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Gluconic acid oxidizing system of Pseudomonas aeruginosa Ramakrishnan, Thekkepat 1955

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THE GLUCONIC ACID OXIDIZING SYSTEM OF Pseudomonas aeruginosa  by  THEKKEPAT RAMAKRISHNAN  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of AGRICULTURAL MICROBIOLOGY  We accept t h i s thesis as conforming to the standard required from candidates for the degree of DOCTOR OF PHILOSOPHY.  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1955  - i i ABSTRACT E a r l i e r work has shown that Pseudomonas aeruginosa 9027  can oxidize glucose to carbon dioxide and water by way of  gluconic, 2-ketogluconic and pyruvic acids.  However, i t has  been found that closely related organisms can phosphorylate gluconic acid.  The object of the present work was t o i s o l a t e  the gluconate oxidizing enzyme, to s o l u b i l i z e i t , p u r i f y i t , determine  the co-factor requirements and ascertain whether or  not any energy was gained or l o s t by the system during the reaction* Cells harvested from a gluconic acid medium were disintegrated i n a 1 0 kc. Raytheon sonic o s c i l l a t o r .  The  enzyme which was s t i l l attached to the c e l l p a r t i c l e s was s o l u b i l i z e d with sodium glycocholate and remaining p a r t i c l e s were removed by the addition of 0 . 3 0 saturation ammonium sulphate.  Nucleoproteins were then removed by the addition  of protamine  sulphate.  Further fractionation with acid and  alkaline ammonium sulphate p u r i f i e d the enzyme 2 0 0 f o l d . F i n a l l y the enzyme was absorbed on t r i c a l c i u m phosphate and eluted with M/5 phosphate buffer of pH 7 » 0 »  The pH optimum  of the p u r i f i e d enzyme was found to be 5 . 6 while i n the whole c e l l s the maximum a c t i v i t y was at pH 7 . 0 * A hydrogen acceptor was necessary f o r l i n k i n g the system to atmospheric oxygen; 2,6-dichlorophenolindophenol and pyocyanine were found to be the most e f f i c i e n t acceptors.  Ferricyanide poisoned the  system, while b r i l l i a n t c r e s y l blue was inactive as a hydrogen  iii acceptor.  Reaction with methylene blue was slow.  Diphospho-  pyridine nucleotide, triphosphopyridine nucleotide, f l a v i n mononucleotide, f l a v i n adenine dinucleotide, cytochrome c, adenosine diphosphate and adenosine triphosphate had no influence on the enzyme a c t i v i t y .  Sodium f l u o r i d e ,  2,4-dinitrophenol,  azide, iodoacetate, arsenite or 8-hydroxyquinoline did not act as i n h i b i t o r s .  Cyanide, glutathione and cysteine activated  the enzyme s l i g h t l y . The enzyme i s specific f o r gluconic acid. glucuronic acid, 2-ketogluconic acid, ribonic acid, arabonic  Glucose,  acid, pyruvic acid, saccharic  acid, fructose, mannose, ribose-  5-phosphate, glucose-6-phosphate or 6-phosphogluconic acid were not oxidized by the enzyme.  No carbon dioxide was evolved  during the oxidation of gluconic acid by the enzyme.  The  product on chromatographic analysis, was found to be 2-ketogluconic acid. The enzyme was routinely stored at -10°C i n M/10 t r i s buffer, pH 7.0. for  several weeks.  Under these conditions i t was stable  At 4°C, under the same conditions, the  enzyme may be kept f o r three to four days without any appreciable loss of a c t i v i t y .  When dialyzed against d i s t i l l e d water,  there was a gradual loss of a c t i v i t y after eight to ten hours, accompanied by p r e c i p i t a t i o n .  Dialysis against neutral buffers  for as long as 24 hours i n the cold produced no loss i n activity. Instead of sodium glycocholate, "Cutscum" can be  iv used, for s o l u b i l i z i n g the enzyme.  P u r i f i c a t i o n can also be  effected from the sonicate through the use of the u l t r a c e n t r i fuge.  The supernatant l e f t after one hour of centrifugation  at 105,000 x G oxidized gluconic acid i n the presence of pyocyanine and showed two peaks i n the electrophoretic apparatus, one of which i s believed to be due to protamine sulphate. Though no phosphorylation of the substrate was demonstrable  as evidenced by the lack of activation by ATP  and the lack of i n h i b i t i o n by f l u o r i d e , the problem further investigated i n the sonic extracts.  was  No increase i n  acid was found either aerobically or anaerobically i n P. aeruginosa as tested by the method of Colowick and Kalckar. Moreover, sonic extracts f a i l e d to reduce TPN i n the presence of gluconate and an excess of phosphogluconic isolated from Brewer's yeast.  dehydrogenase  In contrast to these data, i t  was found that by either of the last two mentioned c r i t e r i a , P. fluorescens A. 312 did phosphorylate gluconate.  p. f l u o r e s -  cens thus possesses an additional phosphorylated pathway f o r d i s s i m i l a t i n g glucose and this i s absent in P_. aeruginosa. No energy was found to be produced i n the i n i t i a l stages of glucose oxidation. to the "zwischenferment" ATP.  Chromatographic  The system could not be coupled  reaction of glucose which requires  analysis f a i l e d to show any ATP formed  during the oxidation of gluconic acid. The significance of these findings i n the l i g h t of the glucose metabolism  by P. aeruginosa i s discussed.  