per mole of glucose and one,atom per mole of gluconate. The lysozyme-versene e x t r a c t s y i e l d e d i d e n t i c a l r e s u l t s . The usefulness of the method however was emphasized, when upon f r a c t i o n -a t i o n i n t o membrane and cytoplasm f r a c t i o n s i t was shown that w i t h glucose as substrate the membranes alone could account f o r the uptake of 1 atom of oxygen wi t h glucose, and secondly, with the membranes very l i t t l e , i f any endogenous uptake of oxygen was observed (Figure: 7). Warburg vessels con-tai n e d T r i s (0.1 M pH 7.!*} 1.0 ml; MgCl 2 (100 /moles/ml} 0.1 ml; KOH (20$Q 0.15 ml; substrate 25ycrmoles/ral) 0,2 ml; enzyme f r a c t i o n , 1.0 ml. The f i n a l volume was made to 3.15' ml wit h water. VI. Radioactive phosphate studies with c e l l extracts; In previous studies with c e l l f r e e extracts, P 3 2 incorporation i n t o charcoal-adsorbable m a t e r i a l was used as a measure of phosphate uptake. The method was not considered to be a s u f f i c i e n t l y accurate measure of Incorporation and a more r e l i a b l e method was sought. The method of choice was that used by Nielsen and Lehninger (1||8) f o r the study of phosphoryla-t i o n coupled to the oxidation of ferrocytochrome c. The method i s i n f a c t a modification of an e a r l i e r method described by Ernster et a l . (105)• The technique employed i n our studies and the experiments designed to determine, the s e n s i t i v i t y of the method is: o u t l i n e d i r i Methods. 1. Incorporation of rad i o a c t i v e phosphorus i n t o c e l l f r a c t i o n s . The incorporation of P 3 2 i n t o organic m a t e r i a l was determined using crude c e l l extracts;, membranes and cytoplasmic f r a c t i o n . Oxygen uptake was followed s i m u l t a n e o u s l y . The rea c t i o n vessels contained Tris: (0.1 M JULITERS OXYGEN CONSUMED -19-JU LITERS OXYGEN CONSUMED -68'-pH 7.1+) 0.8 ml; cytochrome c (1.5 x Kf^M) Q:.1 ml; ADP (i+0 /lmoles/ml) 0.10 ml; KaE (200 pinoles/ml) 0.10 ml; MgCl 2 (1+00 /moles/ml) O'.IO and c e l l f r a c t i o n , 1.0 ml. The trapping system consisted of hexokinase (10 mg/ml) 0.10 ml; fructose (100 pmoles/ml) 0.10 ml. Inorganic phosphate (l+0! yamoles "/ml, containing O.U p c u r i e s of P 3 2 ) 0.2 ml, and substrate 200 /imoles/ml) 0.1 ml were: tipped i n from the side arm at zero time. KOH (20$) 0.15 ml was added t o the centre-well. Reactions were stopped a f t e r 15 minutes i n -cubation at 30°C by t i p p i n g i n TGA (60$) 0'.3 ml from a second side-arm. Water was added to b r i n g the t o t a l volume to 3»15 n i l . Results from a t y p i c a l r e a c t i o n mixture with glucose and glucose-6-phosphate as: substrates are tabulated i n Table 7'» Results were comparable from one experiment to another. With DPNM, malate and. succinate, substrate-dependant phosphory-l a t i o n could not be observed. Table: Phosphorus incorporation i n t o c e l l fractions,. F r a c t i o n C e l l extract Membranes Cytoplasm Cytoplasm membranes' Endogenous 0 2 P P/0 ,30 .01* .13 .15 .01+ .27 .50 .03 .06 Glucose G-6-P 0 2 P P/0 P/0° 0 2 P P/0 p/o( 12.3 . 23 .02 .02 .82 .05 .06 .02 7.1*. .02 - - -.8'* .01+ .05 - .1+3 .01+ .09 -8.7 .16 .02 .02 .11* .01* .30 -*• i n juatoms; • H * i n /imoles o corrected f o r endogenous a c t i v i t y - 6 9 -The P:0 ratios given in the final column are corrected for endogenous oxygen and phosphate uptake, I.e. they represent substrate dependant phosphorylation* The values are of course not high enough to allow a quantitative interpretation* However, i t was felt that since this' repre-sented the first positive data for the presence of a substrate dependant oxidative phosphorylation in this organism,, further work was warranted. 2. The effect of omitting various components from the system. Experiments were first conducted to determine the effect on both oxygen and Y uptake of omitting various, reaction components. The com-plete system refers to that given in Table ?. The concentration of KCN, -3 when present was % x 10 M. Conditions were Identical to those above. Table; 8* The effect of omitting various; reaction components 32 on ]r incorporation. Reaction Endogenous Glucose °2 •5BS- . s P P/0 °2 p p/o P/0° Complete 0.0 0.81* 6.1 0*89 .15 -minus NaF .0.0) 0.31*. 6.2 0.1*5 .07 .02 minus trap 0.0 1.10 k.9 1.10 .22 -minus trap 0.0 1.10 U.T 1.00 .22 mm minus NaF Complete + 0.0 0.91 l.O o.9U .9k .03 KCN f in /latomsj in yumoles p corrected for endogenous: activity -70-The r e s u l t s i n d i c a t e d that i t was very u n l i k e l y that the observed 32 i n c o r p o r a t i o n of YJ was i n d i c a t i v e of an oxidative phosphorylation process. The f o l l o w i n g points are evident r (a) In the endogenous system the l a c k of measurable oxygen uptake rules out an oxidative phosphorylation system as being respon-s i b l e f o r P^ 2 Incorporation. (b) Cyanide, a potent i n h i b i t o r of oxidative phosphorylation, stimu-32 l a t e d P^ uptake by about 8 per cent while i n h i b i t i n g oxygen uptake by 81i, per cent. (c) Incorporation of P'32 was stimulated two f o l d by the omission of the hexokinase trapping system. 