THE UNIVERSITY OF BRITISH COLUMBIA Faculty of Graduate Studies PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of THEKKEPAT RAMAKRISHNAN B.A. (Madras) 1943 M.Sc. (Madras) 1950 WEDNESDAY, APRIL 27th, 1955, AT 11*00 a.: IN ROOM 102, AGRICULTURE BUILDING COMMITTEE IN CHARGE: Dean H.F. Angus - Chairman B.A. Eagles D.G. Laird D.C.B. Duff S.H. Zbarsky  J.J.R. Campbell W.J. Polglase C.E. Dolman E. Morrison  External Examiner - P.G. Scholefield McGill University  THESIS THE GLUCONIC OXIDISING SYSTEM OF PSEUDOMONAS AERUGINOSA Abstract The enzyme w h i c h o x i d i s e s g l u c o n i c a c i d t o 2 k e t o g l u c o n i c a c i d i n P. a e r u g i n o s a has been e x t r a c t e d i n a s o l u b l e f o r m and p u r i f i e d 300 f o l d . A hydrogen a c c e p t o r was n e c e s s a r y f o r l i n k i n g t h e system t o a t m o s p h e r i c oxygen; 2:6 d i c h l o r o p h e n o l i n d o p h e n o l arid p y o c y a n i n e were found to. be t h e most e f f i c i e n t a c c e p t o r s . The enzyme has a pH optimum o f 5.6. DPN, TPN, FMN, FAD, cytochrome c, ADP and ATP had no i n f l u e n c e on t h e enzyme a c t i v i t y . Sodium f l u o r i d e d i d n o t a c t as i n h i b i t o r i n d i c a t i n g t h a t phosphoryl a t i o n o f t h e s u b s t r a t e was n o t i n v o l v e d . DNP, a z i d e , i o d o a c e t a t e o r 8 h y d r o x y q u i n o l i n e a l s o d i d not i n h i b i t t h e activity. The l a c k o f p h o s p h o r y l a t i o n o f t h e s u b s t r a t e was conf i r m e d w i t h t h e s o n i c e x t r a c t o f t h e organism b y measuring t h e i n c r e a s e i n a c i d i t y produced when phosphate t r a n s f e r t o o k p l a c e . No i n c r e a s e i n a c i d i t y was observed w i t h P. a e r u g i n o s a w h i l e a r e l a t e d organism w h i c h was r e p o r t e d t o p h o s p h o r y l a t e g l u c o n i c a c i d p r i o r t o o x i d a t i o n was shown t o produce i n c r e a s e d a c i d i t y under t h e same cond i t i o n s .< No energy was found t o be produced when g l u c o n i c a c i d was o x i d i s e d b y P . a e r u g i n o s a as i n d i c a t e d b y t h e l a c k o f ATP production.- Thus t h e o x i d a t i o n system i n t h i s organism i s d i f f e r e n t f r o m t h a t i n o t h e r organisms i n t h a t i n t h e i n i t i a l stages no u s a b l e energy i s e i t h e r u t i l i z e d or produced.  GRADUATE STUDIES Field of Study - Agricultural Microbiology S o i l Bacteriology Lactic Acid Bacteria Laboratory Methods and Procedures Dairy Mycology  D.G. Laird J.J.R. Campbell J.J.R. Campbell K. Neilson  Other Studies: General Biochemistry Enzyme Kinetics Molecular Structure and Biological A c t i v i t y Current Literature  S.H. Zbarsky W.J. Polglase W.J. Polglase J.J.R. Campbell  i  -  ACKNOWLEDGEMENTS I wish to express my sincere thanks to Dr. J. J . R. Campbell for his encouragement and invaluable assistance throughout the course of this work, and to Dean B. A. Eagles for his helpful suggestions  and assistance.  I also wish to thank the Department of Trade and Commerce, Ottawa, for the award of a Fellowship under the Colombo Plan during the entire period of this work and to the National Research Council of Canada for a grant to carry out part of this study.  TABLE OF CONTENTS Page Historical Introduction  1  Methods  2 7  Experimental  3 4  Methods f o r d i s r u p t i n g c e l l s  3 5  Attempts t o f r a c t i o n a t e t h e s o n i c a t e  3 9  Solubilization  3 9  of t h e enzyme  Ammonium s u l p h a t e f r a c t i o n a t i o n  4 2  N u c l e o p r o t e i n removal  4 3  F i n a l f r a c t i o n a t i o n w i t h ammonium s u l p h a t e  4 5  Purification  4 6  by c a l c i u m phosphate g e l  P r o p e r t i e s o f t h e Enzyme  5 1  Stability  5 1  A c t i v i t y of t h e e l e c t r o n a c c e p t o r s  5 1  Cofactors  5 2  E f f e c t of i n h i b i t o r s  5 2  Substrate s p e c i f i c i t y  5 4  P r o d u c t of t h e enzymic a c t i v i t y  5 4  M i c h a e l i s constant P r o p e r t i e s of t h e Enzyme System  5 6 58  I n f l u e n c e of pH  5 8  Phosphorylation  6 0  TABLE OF CONTENTS (CONTINUED) Page A l t e r n a t i v e Methods of Enzyme P u r i f i c a t i o n  66  Use of Gutscum as s o l u b i l i z e r  66  Use o f t h e u l t r a c e n t r i f u g e  66  Discussion  69  Bibliography  SO  LIST OF FIGURES Figure 1  2  Page C o n s t i t u t i v e n a t u r e o f t h e g l u c o s e enzyme and the a d a p t i v e nature of t h e g l u c o n a t e enzyme i n whole c e l l s h a r v e s t e d from t h e a c e t a t e medium  3 6  Rate of o x i d a t i o n o f g l u c o n a t e by whole c e l l s as a f u n c t i o n of pH  38  3  A c t i v i t y of s o n i c d i s i n t e g r a t e d c e l l s i o n on g l u c o s e and g l u c o n a t e  4  A c t i v i t y of c e l l e x t r a c t s s o l u b i l i z e d w i t h sodium g l y c o c h o l a t e a g a i n s t g l u c o s e and g l u conate  4 4  A c t i v i t y of protamine t r e a t e d enzyme s o l u t i o n on g l u c o s e and g l u c o n a t e  4 7  6  E f f e c t o f pyocyanine c o n c e n t r a t i o n on v e l o c i t y of o x i d a t i o n by g l u c o n i c dehydrogenase .  5 3  7  I n f l u e n c e of r e d u c i n g agents on t h e o x i d a t i o n of g l u c o n a t e .  55  8  M i c h a e l i s c o n s t a n t of g l u c o n i c dehydrogenase  5 7  9  Rate of o x i d a t i o n of g l u c o n a t e by s o n i c e d c e l l s as a f u n c t i o n of pH  59  10  Rate o f o x i d a t i o n of g l u c o n a t e by p u r i f i e d g l u c o n i c dehydrogenase as a f u n c t i o n of pH ..  6 1  11  Gluconate p h o s p h o r y l a t i o n by Pseudomonas f l u o r e s c e n s as measured by T.P.N, r e d u c t i o n i n the presence of excess phosphogluconic dehydrogenase  65  P r o d u c t i o n of h i g h energy phosphate d u r i n g g l u c o s e o x i d a t i o n by Pseudomonas a e r u g i n o s a 9 0 2 7 as measured by T.P.N, r e d u c t i o n i n t h e presence o f hexokinase and glucose-o-phosphate dehydrogenase  67  5  12  suspen-  4 0  1  -  HISTORICAL INTRODUCTION Prior to 1948 our knowledge of the intermediate metabolism  of carbohydrates was r e s t r i c t e d almost e n t i r e l y  to the reactions of the Embden-Meyerhof pathway and the Kreb's tricarboxylic acid cycle.  The importance  attached to the gly-  c o l y t i c pathway was without doubt partly due to i t s almost universal demonstration i n higher plants and animals as well as i n yeasts, molds and bacteria.  The fact that the enzymes  of this system were water soluble and therefore capable of being i s o l a t e d and p u r i f i e d greatly increased the s c i e n t i f i c interest i n this pathway.  The r e s u l t was that many of the  greatest biochemists of this period turned their attention to this problem, perhaps causing the average worker to attach too great a significance to the pathway.  The observation that  animal tissue had an Embden-Meyerhof system and i n addition, could oxidize carbohydrate, l e d to the suggestion that carbohydrate was fermented to the pyruvate stage by t h i s g l y c o l y t i c pathway and then oxidized by way of the t r i c a r b o x y l i c acid cycle to  CO2  and  H20«  This viewpoint was strengthened when  the investigations were extended to bacteria.  The bacteria  studied were f a c u l t a t i v e types such as Escherichia c o l i and Aerobacter aerogenes and the importance  of the Embden-Meyerhof  pathway and i t s p a r t i c i p a t i o n i n the oxidation of carbohydrate were confirmed.  As late as 1951* workers such as Umbreit  (1949) and Colowick and Kaplan (1951) assumed that this glyc o l y t i c pathway was the only important pathway for the break-  down of hexoses.  This was i n spite of the fact that by this  time a great deal of evidence on the occurrence of alternate routes of carbohydrate metabolism had  accumulated.  Warburg and Christian (1931) found that required glueose-6-phosphate  hemolysate  and would not use glucose.  How-  ever, these workers concluded that this pathway was of no importance i n whole c e l l s and could not compete for glucose6-phosphate with the Embden-Meyerhof system.  The f i r s t schema-  t i c presentation of an alternate pathway of glucose metabolism was brought forward i n 1936 by Dickens.  In this preliminary  note he outlined a scheme of oxidation of carbohydrate i n which the f i r s t stages were the e s t e r i f i c a t i o n of the hexose to hexosemonophosphate; oxidation of this f i r s t to the phosphohexonatej then to 2-ketophosphohexonate; decarboxylation to a pentose phosphoric acid and a continuation of these processes eventually producing pyruvic acid, which would then be comp l e t e l y combusted. In the same year Lipmann (1936) found that the oxidation of one mole of phosphogluconic acid by ground yeast resulted i n the consumption  of one atom of oxygen and the  production of one to 1.5 moles of C02»  The amount of CO2  w a s  reduced to about one mole i n the presence of monobromoacetate. Lipmann then advanced  the theory that  2-keto,6-phosphogluconate  was f i r s t formed (§ O2 consumed), this was then decarboxylated (1 CO2 liberated) and the further process consisted of the fermentation of the t h e o r e t i c a l l y expected d-arabinose phosphate.  Some evidence tending to show keto-acid formation was  provided by experiments i n which HCN appeared to act as a ketone f i x a t i v e .  In other experiments, addition of carboxy-  lase to a carboxylase-free enzyme preparation caused a further oxidation and decarboxylation of phosphohexonate,  Warburg and  Christian (1936) meanwhile p u r i f i e d glucose-6-phosphate dehydrogenase and with i t showed that the end-point i n Lipmannts experiment was not v a l i d and was, i n r e a l i t y , due to a gradual destruction of enzyme a c t i v i t y ,  Lipmann's experiments with  HCN and carboxylase, apparently were too i n d i r e c t and could not be r i g i d l y interpreted. Later Dickens (193&) established conclusively that pentose phosphoric acids arose from the oxidation and decarboxylation of hexose phosphoric acids.  Subsequently, i t was  shown that pentose phosphate was readily fermented by yeast enzymes i n the presence of coenzyme I and inorganic  phosphate.  The important point which arose during this work was that the pentose phosphoric acid most r e a d i l y dissimilated both oxidat i v e l y and by fermentation was not arabinose-5-phosphate which would be expected to arise from simple oxidation and decarboxyl a t i o n of carbon-1 of glucose but was ribose-5-phosphate, Ribose i t s e l f was not fermented either by yeast c e l l s or yeast extract, nor was i t oxidized by brain s l i c e s or by yeast extract.  Dickens advanced the theory that ribose-5-phosphate  originated from hexoses by a process of phosphorylation, oxidation and decarboxylation. Shorr e_t a l . (1938) observed that respiration i n  -  4  -  yeast and mammalian tissue continued even after fermentation has been i n h i b i t e d by iodoacetate.  This suggested that f e r -  mentation and respiration proceeded by two d i f f e r e n t pathways and indicated that the iodoacetate-insensitive system might be of some importance. Although Dicken's work gave the f i e l d of aerobic metabolism a tremendous impetus, i t remained for Horecker, Smyrniotis and Seegmiller to carry this line of thought to i t s l o g i c a l conclusion,  Horecker and Smyrniotis ( 1 9 5 0 )  puri-  f i e d , from yeast, the enzyme which catalyzed the oxidation of 6-phosphogluconate.  They found that pentose phosphate  was  produced quantitatively according to the equation: 6-phosphogluconate + TPN —>  pentose phosphate + C O 2 t  TPNH + H * In addition, 85 per cent of the pentose phosphate, on a paper chromatogram, corresponded to ribose-5-phosphate. Various other procedures such as the enzymic removal of phosphate and paper chromatography with four solvent systems confirmed the presence of ribose.  Their results placed ribose-5"phosphate  in the direct pathway of phosphogluconate oxidation. This alternative pathway has been studied i n t e n s i v e l y by these workers i n recent years using a p u r i f i e d yeast preparation.  Glucose was shown to be oxidized by way of glucose-  6-phosphate and 6-phosphogluconate,  The l a t t e r was then  oxidized with the formation of ribulose-5-phosphate (Cohen and Scott, 1 9 5 0 ; Horecker and Smyrniotis, 1 9 5 1 ; Horecker,  -  5  Smyrniotis and Seegmiller, 1 9 5 1 ) .  -  In order to account for  this reaction a hypothetical intermediate, 3-keto,6-phosphogluconate was postulated and this has now been i s o l a t e d as the c r y s t a l l i n e calcium salt by MacGee and Doudoroff It was with  (1954).  shown that ribulose-5-phosphate was i n equilibrium  ribose-5-phosphate. Independent work by Seegmiller and Horecker  (1952)  on an enzyme preparation from rabbit l i v e r , confirmed the observation that 6-phosphogluconate phosphate and carbon dioxide.  