3. E f f e c t of magnesium. D i a l y s i s of the crude c e l l e xtract against M/100 T r i s b u f f e r pH 7'.U f o r two hours at in°C r e s u l t e d i n a 90 per cent l o s s i n the incorporation 32 -3 of p- . Addition of MgCl2 to a f i n a l concentration of 3 x 10 M com-p l e t e l y restored a c t i v i t y . U. The e f f e c t of ADP concentration. In view of the above r e s u l t s , the e f f e c t of ADP concentration on P^ 2 incorporation was examined. Reaction vessels contained T r i s (0.1 M pH 7.U) 0.80 ml; hexokinase (10' mg/ml) 0 .1 ml; ADP (100 ^xmoles/ml) as given i n Table 9; MgCl^ (100/jmoles/ml) 0.10 ml; fructose (100 jumoles/ml) 0.10 ml; glucose (200jumoles/ml) 0.10 ml; inorganic phosphate (50/mioles/ml, con-t a i n i n g 0.1 / i c u r i e P 3 2 ) 0.20 ml; TCA (60$) 0.30 ml; enzyme (crude; c e l l extract) 1.0 ml; water to 3.0 1 ml. Incubation time and temperature were 1$ minutes and 3Q°C. Results are tabulated i n Table 9 . -71-Table 9. The e f f e c t of ADP concentration on phosphorus incorporation. ADP' concentration ~ Endogenous; Glucose (umoles) 0* P** P/0 0 2 P P/0 0' 1.1 .06 .05 5.7 .05 .01 5 1,2 .16 .13 5.5 .18 .03 10 1.2 .50 .1*2 5.9 .58 ; .10^ 20 1.0 .96 .96 6.0 .88 .15 1*0 1.1 .62 . 52 6.0 . 58 .10 I n /latoms •K-if- i n yumoles A d e f i n i t e r e l a t i o n s h i p was shown to e x i s t between the amount of ADP added and the yumoles of phosphate taken up. The phosphate uptake was not a f u n c t i o n of substrate oxidation and therefore we were not measuring an oxidative phosphorylation process. That we were probably measuring an exchange of r a d i o a c t i v e inorganic phosphate with bound organic phosphate was i n d i c a t e d . This view was strengthened by the f i n d i n g that although the r e a c t i o n mixture containing 20 jumoles of ADP had taken up close to one /imole of inorganic phosphate, as determined by the s p e c i f i c a c t i v i t y of the organic P32 , no net l o s s of inorganic phosphate from the external medium: could be; shown. The evidence i n favor of an exchange r e a c t i o n i n t h i s system may be summarized as follows: (a) Radioactive inorganic phosphate i s incorporated i n t o an organic -72-form. (b) No net uptake of inorganic phosphate could be shown. (c) Magnesium i s required f o r the i n c o r p o r a t i o n of P 3 2 . (d) Incorporation of P'3^ increased with i n c r e a s i n g concentrations, of ADP. (e) ATP' i s formed. ( f ) Incorporation of P 3 2 ; i s not dependant on the oxidation o f substrate. . I t was mentioned, i n the review of the l i t e r a t u r e t h a t considerable data has been obtained on the mechanisms of oxidative phosphorylation through studies of adenine polyphosphate-p 3 2 exchange rea c t i o n s . Since an exchange r e a c t i o n was i n d i c a t e d i n our system, and because of the f a i l u r e to demonstrate coupled phosphorylation by the usual methods, the exchange r e a c t i o n appeared t o be the only available approach leading; to the e l u c i d a t i o n of the process of oxidative phosphorylation i n t h i s organism. Exchange-reactions between adenine nucleotides and inorganic phos-phate are not however p e c u l i a r to oxidative phosphorylation processes and as new information was obtained, other p o s s i b i l i t i e s were considered. Compounds incorporating radioactive phosphate i n c e l l extracts;. Large scale r e a c t i o n mixtures were prepared by i n c r e a s i n g the usual mixtures s i x foldi. F i n a l concentrations of r e a c t i o n components were i d e n t i c a l to those i n the previous experiment. The ADP' concentration chosen was that g i v i n g maximumi inco r p o r a t i o n i n Table 9* The object was to determine q u a n t i t a t i v e l y the l o c a t i o n of the radioactive organic P'32, i n the absence of an oxidizable substrate, by separation of the phosphate -73-esters and determination of t h e i r s p e c i f i c a c t i v i t i e s . A d e t a i l e d account of the procedure i s given i n Materials and Methods. Isolated peaks corresponding to inorganic phosphate, F-6-P, AMP, ADP and ATP'were obtained from ion-exchange columns. The compounds' were con-centrated and i d e n t i f i e d by paper chromatography and t h e i r respective con-centrations determined. Table 10. Concentration of r e a c t i o n components-.. Compound Concentration i n nmoles based on p 3 2 g*« F-6-p 1.^0 AMP' O.Ol* 5.12 -ADP 2.65 8.56 ATP 1.90- 3.56 P 22.20 - 39.6 •a- e x t i n c t i o n c o e f f i c i e n t -:s-* inorganic phosphate As may be seen i n Table 10, the concentration of AMP'plus ATP (8.68) i s very close t o that of ADP (8.56) , i n d i c a t i n g the presence of adenylate kinase a c t i v i t y i . e . , 2 ADP » ATP f AMP (11) I t i s also evident that the AMP d i d not contain r a d i o a c t i v e phosphate. The Inaccuracy i n determining concentration of inorganic phosphate and nucleotides by t h e i r s p e c i f i c a c t i v i t i e s w i t h p32 i s also shown. -1k-Thus, s p e c i f i c a c t i v i t y measurements, indicated!, that 1.9- /imoles of ATP were formed whereas o p t i c a l determinations gave a value of 3.56, i n d i c a t i n g that some of the ATP formed contained l a b e l l e d phosphate i n both the beta and gamma p o s i t i o n s . Again, s p e c i f i c a c t i v i t y measurements on inorganic phos-phate i n d i c a t e d the presence of 22.2 /moles, whereas when determined a n a l y t i c a l l y the value was 39.6 yumoles.. This would mean that the inorganic phosphate pool had been d i l u t e d by non-labelled (cold) phosphorus. E i t h e r or both of these observations might explain the "Inhibition' 1 of P32 i n c o r -poration by the addition of substrate. VII. Studies on the exchange of inorganic and organic phosphate. No explanation f o r the incorporation of F32 i n t o the nucleotide f r a c t i o n by an exchange system was as yet evident. Two p o s s i b i l i t i e s were considered:: (a) An ADP-P 3 2 exchange followed by equation 11. 2ADP ATP + AMP (11) (b) Equation 11 followed by an ATP-P 3 2 exchange. Both a c t i v i t i e s could explain the formation of l a b e l l e d ADP,'ATP and cold AMP. 1. Standard r e a c t i o n conditions : v^: To allow a comparison of incorporating a c t i v i t y i n the experiments to follow, standard r e a c t i o n conditions were required. I t was shown, by experiment, that there was a s t r a i g h t - l i n e r e l a t i o n s h i p between the volume of c e l l extract added to the re a c t i o n mixture and the incorporation of P32 over the range 0.1 to 0,$ ml of extract per 3«0 ml rea c t i o n volume i n 1$ minutes at 30<°C (1.7 to 8.6 mg p r o t e i n per cup). The remaining experiments i n t h i s study were therefore conducted with 0,2$ ml of crude extract or an -75-enzyme f r a c t i o n containing approximately h mg of p r o t e i n . 2. Radioactive phosphorus inc o r p o r a t i o n i n t o c e l l f r a c t i o n s . Indications at t h i s time were that the reactions responsible f o r the. inco r p o r a t i o n of p-32 were associated w i t h the soluble cytoplasm of the c e l l . Centrifugation of the crude c e l l extract (CFX) at 100,000xg f o r 2 hours (SC) followed by d i a l y s i s j protamine sulphate treatment and. acid p r e c i p i t a t i o n to pH 5.0 (see Methods) gave f r a c t i o n s with various degrees of a c t i v i t y (Table 1 1 ) . Reaction vessels contained T r i s ( 0 . 1 M pH 7.h) 1.0 ml; MgCl2 (100 /mioles/ml) 0 .10 ml; ADP (25 /xmoles/ml) 0 .20 ml; K^HPO^ (25 /imoles/ml containing 0.5 /lcuries: of P^ 2) 0 .20 ml; TCA (60#$ 0 . 3 0 ml; enzyme, 0.25 ml. Water was added to bring the f i n a l volume to 3*0 ml. Table 11. Acid p r e c i p i t a t i o n of E ^ 2 incorporating a c t i v i t y . F r a c t i o n yumoles P /imoles P /imoles 7'P Aimoles 7'P e s t e r i f i e d e s t e r l f i e d l o s t lost/mg per mg p r o t e i n p r o t e i n CFX 0.82 ©.19 2.11 0.77 SC 0.k2 ©.16 2.00 1.01 SC. a f t e r d i a l y s i s 0.37 0.21 2.00 1.11 SC a f t e r d i a l y s i s and protamine treatment 0.17 0.13 2.13 1.6h pH 5.0 p r e c i p i t a t e 0.k9 0.27 0.52 0.29 pH 5.0 supernatant 0.03 0.0k 2.20 1.23 Although some p u r i f i c a t i o n had been obtained i t was apparent that about 5 0 per cent of the a c t i v i t y was l o s t by c e n t r i f u g a t i o n at 1 0 0 , 0 0 0 x g . Also, treatment with protamine sulphate, t o remove nucl e i c acids, r e s u l t e d i n a f u r t h e r l o s s i n enzyme a c t i v i t y . This pointed to the p o s s i b i l i t y of the exchange a c t i v i t y being associated with the p a r t i c u l a t e c e l l f r a c t i o n s . Adenylate kinase assays demonstrated that t h i s enzyme was predominantly i n the p r o t e i n remaining i n s o l u t i o n a f t e r removing the pH $ . 0 p r e c i p i t a t e . 3 . ATP' or ADP as acceptor f o r the exchange a c t i v i t y ? Employing the f r a c t i o n s l i s t e d above, reactions were prepared with equimolar concentrations: of ATP: s u b s t i t u t i n g f o r ADP i n the incubation mixture. In each case i t was shown that ADP allowed s i x times more i n c o r -poration of P 3 2 than ATP. The a c t i v i t y w i t h ATP was assumed to be due t o the presence of adenylate kinase i n these f r a c t i o n s . At t h i s time, evidence was i n favor of an ADP-P 3 2 exchange rea c t i o n accounting f o r the observed i n c o r p o r a t i o n of P-^ . Doubt s t i l l remained however as to whether the enzyme liras soluble or p a r t i c u l a t e i n nature. Adenylate kinase a c t i v i t y contaminated a l l f r a c t i o n s and actigraph r e -cordings of the incubation mixtures y i e l d e d three spots: absorbing at 260-ITJU, corresponding to AMP, ADP and ATP', the l a t t e r two of which were l a b e l l e d with radioactive phosphate:. The data was i d e n t i c a l to that pre-sented by Brummond et a l . f o r the a c t i v i t y of polynucleotide phosphorylase i n b