i s oxidized to pentose  The keto-pentose, D-ribulose-  5-phosphate was i d e n t i f i e d as-the product.  The enzyme prepara-  tions contain also a phosphopentose isomerase which catalyzes the reversible conversion of D-ribulose-5-phosphate  to D-  ribose-5-phbsphate reaching an equilibrium r a t i o of approximately one to three.  An enzyme preparation from rabbit bone  marrow catalyzed the formation of glucose-6-phosphate phosphogluconate,  from 6 -  thereby demonstrating a reversal of the  direct oxidative scheme, A variation of this scheme has been elucidated by Entner and Doudoroff  (1952)  i n Pseudomonas saccharophilia.  Instead of being oxidized, the 6-phosphogluconate into one mole of 3 phosphoglyceraldehyde  is split  and one of pyruvate,  and these are metabolized by the usual pathways. These phosphorylated schemes have been generally accepted as being almost universally distributed i n nature. But, of l a t e , s t i l l another pathway of carbohydrate  metabolism  not involving the formation of any phosphorylated  intermediates  in the e a r l i e r stages of oxidation has assumed some importance in the biochemical f i e l d .  As early as 1928 Muller made the  observation that the enzyme Notatin, obtained from young c u l tures of A. niger and other molds, oxidized glucose to gluconic acid,  K e i l i n and Hartree (1948) established that f l a v i n  adenine dinucleotide was the prosthetic group of this enzyme and that the enzyme system was  s p e c i f i c for glucose.  Further  work by Bentley and Neuberger (1949) established that Notatin was a dehydrogenase and that the product of glucose oxidation was A glue onolac t one, The f i r s t observation that a somewhat similar enzyme was present i n other tissues was made by Harrison i n 1931 when he noted that glucose could be converted to gluconic acid i n liver.  He isolated the glucose dehydrogenase catalyzing this  reaction i n crude form.  However, biochemists viewed the  importance of these findings with some skepticism.  Since no  energy transfer could be demonstrated i n the conversion, the reaction was considered to be p h y s i o l o g i c a l l y There were other reasons why a scheme of metabolism.  unimportant.  t h i s scheme was not brought into  Since the reaction was studied as an  i s o l a t e d system, and since no e f f o r t was made to determine whether or not gluconate was an intermediate i n glucose oxidation, the gluconate was pictured as an end-product rather than an intermediate.  Moreover, i t i s now known that glucose i s  not oxidized by way  of this pathway i n l i v e r ,  Strecker and  7 Korkes (1952) have recently i s o l a t e d this glucose dehydrogenase in a f a i r l y pure form and showed that i t oxidizes beta-glucose p r e f e r e n t i a l l y with A glueonolactone as a probable product, E i c h e l and Wainio (1948) found that DPN,  cytochrome c and cyto-  chrome oxidase were involved i n the oxidation of glucose by this enzyme and Renvall (1950) reported that diaphorase  was  the l i n k between the dehydrogenase and cytochrome c. The formation of the lactone as the f i r s t step i n the oxidation of glucose has been stressed by Brodie and Lipmann (1954). vinelandii  f  They isolated two enzymes from Azotobacter  one which oxidizes glucose to  D-glucono-&-lactone  and the other which hydrolyzed t h i s lactone to the corresponding acid.  Previously the reaction involved i n the overall  oxidation of glucose to gluconic acid or  glucose-6-phosphate  to 6-phosphogluconate has been assumed to involve a nonenzymatic  hydrolysis of the lactone to the acid.  Lactone  formation has also been demonstrated by Cori and Lipmann (1952) during the oxidation of glucose-6-phosphate  by using hydroxy-  lamine as a trapping agent. Many observations were recorded which indicated that the direct oxidation of glucose might be carried out by a variety of bacteria.  In 1929 Bernhauer and his associates  showed the production of gluconic acid by Bacterium xylinum and a few years l a t e r demonstrated the formation of gluconic and 2-ketogluconic acids by other pseudomonads.  In 1935  Bernhauer and Knoblock reported that when Acetobacter  8  suboxydans was carbonate,  -  grown on a glucose medium containing calcium  2-ketogluconate  and 5-ketogluconate  accumulated.  These data were c r i t i c i z e d on the grounds that an impure c u l ture must have been employed, since no known organism produces both intermediates.  Bernhauer and Gorlich (1940) also noted  the formation of 2-ketogluconic  acid during glucose  dissimila-  tion by Pseudomonas glueonicum. On this continent Lockwood (1940) reported that large quantities of gluconic and 2-ketogluconic  acids accumula-  ted during the growth of P. fluorescens on a glucose medium. Moreover, Barron and Friedman (1941), using whole c e l l s of P. aeruginosa, could not f i n d any i n h i b i t i o n of glucose oxidation by sodium f l u o r i d e and therefore concluded that the reaction did not involve phosphorylation.  However, with the exception  of Barron and Friedman, workers were interested only i n the commercial production of organic acids andwere not concerned with pathways of metabolism.  They therefore made no e f f o r t  to determine whether or not the compounds i n question were intermediates i n the d i r e c t pathway of carbohydrate  oxidation.  Norris and Campbell (1949) found detectable amounts of gluconic and 2-ketogluconic acids i n 16 and 24 hour cultures of P. aeruginosa when glucose was used as the growth substrate. Since the resting c e l l s harvested at these same periods of time were shown to have strong systems for the oxidation of these two acids i t was  concluded that gluconate and 2-keto-  gluconate were being continuously formed and continuously  oxidized  and  oxidation. glucose by  thus  m e d i a t e s were n o t  without  to  take  up  involved.  was  failed  6 - p h o s p h a t e and  t o remove any  degradation.  P.  aeruginosa.  the  cells  and  I t was  grown on  conditions, resting  also  or f r u c t o s e or  found  or g l u c o n a t e  results  (1952)  He  between r u n s ,  the  two  their  makeup as  found  t h a t no  of c e l l s  indicated  by  not  Norris  p a t t e r n of  t h a t of c e l l s  showed no  (1950)  operative i n  enzymic  their  other  intermediate i n  that a l l o w i n g f o r normal  types  cell  quantitative  l e d C a m p b e l l and  compared t h e  absent  that dried  with  i s an  glucose-  t o be  indicating  g l u c o s e medium w i t h  2-ketogluconate.  enzymic  Barron's  oxidize glucose  were f o u n d  thus  inter-  information against  t h a t a c o n v e n t i o n a l scheme was Hill  with  glucose  that gluconate  A l l these  conclude  inhibited  Moreover, g l u e o s e - l - p h o s p h a t e ,  of 2-ketogluconate,  to  not  agree  anaerobic  accumulation  pathway.  o x i d a t i o n of  was  aeruginosa  fructose-6-phosphate  active  glucose  that phosphorylated  Additional  o x i d i z e d glucose  the  organism  of P.  preparations  pathway was  t h a t the  These d a t a  t h a t under  phosphate.  glucose  found  indicating  phosphorylating i t .  suspensions  during  thus  t h a t whole c e l l s  phosphorylation cell  also  e x t r a c t s of t h i s  sodium f l u o r i d e ,  conclusion  -  were i n t e r m e d i a t e s i n a pathway of  These w o r k e r s  by c e l l  9  grown  variations  difference  ability  on  in  to a t t a c k  glucose,  g l u c o n i c , 2 - k e t o g l u c o n i c , p y r u v i c , <K - k e t o g l u t a r i c ,  fumaric,  succinic,  citric  and  acetic  acids.  conclusion that 2-ketogluconic  firmed  the  direct  pathway o f g l u c o s e o x i d a t i o n .  These d a t a  acid  was  in  con-  the  10  Warburton, pyruvate  16,  at  28  when g r o w i n g  in a  the  was  organism  oxidizing was  found  harvested  from  source  carbon,  aconitate,  a  a  resting  cells  ability  to  oxidize  malate,  citric  acid  of  were  the  enzyme  resting medium  no  of  to  i t was  organism  the  oxidized  rapidly  pyruvate  Campbell  same  and  organism, as  the  sole  citrate, c i s -  s u c c i n a t e or when  that  glucose  of  that  these  they  substrates immediately  Tanenbaum  suspensions  or  acids  (1953)  by  of  a  common  by  fumarate fresh  then and  pyruvate. way  had  the  also  to  They  con-  of  acid  was  acid  gluconic not  being  Krebs*  of  glucose,  found  i s part  of  gluconic  a major They  organism  acid  and  2-  pathway claimed  5-ketogluconic  of that  Foda  dissimilates  intermediate.  or  supporting  intermediate while  this  that  Acetobacter  i n t e r m e d i a t e , thus  the  an  (1953)  extracts  i n many m i c r o o r g a n i s m s .  reported that  glucose,  Tatum  oxidized  2-ketogluconate  oxidation  Vaughn  to  and  cell-free  quantitatively  that  ketogluconic  found  interval  capable  oxidize  However,  aeruginosa  this  containing acetate  adaptation.  these  P.  oxidation.  cells  ability  dried,  of  concluded  a c e t a t e , o x a l a c e t a t e and  2,5-diketogluconic  way  an  determined  cycle.  ketogluconic  glucose  culture  therefore,  Katznelson,  melanogenum  view  that  (1941)  Throughout  possess  They,  had  oxidize  that  the  medium.  to  growth  period  cluded  cell  in a  i s o - c i t r a t e , **_-ketoglutarate,  without  aged  Campbell  hours  glucose shown  and  intermediate i n glucose  (1951)  of  40  and  pyruvate.  a normal  Stokes  Eagles  *>  and maltose  acid,  Results i n  2-  11  agreement with Foda and Vaughn have been obtained by Khesghi et a l . (1954) working with A . suboxydans.  However, other  workers obtained data that l e d them to conclude that this direct oxidative pathway f o r the degradation of glucose either i s a shunt mechanism or does not e x i s t .  Using E. c o l i which  had been adapted to gluconate, Cohen and Scott (1950) extracted an enzyme, gluconokinase, which catalyzed the phosphorylation of gluconate to 6-phosphogluconate,  However, they could not  obtain evidence for the existence of this enzyme i n the un~ adapted c e l l s .  When E, coli.was adapted to gluconate, the  c e l l s were not simultaneously adapted to D-arabinose, arabonate or 2-ketogluconate,  The phosphogluconate  was  appar-  ently degraded exclusively v i a the oxidative pathway through pentose phosphate and presumably no non-phosphorylative oxidation of gluconate occurred, Entner and Stanier (1951), studied glucose and gluconate oxidation by P. fluorescens after growth on media containing either asparagine or glucose.  The asparagine grown  c e l l s were unadapted to gluconic and 2-ketogluconic acids but were immediately able to oxidize glucose.  One atom of oxygen  was taken up per mole of glucose before the lag period set i n , showing that glucose was converted to gluconic acid but that the enzyme to oxidize gluconic acid was absent. ditions which prevented subsequent  Under con-  adaptation to gluconate  and 2-ketogluconate, i t was found that glucose grown c e l l s oxidized glucose and gluconate immediately, whereas 2-keto-  -  12  gluconate was i n i t i a l l y oxidized slowly with the rate i n creasing s l i g h t l y thereafter.  These observations were i n t e r -  preted as showing that glucose d i s s i m i l a t i o n proceeds v i a gluconic acid. excluded.  largely  The p o s s i b i l i t y of glycolysis was not  Although 2-ketogluconate  accumulated  during the  oxidation of glucose or gluconate, the manometric data were interpreted to show that the oxidation of these could not proceed v i a 2-ketogluconate.  substances  However, the data  could also be interpreted as indicating only that the p a r t i cular organism  studied has an unusually weak system beyond 2-  ketogluconate. A major c r i t i c i s m of the nonphosphorylated  pathway  was answered by the work of Wood and Schwerdt (1953,1954). Previously i t was often suggested that gluconic and 2-ketogluconic acids occurred merely as dephosphorylated  products  of the active compounds and were not on the pathway of metabolism.  