a c t e r i a l c e l l e x t r a c t s (29). Further proof that t h i s was indeed the enzyme responsible f o r P 3 2 incorporation came when inosine diphosphate (IDP) was sub s t i t u t e d f o r ADP i n the r e a c t i o n mixtures. Adenylate kinase: a c t i v i t y now d i d not i n t e r f e r e with the incorporating a c t i v i t y and recordings gave only one r a d i o a c t i v e peak corresponding to LDP. The pr e v i o u s l y observed in c o r p o r a t i o n of P 3 2 i n t o ADP and ATP could therefore be explained by two enzyme reactions v i z . a polynucleotide phos-phorylase-catalyzed ADP-P 3 2 exchange followed by adenylate kinase a c t i v i t y (equations 12 and 13) (AMP) n + n P 3 2 ^ ^ nAMP^P 3 2 (12) 2 AMP'^P32 ^ AMP + AMP~P 3 2-v. P 3 2 (13) V I I I . The p a r t i c u l a t e nature of the polynucleotide phosphorylase enzyme 1. Ammonium sulphate f r a c t i o n a t i o n . Ammoniumi sulphate f r a c t i o n a t i o n of the soluble cytoplasm f r a c t i o n was, f o r the most part, unsuccessful. Considerable a c t i v i t y was again l o s t by the high speed c e n t r i f u g a t i o n step: (Table 12). Table 12. Exchange a c t i v i t y of c e l l f r a c t i o n s Fra c t i o n /moles P' /moles P Incorporated Incorporated per mg protein. crude extract O.87 0.19 cytoplasm 0.82 0.23 membranes Q:Q$> 0.03 soluble cytoplasm 0.30 0.l5< ribosomes 0.61 0.21 35/60 cut 0.26 0.51 -78-The a c t i v i t y remaining i n the cytoplasm a f t e r c e n t r i f u g a t i o n at 1 0 0 , 0 0 0 x g could be removed by c o l l e c t i n g the p r o t e i n p r e c i p i t a t i n g be-tween 3 5 and 60 per cent ammoniumi sulphate saturation. Seventy-five per cent of the t o t a l exchange a c t i v i t y was however removed by the c e n t r i f u -s gation at high speed. 2. ADP and inorganic phosphate concentration. ' Working with the soluble and ribosomal f r a c t i o n s from the above experiment, the e f f e c t of ADP concentration was determined. In the soluble cytoplasm, reducing the concentration of ADP' by one-half lowered exchange a c t i v i t y by the same amount. With the p a r t i c u l a t e f r a c t i o n , however, lowering ADP concentration by one-half brought about a two-fold increase i n exchange a c t i v i t y . When the r e a c t i o n supernatant from the vessels containing ribosomal and soluble enzymes were removed, and treated f o r electrophoresis studies and P 3 2 assay, only one 260) pm. absorbing, radioactive spot was. present i n the ribosomal supernatant instead of the usual three spots as i n the super-natant form the soluble cytoplasm r e a c t i o n . The ribosomal f r a c t i o n i s therefore r e l a t i v e l y f r e e of adenylate kinase a c t i v i t y . On numerous occasions i t was found d i f f i c u l t to evaluate the a c t i v i t y of a c e l l f r a c t i o n i n terms of t o t a l a c t i v i t y of the crude extract. The above observation with 2 d i f f e r e n t ADP concentrations Indicated that the presence of any contaminating enzyme rea c t i o n that e i t h e r d i r e c t l y or i n d i r e c t l y a f f e c t s the concentration of ADP (adenylate kinase, ATP'ase, or oxidative phosphorylation, etc.)V would e x h i b i t a marked e f f e c t on the exchange enzyme. The. optimum! r a t i o of ADPrinorganic phosphate was determined, with the -7 /9 -ribosomal and soluble cytoplasmic f r a c t i o n s . The results- are shown i n Figure 8 . I t i s evident that very s l i g h t changes i n the ADPrinorganic phosphate r a t i o r e f l e c t large differences I n the Incorporation of The optimum, r a t i o was shown to be d i f f e r e n t , f o r the two enzyme f r a c t i o n s (probably because of the adenylate kinase a c t i v i t y of the soluble cyto-plasm.) 3. F r a c t i o n a t i o n by u l t r a c e n t r i f u g a t i o n . The exchange a c t i v i t y of the soluble cytoplasm (SC) and the ribosomal (R) f r a c t i o n s was shown to be a f u n c t i o n of the c e n t r i f u g a t i o n speed and the RNi content of the f r a c t i o n s (Table 13}. Table 13. RNA, p r o t e i n and exchange, a c t i v i t y as a f u n c t i o n of g r a v i t a t i o n a l f o r c e g x I O 3 %. of t o t a l a c t i v i t y mg RNA In mg p r o t e i n (average) SG; R p e l l e t In p e l l e t 27 83 17 3 18 UO & hi 13 31 6o 32 68: 16 33 80 21 79 17 28: 100 15 85 18; 30: No s i g n i f i c a n t change was observed i n the protein, content of the p e l l e t between UO^OOOxg and 100,000xg. Although the r a t i o of protein, to RNA might be higher than expected f o r ribosomes, repeated, washing of the 100,000xg p e l l e t with O'.Ol M T r i s b u f f e r pH 7.1+ containing 0.01 M - 8 0 -1500 1000 Q tu I— < tr o Q . QT O U z 0. 500 U CT X) .50 1.0 ADP/Pi 2.0 FIG. 8 E f f e c t of the ratio of ADP to inorganic phosphate on the A D P - P i ,32 exchange -81-MgCl2 did. not a l t e r t h i s r a t i o . Nine per cent of the t o t a l RNA of the c e l l resides i n the 100,000: x g supernatant which might explain the observed, exchange a c t i v i t y of t h i s f r a c t i o n . 