Wood and Schwerdt were able to demonstrate that the sonic  extract of P. fluorescens had the a b i l i t y to oxidize glucose6-phosphate, 6-phosphogluconate, ribose-5-phosphate, and gluconic acid.  glucose  Using ammonium sulphate fractionation they  were able to separate the glucose-gluconic oxidizing system from the soluble enzyme system which oxidized the phosphorylated compounds.  The nonphosphorylated  glucose-gluconate  system was not a flavoprotein oxidase and did not involve a DPN or TPN s p e c i f i c glucose dehydrogenase.  Cytochrome  carriers apparently are involved and so this glucose dehydro-  genase d i f f e r s (1931)•  The  appears t o  soluble  be  The  coses-phosphate  i n P.  pect and to  Apparently  usual  have a  The  and  that  strain  In Claridge  and  containing  before  was  demonstrated.  its  point  that is  The  the  kinase.  only  report  glucose  Wood's l a b o r a t o r y ,  to  a or-  be  considered studied  by  found  g l y c e r o l without strain  res-  pseudomonads  aerogenes  studies  data to  prior L-o(_  formed  aldolase  had  at  pH  however,  only 8.3.  this  glucose  and  P.  This  would  and  contains  laboratory  phosphorylation  had  indicate  organism.  organism  and  aldolase  weak a c t i v i t y  both i n our  show no  subject,  indicate that  phosphorylate  a m a j o r pathway i n t h i s  conducted  on t h e  d i s s i m i l a t i n g mechanisms.  phosphate dehydrogenase  i n d i c a t i n g that  Experiments  for  probably serves  These workers  a l l other  presented  o f optimum a c t i v i t y  g l y c o l y s i s i s not  system  o r g a n i s m s may  (1953).  e x t r a c t s were r e p o r t e d of t r i o s e  be  dehydrogenation.  Werkman ( 1 9 5 4 )  presence  could  i s similar i n this  noncapsulated  c o n t r a d i c t i o n to  the  Dou-  metabolism of the  dehydrogenated  a e r u g i n o s a 9 0 2 7 p o s s e s s e s two Cell-free  312  A,  s t r a i n s o f A.  Karibian  whereas the  glycerophosphate  hexokinase  nonphosphorylated  intermediate  i n the  Magasanik, Brooke  phosphorylation  no  compounds  E n t n e r and  d i f f e r e n c e s between t h e s e  hexokinase  a capsulated  by  Harrison  system f o r o x i d i z i n g g l u -  aeruginosa 9027  P.  counterpart  the  fluorescens  i n the  (Wood, 1 9 5 4 ) . the  as  by  phosphorylated  reported  presence of the  necessary function ganism.  one  studied  s a c c h a r o p h i l i a but  as w e l l  oxidation  from t h a t  enzyme f o r t h e  f o r P.  demonstrated.  glucose  or  s i m i l a r to the  (1952)  doroff  from N o t a t i n  13  of  and the  This hexoin  substrate gluconic  i n the acid  efficiency  the  without  of the  conceivable to  early  t h a t the  (1948)  system.  By  ferent P.  The  be  reduced  has  energy.  substrates i s  coupled  such  with  as t h i s ,  phosphorylation glucose  acid  phosphorylated  and  while  an  d u r i n g the  been shown t o p o s s e s s  Werkman (1954) have  entirely  second acid dif-  oxidation.  a tricarboxylic  shown t h a t t h e  Since acid  stages. aerobic  o f 2 - k e t o g l u c o n a t e p r o d u c e s members o f t h e  citric  cycle. Moreover, recent  does o c c u r  at t h e  f l u o r e s c e n s has  data  t h a t the  gluconate  former  With crude  gluconate  metabolism from t h a t  i s apparently Narrod  and  They used the acid  formation  ATP  but  of  similar  to  2-keto-  formation as the  preparations they  and  of  Wood (1954)  found  r e a c t i v e k e t o - a c i d was  of 2-ketogluconate  alone.  The  phosphorylation  a magnesium-dependent  enzyme.  a 2,4-dinitrophenylhydrazine presence  (1953).  ( e s t e r ) p h o s p h a t e and  for phosphorylation.  that  different  s t a g e s , and  contained  phosphorylating  acid-stable  level.  b e e n shown t o be  s y s t e m p o s t u l a t e d by DeLey  found  indicate  2-ketogluconate  P.. s a c c h a r o p h i l i a i n l a t e r  the  and  gluconic  C l a r i d g e and  the  similar  of a  Also,  P.  the  shown t h a t t h e h y d r o g e n t r a n s p o r t between  (1950) p h o s p h o r y l a t i o n d o e s t a k e p l a c e i n l a t e r  acid  on  Friedkin  cycle  dissimilation  and  However, i t i s  form o f DPN.  o x i d i z e d to 2-ketogluconic compound was  c a s t s some d o u b t  o x i d a t i o n o f these  a system  aeruginosa  o x i d a t i o n of glucose  phosphorylation  have  oxygen can  be  -  system i n y i e l d i n g  DPNH and  may  stages.  o x i d a t i o n of the  Lehninger  14  not w i t h  of  criteria that  formed i n  2keto-  Although cell  s u s p e n s i o n may  relating  of these  some t y p e  of  studies  allowing  o f the  inhibitor will  have l o s t  synthesize the  cell  any  on  a  their cell  with  one  intensive to  the  to  freely  advantage t h a t  the  been d e s t r o y e d  thus  come i n t o c o n t a c t  change i n p e r m e a b i l i t y  sounder b a s i s .  ability  to  In  adapt  to  c o n s t i t u e n t s , thus  to  cell  have the  membrane has  This  m a t e r i a l to  then, allows tained  cell  cell.  studies  problems  use  preparation.  a l l substrates  enzymes o f t h e  more  problems almost i n v a r i a b l y l e a d s  cell  resting  e l u c i d a t i o n of  pathways, n e v e r t h e l e s s ,  These p r e p a r a t i o n s permeability  or experiments with  g r e a t l y a i d i n the  to metabolic  treatment of  growth  c a r r y out  preparation  the  can  exchange v a l u e s a rigid  cells or  to  ability  assimilation.  given  the  puts  substrates  removing the  gas  be  also  a d d i t i o n , the new  oxidative  assume t h a t  with  semi-  of  This, ob-  quantitative  interpretation. Moreover, o f t e n treatment o f an  cell  intermediate  vantage tion  o f the  and  with  in  quite  specific  tissue  form, the  source  similar techniques  and  This,  techniques  grinding  isolated  fortunately  product.  enzyme i n t a c t  Certain  cell  will  to  of of  must be  and  course, securing  the  during  i s employed. produce  i s a mixed a cell  a c t i v i t y as  prepara-  produce,  long  However, o t h e r  still  p r e d i c t , with  ad-  found.  