1*. S o l u b l i z a t i o n of the. ADP-P 3 2 exchange a c t i v i t y . The ease with which the exchange a c t i v i t y may be s o l u b l i z e d was demon-strated by homogenizing the p a r t i c u l a t e f r a c t i o n i n O.i M T r i s pH 7.It, ce n t r i f u g i n g again at 100,000 x g, and examining the p e l l e t and supernatant f o r a c t i v i t y . By t h i s treatment the a c t i v i t y decreased from 750> cpm per mg of p r o t e i n i n the o r i g i n a l p a r t i c u l a t e f r a c t i o n to 55 cpm. per mg .protein i n the 100,000) x g p e l l e t . The a c t i v i t y of the supernatant from t h i s treatment was; 1395 cpmiper mg protein,, i n d i c a t i n g almost complete s o l u b l i z a t i o n . of the exchange enzyme. A f t e r homogenization the r a t i o of ,:RNA t o p r o t e i n i n the supernatant was however almost the same as that given f o r the o r i g i n a l p a r t i c u l a t e f r a c t i o n , i n d i c a t i n g that the p a r t i c u l a t e f r a c t i o n had been s o l u b l i z e d and that the ADP-P 3 2 exchange a c t i v i t y was associated with t h i s ribosomal f r a c -t i o n . 5. Treatment of the p a r t i c u l a t e f r a c t i o n s w i t h RN.ase. and adenylate kinase. Since the 1*0,000' x g p e l l e t contained a considerable amount of the cytoplasmic RNA i t was thought that hydrolysis of t h i s RNA i n t o smaller units might stimulate the exchange a c t i v i t y . Incubation of t h i s f r a c t i o n (0.25 ml) f o r 15 minutes at 30^ C with 1 mg of c r y s t a l l i n e RNase however brought^ about an 87' per cent l o s s i n the exchange a c t i v i t y . To determine i f t h i s l o s s i n a c t i v i t y might be due to destruction of the exchange enzyme by contaminating p r o t e o l y t i c enzymes i n the RNases preparation, -82-i s o c i t r i c dehydrogenase a c t i v i t y was also determined with and without added. RNfise . Ho change i n the a c t i v i t y of t h i s enzyme was observed under the same conditions as those employed f o r the exchange enzyme. The 100,000' x g p e l l e t was shown to be r e l a t i v e l y f r e e of adenylate kinase a c t i v i t y . I t was therefore now p o s s i b l e t o determine the e f f e c t of t h i s enzyme on the exchange a c t i v i t y . C r y s t a l l i n e myokinase (250f) was added to a r e a c t i o n mixture i n the usual assay procedure f o r deter-mining exchange a c t i v i t y . As might be expected, there was a 22 per cent increase i n the exchange a c t i v i t y over the c o n t r o l system containing no added myokinase. This would i n d i c a t e t h a t exchange a c t i v i t y of the soluble supernatant f r a c t i o n i s , i n f a c t , probably lower than that which was. ob-served experimentally. I f we consider the exchange a c t i v i t y as a two-step process (equations lU and 15): ADP ^ .>= polyadenylic a c i d 4- P (ll*) polyadenylic acid + P 3 2 ^ """ ADP 3 2 (l£) The removal of ADP' by adenylate kinase (equation 11) would act to stimulate the incorporation due to the exchange r e a c t i o n . 6. Exchange a c t i v i t y with various nucleoside diphosphates. Due to the high cost of the nucleoside diphosphates and the number of reactions being c a r r i e d out, 1.0 ml re a c t i o n volumes, were now employed. Reactions were conducted d i r e c t l y In the glass-stoppered, pyrex e x t r a c t i o n tubes (see Methods) t o eliminate t r a n s f e r r i n g from Warburg rea c t i o n vessels and c e n t r i f u g i n g a f t e r TCA treatment. The small amount of p r o t e i n now employed d i d not i n t e r f e r e with the extraction of Inorganic phosphate. Exchange a c t i v i t y with f i v e nucleoside diphosphates was compared employing; optlmumi conditions; f o r the ABP-P 3 2 exchange. The reactions contained the fol l o w i n g components i n /moles:. T r i s b u f f e r , pH 7>'.l*, $0; MgCl2, f>J K^HPO^ -83-(containing 100,000 cpmiP 3 2), 5; nucleoside diphosphate, 2.5; ribosomal enzyme f r a c t i o n , 0.1+6 mg, the f i n a l volume being 1.0 ml. Reactions were! stopped by the addition of 0.1 ml of 60 per cent TGA. The r a t i o of nucleoside diphosphate to inorganic phosphate was 0.50 (optimum r a t i o f o r ADP-P 3 2 exchange at t h i s magnesium: concentration).. The r e s u l t s of t h i s experiment are given i n Table l i t . TaMe: l i t . Exchange a c t i v i t y of nucleoside diphosphates Nucleoside diphosphate ADP GDP I D P U D P GDP 7. The synthesis of polyadenylic a c i d . As a f i n a l confirmation of the presence of polynucleotide phosphorylase the synthesis of polyadenylic a c i d was undertaken. The method employed i s given i n d e t a i l i n Materials and Methods.. The r e a c t i o n was followed by measuring the appearance of inorganic phosphate from. ADP (Figure 9). A f t e r only 60 minutes Incubation, at which time about 50 per cent of the ADP' had been converted to polyadenylic a c i d and. inorganic phosphate, the r e a c t i o n mixture was extremely viscous. The ribosomal f r a c t i o n was used to catalyze the polymerization. The r e a c t i o n was; 72 per cent complete at the end of cpm P-3 incorporated per mg p r o t e i n 1285 2565 1520 385 763 T I M E ( h o u r s ) F IG - 9 O r t h o p h o s p h a t e l iberat ion during polynucleot ide s y n t h s i s f r o m A D P . > -85-2 hrs. Incubation.. This value i s considerably higher than that given by Grunberg-Manago et a l . f o r the azotobacter system i n which the polymeri-zation at t h i s time was only 35 per cent complete (25). Our value of 83. per cent f o r the f i n a l conversion of ADE to polyadenylic acid compares favorably w i t h the above authors* value of 78 per cent.. The product of the reaciiion was characterized by hydrolysis and i d e n t i f i c a t i o n of the AME! residues as o u t l i n e d i n Methods. VIII.. The e f f e c t of the ADP-B 3 2 exchange on. oxidative phosphorylation. Systems i n t e r f e r i n g or competing with ADE' have been shown to r e f l e c t t h e i r a c t i v i t y on the ADE-E 3 2 exchange r e a c t i o n . The observed i n h i b i t i o n of E' 3 2 i n c o r p o r a t i o n on the addition of an oxidizable substrate t o a crude c e l l e xtract might then be due to an oxidative phosphorylation, pro-cess competing f o r ADP. Although i t i s u n l i k e l y t h a t the amount of I n h i b i t i o n could be q u a n t i t a t i v e l y Interpreted, i t might serve as an I n d i c a t i o n of the presence of oxidative phosphorylation i n the extract. 1. The e f f e c t of added ATP on the ADF-P-32 exchange. Adenylate kinase a c t i v i t y was shown to stimulate the ADP-P 3 2 exchange i n the ribosomal f r a c t i o n . The i n h i b i t i o n of t h i s a c t i v i t y might therefore be expected to suppress exchange. The i n h i b i t o r y e f f e c t of AMB1 on adenylate kinase, by f o r c i n g equation 11 t o the l e f t was demonstrated with the myo-kinase assay given i n Methods. Assuming that ATE would act t o suppress the r e a c t i o n i n the same way, a s i m i l a r decrease i n exchange should be evident. Reactions were prepared and treated as o u t l i n e d e a r l i e r . The enzyme source was the crude c e l l e x t r a c t . I n addition to ADE and B 3 2 the s i d e -arms of the Warburg vessels contained Increasing concentrations- of ATE'. The r e s u l t s are tabulated i n Table 15. -86-Tahle l g . The e f f e c t of ATE concentration on exchange a c t i v i t y . Per cent of ATE' (umoles) i n i t i a l a c t i v i t y 0.0' 100 1.0 10£ 2S 91 5.0 65 : 10.0 k9 25.0 U7' The s l i g h t s t i m u l a t i o n with one umole of ATE could be explained by ATP'ase a c t i v i t y i n creasing the concentration of ADE. Thus i f an oxida-t i v e phosphorylation system were functioning, and ATP was being produced i n excess of one /nnole, i n h i b i t i o n of exchange a c t i v i t y might' occur. 2. The e f f e c t of cyanide. The hypothesis given above f o r the e f f e c t of an oxidative phosphory-l a t i o n system on > exchange a c t i v i t y was extended when the e f f e c t of KGH was observed. Reaction mixtures- contained T r i s (0*1 M p i 7".It) 1.0' ml; MgCl 2 (100/3moles/ml) O'.l ml; ADP (25/moles/ml) 0.2 ml; KgHPOj^ (containing 100,000 cpm. P 3 2 ) 0.2 ml; malate (200: jumoles/ml) 0.2 ml; KCNi (0.15 M) 0.2 ml; TCA (60%) 0.3 ml; KOH (20$) 0.15 ml, i n centre w e l l ; crude c e l l extract, 0.25 ml; water to 3.15 ml. The r e s u l t s are tabulated i n Table 16. As shown i n Table 8, the addition of KCM to the c e l l extracts- brings-about a s l i g h t stimulation of the exchange a c t i v i t y . - 8 7 -Table 1 6 . The e f f e c t of cyanide on exchange a c t i v i t y cmp E 3 2 incorporated /latoms 02' consumed No additions 12,500 0.8; KC1 ll+,l+50 0.1* malate 12,100 3 . 9 malate +• KCN 15,200 1.2 To summarize, the work to t h i s stage gave evidence i n favor of a functioning oxidative phosphorylation system i n t h i s organism. This evidence however was only I n d i r e c t and based on observations of E3"'*^ incorporation i n t o whole c e l l s , and on the i n h i b i t i o n of an ADP-P 3 2 exchange enzyme i n c e l l f r e e extracts by e i t h e r oxidizable substrate or ATP. IX. Studies on oxidative phosphorylation. 1. The e f f e c t of vitamin K-j_. The r e a c t i v a t i o n o f oxidative: phosphorylation i n i r r a d i a t e d extracts o f M. p h l e i by the addition of vitamin. K]_ reported by Brodie et a l . ( 8 3 ) , prompted us to in v e s t i g a t e the e f f e c t of t h i s vitamin i n the pseudomonas Crude c e l l e x t r a c t s were prepared by the lysozyme-versene method. Reaction vessels c o n t a i n e d the following concentrations of components, i n ml: T r i s (0.1 M pE7.U) 1.1+5; Mg (100 /iraoles/ml) 0.1$; inorganic phosphate (0.1 M) 0.10; ADP (25 jumoles/ml) 0.20; Ni'aF (200 pmoles/ml) system. -88-0.10; fructose (100 yumoles/ml) 0.20; hexokinase (10 rag/ml) 0.20s; malate (200 /moles/ml) O.iO; DPN (2.5 wmoles/ml) 0.10; K0M (20$) 0.15; crude c e l l extract 0.50; water to 3«l5 ml* When vi t a m i n K! was used i t was added d i r e c t l y to the c e l l e xtract, and a f t e r the extract had been saturated the excess vitamin was removed. Reactions were stopped by the addition of 0.3 ml of col d , 60 per cent TCA, the mixture was centrifuged at lt°C and the supernatants analyzed f o r loss of inorganic phosphate (table 17). Table 17. Uptake of inorganic phosphate and oxygen i n the presence and absence of vitamin. Kj.. P i 0 2 PrO fmoles patoms Endogenous .58 1.5 .39 Malate .61 6.3 .10 Endogenous ( ^ + DPN;) 2.1*9 5;.9 .1*2 Malate (K *• DPN) 2.5.6 10.!