a complex o f  are  the  accumulation  e x t r a c t i o n techniques  required  preparations  i t i s impossible  destroyed  suspension thus a l l o w i n g  eventually  this  some enzymes are  as  a  very  activities.  Un-  e m p i r i c a l l y produced  any  degree of  certainty  16  which a c t i v i t i e s can be e x t r a c t e d by any g i v e n procedure.  A  d e s c r i p t i o n o f some o f the g e n e r a l t e c h n i q u e s a v a i l a b l e a r e presented  here. 1.  Drying:  The s i m p l e s t method f o r making  cell  p r e p a r a t i o n s o f b a c t e r i a i s t o spread a s u s p e n s i o n t h i n l y and d r y i n vacuo over a d r y i n g agent. working  Doudoroff  e_b a l . (1943)  on p h o s p h o r o l y s i s and s y n t h e s i s o f sucrose by P.  s a c c h a r o p h i l i a d r i e d t h e c e l l paste over P2O5. the c e l l s were powdered and resuspended. (1945)  B e f o r e use  F o s d i c k and Dodds  spread t h e c e l l s o f A e r o b a c t e r aerogenes t h i n l y on a  porous p l a t e , d r i e d them i_n vacuo and then ground the c e l l s l i g h t l y when d r y . degradation.  These c e l l s were used i n s t u d i e s on g l u c o s e  S l e e p e r e_t a l . (1950) s t u d y i n g t h e o x i d a t i o n o f  a r o m a t i c compounds, spread a c e n t r i f u g e paste i n a t h i n and d r i e d s l o w l y i n vacuo a t room t e m p e r a t u r e .  Dried  layer  cells  were f i n e l y ground and s t o r e d i n t h e f r e e z i n g compartment o f the r e f r i g e r a t o r .  They found t h a t t h e endogenous r e s p i r a t i o n  o f such p r e p a r a t i o n s i n c r e a s e d f o r f o u r t o f i v e days, t h e r e a f t e r dropping to a low l e v e l .  C e l l s d r i e d t o o r a p i d l y had  the u n d e s i r a b l e c h a r a c t e r i s t i c s o f f r e s h c e l l s .  T h i s method  p r o b a b l y r e s u l t s i n i n c r e a s e d p e r m e a b i l i t y o f the c e l l s , and d u r i n g prolonged  d r y i n g some l y s i s w i l l o c c u r .  c e l l s a r e dead a f t e r t h i s t r e a t m e n t . (1949),  Most o f t h e  Stadtman and B a r k e r  i n t h e i r s t u d i e s o f t h e metabolism  of fatty acid  s y n t h e s i s by C l o s t r i d i u m k l u y v e r i d r i e d t h e c e l l s i n vacuo over c a l c i u m c h l o r i d e and e x t r a c t e d t h e enzymes which c o n v e r t e t h a n o l and a c e t a t e t o b u t y r a t e and caproate w i t h d i l u t e  sodium  -  17  -  s u l p h i d e s o l u t i o n at pH 7.0. Hughes and W i l l i a m s o n prepared t h e glutaminase  (1952)  o f C l o s t r i d i u m w e l c h i i by d r y i n g the  f r o z e n c e l l s i r i vacuo over phosphorus p e n t o x i d e f o l l o w e d by e x t r a c t i o n o f t h e d r i e d powder w i t h Na2HP0/ ./KCl s o l u t i o n f o r )  15 hours by r o t a t i n g at 2°C i n a stoppered c o n i c a l f l a s k  con-  t a i n i n g 5 mm. diameter g l a s s beads. 2.  Acetone d r y i n g :  The method c o n s i s t s o f m i x i n g  t h e t i s s u e mince o r b a c t e r i a l c e l l s w i t h c o l d a c e t o n e , t e r i n g and d r y i n g i n vacuo. of  fil-  A c c o r d i n g t o t h e o r i g i n a l method  Bernheim ( 1 9 2 8 ) , 3 0 0 ml. o f acetone were used f o r 1 l b . o f  l i v e r mince t o o b t a i n c i t r i c a c i d dehydrase and t h e procedure repeated four times before d r y i n g i n vacuo. Schlenk  W a l d v o g e l and  (1949) i n t h e i r s t u d i e s on t h e enzymic c o n v e r s i o n o f  r i b o s e t o hexosemonophosphate, recommend an excess o f a c e t o n e . Wood e_b a l . ( 1 9 4 7 ) washed t h e E. c o l i c e l l s w i t h e t h e r t o ext r a c t tryptophanase  w h i l e Epps ( 1 9 4 4 ) used a c e t o n e - e t h e r  t u r e on S t r e p t o c o c c u s f a e c a l i s t o o b t a i n t r y o s i n e  mix-  decarboxylase.  Hochster and Q u a s t e l ( 1 9 5 1 ) employed t h e acetone d r y i n g t e c h n i ques i n t h e i r s t u d i e s on t h e e f f e c t o f n i c o t i n a m i d e on fermenta t i o n s by p r e p a r a t i o n s o f Baker's y e a s t .  Acetone t r e a t m e n t  a p p a r e n t l y does not d e s t r o y t h e c e l l s t r u c t u r e but d e h y d r a t e s it  r a p i d l y and i n c r e a s e s t h e p e r m e a b i l i t y o f t h e c e l l membrane.  Some enzyme systems a r e s e n s i t i v e t o t h i s t r e a t m e n t .  A l l the  enzymes which we have s t u d i e d i n Pseudomonas a e r u g i n o s a , f o r i n s t a n c e , a r e i n a c t i v a t e d by acetone 3.  Grinding technique:  treatment. Grinding i s a very widely  used method o f making c e l l p r e p a r a t i o n s .  The t e c h n i q u e s o f  -  18 grinding vary i n t h e i r mortar to  complexity from simple g r i n d i n g i n a  g r i n d i n g i n a machine  s u c h as t h e  F o r m e r l y a meat g r i n d e r w i t h a f i n e (1944)  u s e d t h i s method t o e x t r a c t  system  from b e e f h e a r t .  Now i t  extract for  hexokinase;  studies  Claude  et  al.  to  obtain pyruvate  (1939)  extracted  muscle w i t h R i n g e r ' s the  adenylic  effective is  since  i n rat  0.85  per  w i t h 0.9 p e r  study the  are  to  saline Banga  cent  KC1  ground  (1943)  phosphorylation  Grinding i n a mortar i s  few c e l l s  Colo-  cent  tumour e x t r a c t s ;  and B o y e r and L a r d y  solution to  system.  used  brain tissue  oxidase;  apparatus.  i n a mortar with water  (1944)  on n u c l e i c a c i d s  Claude  succinate-fumarate  i n another  ground muscle  (1947)  the  was u s e d ,  mill.  i s used o n l y as a p r e l i m i n a r y  t o more t h o r o u g h h o m o g e n i z a t i o n w i c k e_t a l .  cutter  Booth-Green  disintegrated  not  of  particularly  and l i t t l e  activity  freed. Greater  result  if  method f o r  B o y e r and L a r d y studying the  Diatomaceous of maceration mixture. are  since  earth  T h i s method i s  they  are too  cells.  1926)  c a n be j u d g e d  f a i r l y large,  ( 1 9 4 2 ) ,  for  instance,  phosphorylation of  (Miiller,  of  activity  by t h e  fine  but  is  e_t a l .  muscle.  