+ .25 A marked stim u l a t i o n i n both phosphorus and oxygen uptake occurred i n the vessels containing added vitamin and DPN. However, no substrate-depen-dant phosphorylation was observed. In an attempt to reduce the endogenous r e s p i r a t i o n of the c e l l e xtract, succinate-was substituted f o r glucose i n the growth medium. C e l l extracts were prepared i n the usual way and the re a c t i o n mixtures were i d e n t i c a l t o those described above. The e f f e c t of added DPNI and vitamin K-j_ was also determined (Table 18). -89-Table 18.. The e f f e c t of DPN and vitamin. K-j_ on oxygen and phosphate uptake with a crude c e l l extract of succinate-grown c e l l s . P i yumoles; 02 /latoms P:0 P::0 Endogenous 2.70 , 1.13 2.39 -+ K l lt.90) l.Og U.67 -•» DPN 6.50 1.78- 3.65; -+ K - L + DPN ii 7.50' 1.U7 5.10 -Succinate 7.16 6.01 1.19 .92 + K l 8.72 6.00; IM .lh • DPN.! 9;.07 6.26 I.b5 .57 + K + DPN 9.27 6.73 1.38 ,2k Malate a.36 8.10 1.03 .81 # Corrected f o r endogenous a c t i v i t y The data recorded i n Table 18': are the f i r s t convincing demonstration of oxidative phosphorylation i n extracts of P. aeruginosa. I t i s i n t e r e s t i n g that the important diff e r e n c e between these data and a l l that of our other experiments i s that i n the present experiment succinate was the growth medium. I t i s d i f f i c u l t to understand why extracts obtained from, c e l l s grown i n a glucose medium f a i l to e x h i b i t oxidative phosphorylation. Vitamin K, and DPN^although both stimulated phosphorus; uptake^ reduced sub-strate-dependant oxidative phosphorylation. 2. Factors lowering the P:0 r a t i o s , (a) Fructose-6-phosphatase a c t i v i t y . The e f f i c i e n c y of the hexokinase trapping system, was i n v e s t i g a t e d by - 9 0 -determining changes i n the inorganic phosphate concentration of the system i n the absence of the hexokinase trap but with added F-6-E /umoles). Reaction conditions were i d e n t i c a l to those described f o r oxidative phos-phor y l a t i o n s t u d i e s . An increase i n l»hk /imoles of P^ over the c o n t r o l r e a c t i o n i n a period of 60< minutes was noted thus i n d i c a t i n g the presence of a fructose - 6-phosphatase i n the crude c e l l e x t r a c t s . The importance of t h i s observation to P:;0 r a t i o s may be r e a l i z e d by c o r r e c t i n g the phosphorus uptake, i n the presence of succinate, f o r F-6-P'-ase a c t i v i t y . When t h i s i s done the amount of inorganic phosphorus going to organic phosphate increases by l.hk/umoles and the P : 0 r a t i o increases-, from 0 . 9 2 to 1 . 0 (b) Adenosine triphosphatase a c t i v i t y . ATP'ase a c t i v i t y was i n v e s t i g a t e d i n the absence of the hexokinase trapping system. Since an active fructose - 6-phosphatase i s present a low ATP'ase a c t i v i t y would favor the estimation of phosphorus uptake as; ATE phosphorus. Conditions were i d e n t i c a l t o those employed f o r the oxidative phosphorylation studies j ATE was substituted f o r ABE" and hexokinase;, fructose and FaF were omitted from the r e a c t i o n mixtures. S u f f i c i e n t controls were conducted to account f o r non-enzymatic release of inorganic phosphate from ATE. During the incubation p e r i o d of 6 0 minutes the inorganic phosphate; concentration of the system increased by 1.7;6 /imoles, i n d i c a t i n g the pre-sence of an active ATP'ase. Corrections f o r a lower observed phosphorus uptake by ATE?ase a c t i v i t y i n the presence of a hexokinase trap would not be p o s s i b l e since i t would depend on the r e l a t i v e a f f i n i t i e s of the 2 enzymes (hexokinase and. ATP'ase) -91-f o r ATP. The ATP'ase a c t i v i t y does however bring about a f a s t e r release of P^ from ATP' than the fructose-6-phosphatase from. F-6-P. For t h i s reason and also since F-6-F i s l e s s s e n s i t i v e to h y d r o l y s i s by a c i d than ATP, i t was decided to convert ATP' to F-6-P rather than allow ATP to accumulate as such. 3. Oxidative phosphorylation withi succinate-grown c e l l s . Demonstration of oxidative phosphorylation with the crude c e l l extracts was not always p o s s i b l e . Numerous v a r i a t i o n s were made i n the method of c e l l preparation as w e l l as i n the assay procedure but they f a i l e d to i n d i c a t e c l e a r l y why active extracts could not be obtained at a l l times. I t was noted th a t when a preparation d i d not demonstrate substrate-depen-dant oxidative phosphorylation,, DPN and vitamin K-j_ additions also f a i l e d to stimulate phosphorus uptake. The degree of a c t i v i t y was also shown to vary considerably among active preparations. Thus, P:0 r a t i o s v a r i e d from as low as: 0.1 to as high as 2.01 with succinate and from 1.0 to U.3 w i t h ©t -ketoglutarate. Accurate r a t i o s with ©<, -ketoglutarate were d i f f i c u l t to obtain due to the very l i m i t e d a b i l i t y of the crude extracts^ to oxidize t h i s substrate. In a l l instances however, preparations that were i n a c t i v e with succinate as: substrate allowed a P:0 r a t i o of approximately 1.0 with ©C -ketoglutarate, i . e . the single substrate l e v e l phosphorylation associated with t h i s substrate. Results obtained from a representative number of positive: experiments are given In Table 19. A P:0 r a t i o of 2.0! f o r succinate i s considered maximum' i n animal mito-chondria (33). In one instance we d i d succeed! in. obtaining a r a t i o of 2.0', however, values of. 1.0 or greater were obtained a number of times. Since a P'r.O r a t i o of I»0i to 2.0! i s i n t e r p r e t e d to mean t h a t the actual, value is: 2.0, the pseudomonas system would appear t o hehave s i m i l a r l y to mitochon-d r i a . • Table. 19. Oxidative phosphorylation i n crude; extracts of succinate-grown c e l l s . Experiment number Substrate 0 2 josatoms. * i . /imoles: P K O * I succinate 1.6 3.2' 2.0-2 succinate 2.1 2.1, 1.0) 3 succinate t».0> 0.8) 0).2 malate 1.6 0.1* It succinate 2.6 1.2 0.5 succinate 1.8: 1.6 0.9 o( -ketoglutarate; 1.1 1+.6 1*.3 6 succinate 2.7 0.1* 0).2 * -ketoglutarate 0>.7 0.8 1.1. * Corrected f o r endogenous a c t i v i t y Again, in: mitochondria, , B., Sudduth, H. C , and Lehninger, A. 'L. Phosphorylation coupled to the reduction of cytochrome e by @ -hydroxybutyrate. J . B i o l . Chem. 215, 571 (1955). 1+8-. Nielsen, S. 0. and Lehninger, A. L. Phosphorylation coupled to the oxidation of ferrocytochrome c. J . B i o l . Chem. 215, 555 (1955). ' 1+9. Copenhauer, J . B. and Lardy, H. A. Oxidative phosphorylation pathways and y i e l d i n mitochondrial preparations. J . B i o l . Chem. 217, 1+29 (1955). 5'0. Chance, B. and Williams, G. R. Respiratory enzymes i n oxidative phosphorylation. IV. The r e s p i r a t o r y chain. J . B i o l . Chem. 2TT, 1+29 (1955). 51. 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Bacterial, p r o t o p l a s t s . In. "The Bac t e r i a " 1, 21*9. I. C. Gunsalus and R. Y. Stanier,. e d i t o r s . 'Academic Press. New York. (I960). 122. Repaske, R. Lys i s of Grami-negative b a c t e r i a by lysozyme. Biochim. et Biophys. Acta. 22, 189 (1956). ' 123. Mahler, H... R. and Fraser, D. Reproduction of bacteriophage T3 i n protoplasts of E s c h e r i c h i a c o l i , s t r a i n B. Biochim 5. et Biophys. Acta. 22, 197 (1956).. 12i+. Stuy, J . H. The nuc l e i c acids of B. cereus. J . B a c t e r i o l . 76, 179 (1958). 125. Burton, K. A study of the conditions and mechanisms of the diphenylamine reaction f o r the c o l o r i m e t r i c e x t r a c t i o n o f DNA from E. c o l l . . Biochem.- J . 62, 315 (1956). -111-126. Gornall, A. G., Bardlawill, G. S., and David, M. M. Determination of serum proteins by means of the b i u r e t r e a c t i o n . J . B i o l . Chem. 1777, 751 (19U9).. \ 127. Campbell, J . J . R., Ramakrishnan, T., Linnes, A. G., and Eagles,. B. A. Evaluation of the energy gained by Pseudomonas: aeruginosa during the oxidation of glucose t o 2-ketogluconate. Can. J . M i c r o b i o l . 2 , 3 0 % (1956). 128. Strasdine, G. A., Hogg, L. A., and Campbell,. J . J.. R. A ribosomal polynucleotide phosphorylase i n Pseudomonas 8aeruginosa, ( i n Press)• 129. Spahr, P. F. and Hbllingworth, B. R. P u r i f i c a t i o n and mechanism of action of ribonuclease from E s c h e r i c h i a c o l i ribosomes. J . B i o l . Chem. 236,, 823 (193111 130'. Jacobsen, B. K. and Dam, H. Vitamin; K i n b a c t e r i a . Biochim. et Biophys. Acta. hO', 211 ( I 9 6 0 ) . Biographical Information (continued;)) r e : G. A. Strasdine PUBLICATIONS r 1. Strasdine, G. A. and Campbell, J . J . R. Phosphate uptake i n Pseudomonas aeruginosa, Proc. of the B. C. Academy of Science. J (I960). 2. Strasdine, G. A. and Campbell, J . J.. R. Phosphorus inco r p o r a t i o n i n t o c e l l fractions, of Pseudomonas! aeruginosa. Abstracts of the 10th annual meeting of the Canadian Society of M i c r o b i o l o g i s t s . ( I 9 6 0 ) . 3;. Strasdine, G. A., Campbell, J . J . R.,. Hogenkamp,, H: P. C , and Campbell, J . M. Substrate dependant phosphorylation i n r e s t i n g c e l l s of Pseudomonas aeruginosa. Canadian J . of M i c r o b i o l . 7, 91 (1961). l u Campbell, J . J . R., Hogg, L. A., and Strasdine, G. A. L o c a l i z a t i o n of enzymes i n Pseudomonas aeruginosa, ( i n Press). g. Strasdine, G. A., Hogg, L. A., and Campbell, J . J.-.R. A ribosomal polynucleotide; phosphorylase i n Pseudomonas aeruginosa, ( i n Press).