The  of  bacterial  l i t t l e use f o r  is  degree  i n f l u i d i t y of cells  (1940)  o f powdered  in  for animal tissue  t o be b r o k e n  paste  the  since cells  easily.  w i d e l y used f o r  I n t h i s method a b a c t e r i a l  m i x e d w i t h 2 5 gms.  this  be u s e d .  increase  or  used  creatine  can a l s o  s m a l l and r e f r a c t o r y  The method o f W i g g e r t terial  release  sand i s u s e d i n t h e m o r t a r a l o n g w i t h t h e w a t e r  extractant.  they  m a c e r a t i o n and i n c r e a s e d  ( 3 gms.)  bacis  g l a s s and 7 m l . o f pH 7 . 0 M / 1 5  -  phosphate.  19  -  The p a s t e i s ground v i g o r o u s l y i n a m o r t a r f o r not  longer t h a n . f i v e minutes.  K a l n i t s k y and Werkman ( 1 9 4 3 )  studied  t h e a n a e r o b i c d i s s i m i l a t i o n o f p y r u v a t e by E. c o l i by t h i s method.  Lee e_t a l . ( 1 9 4 2 ) used t h e same method t o o b t a i n hydro-  genase from A z o t o b a c t e r but used one p a r t o f c e l l s t o two p a r t s o f powdered g l a s s and s u f f i c i e n t b u f f e r t o make a t h i c k  batter.  M c l l w a i n ( 1 9 4 8 ) found a slow c u t t i n g , p o l i s h i n g alumina was superior to glass.  He used 2 . 5 t i m e s t h e c e l l weight o f alumina  i n a c h i l l e d tube w i t h a c h i l l e d g l a s s r o d .  The m a t e r i a l i s  rubbed w i t h maximum hand p r e s s u r e f o r 3 0 seconds, f r a g m e n t a t i o n being  e v i d e n c e d by an i n c r e a s e i n f l u i d i t y and d a r k e n i n g .  E x t r a c t s were p r e p a r e d from s t r e p t o c o c c i i n t h i s way, which were capable o f g l y c o l y s i s , t h e d e a m i n a t i o n o f adenosine d i phosphate and t h e p r o d u c t i o n o f ammonia from a r g i n i n e .  The  mass i s washed from t h e tube w i t h e x t r a c t a n t and c e n t r i f u g e d . The enzyme a c t i v i t y i s found i n t h e c l e a r s u p e r n a t a n t .  Barnard  and H e w l e t t ( 1 9 1 1 ) d e v i s e d a m i l l f o r g r i n d i n g b a c t e r i a l and other c e l l s .  The m i l l was a phosphor-bronze  f i v e hardened s t e e l b a l l s .  cup c o n t a i n i n g  The i n s i d e d i a m e t e r o f t h e cup was  s l i g h t l y l e s s t h a n the sum o f t h e d i a m e t e r s o f t h r e e o f t h e balls.  A c e n t r a l c o n i c a l s t e e l shaft f i t t e d i n t o the centre  kept t h e b a l l s at t h e p e r i p h e r y and a c t e d as a r o t o r .  The  s h a f t c o u l d be f o r c e d down t o b r i n g c l o s e c o n t a c t between t h e s h a f t and the b a l l s .  G r i n d i n g was c a r r i e d out a t 1 5 0 0 r.p.m.,  t h e chamber b e i n g c o o l e d  by a stream o f CO2 from l i q u i d CO2  o r by b e i n g p l a c e d i n an i c e - s a l t b a t h . (1935)  Ogston and Green  used a m i l l o f s i m i l a r s t r u c t u r e f o r p r e p a r i n g t h e  2 0  -  succinic,oC-glycerophosphoric,  lactic,  hydrogenases  from  i n which  extract The with  round  varying  cuated with  t o be  filled  with  grinding,  light  90  taining  glass  internal  with  Woods m e t a l rubber  vibrator hours.  The  The whole  rate  were ball  into  may  of tubes  down w i t h  i s mounted Bacterial  on a  funnel  destroyed. mill  f o r bacterial  a V-shape  and  and b e a d s i s placed  be a c c e l e r a t e d  of a  are broken  by a d d i t i o n  con-  as the may  be  i n a  a rod covered  on t h e stage  cells  filled  or overnight  The t u b e s  series  h o l d e r and clamped  i n the cold. This  hours  a micro  of the tubing.  eva-  attached to a  a p p r o x i m a t e l y o f t h e same d i a m e t e r  fluoride.  hose.  i s then  bent  be  be, i f d e s i r e d ,  of the c e l l s  tubes  faecalis.  and t h e  can then  supported  eight  designed  of pyrex  beads  diameter  After  p e r cent  (1951)  consisting  The neck  i tto  G l a s s beads o f  of the flask  I t may  and t h e f l a s k  o i l .  t o 95  Heden  etched  motor  used  stopper i s f i t t e d  The f l a s k  closed.  or other gas.  rotating  glass  t o the bottom  They  ball  Streptococcus  a t one e n d .  i s added.  and t h e s t o p c o c k  devised a glass  from  a ground  a stopcock  ground  slowly  and m a l i c d e -  c a n be d o n e .  enzymes  with  a r e added  nitrogen  cells,  flask  having  sizes  material  oxidizing  bottom  a tube  (1945)  and Umbreit  anaerobic grinding  glycerol  glucose  yeast.  Gunsalus mill  -  50  with c.p.s.  i n four of  glass  powder. Paege  and S c h l e n k  apparatus  of Utter  deaminase  from  (1950)  have  used  a n d Werkman t o e x t r a c t  E. coli.  The m i l l  consists  the  grinding  cytosine  nucleoside  o f an i n n e r  glass  21  -  cone connected t o a motor and f i l l e d w i t h i c e - s a l t , which f i t s c l o s e l y i n a f i x e d o u t e r cone at t h e end o f which i s a chamber f o r t h e g r i n d i n g m i x t u r e .  The m i x t u r e o f 1 gm. o f  c e l l p a s t e t o 2 gm. ground g l a s s i s f e d t o t h e cones by a plunger.  The ground m a t e r i a l i s caught i n a beaker which i s  kept c l o s e d . diameter.  The g l a s s p a r t i c l e s used average two m i c r o n s i n  Carborundum i s a l e s s e f f i c i e n t g r i n d i n g agent.  One o f t h e more w i d e l y used g r i n d i n g m i l l s i s t h e P o t t e r E l v e j h e m homogenizer.  I t i s c h e a p l y and e a s i l y made and  q u i c k l y and e f f i c i e n t l y d i s i n t e g r a t e s l a r g e c e l l s .  A motor-  d r i v e n p e s t l e made from a c a p i l l a r y tube whose end has been s e a l e d , i s blown i n t o a 2 0 mm. b u l b and ground t o f i t a 1 5 0 x 16 mm. p y r e x t e s t t u b e .  The p e s t l e has 1 2 o r so beads  fused  onto t h e bottom edge and i s d r i v e n at 1 , 1 0 0 t o 1 , 2 0 0 r.p.m. by a cone motor.  The degree of d i s i n t e g r a t i o n o f t i s s u e can  be governed by t h e t i g h t n e s s o f the f i t o f t h e p e s t l e i n t h e tube.  G r i n d i n g agents such as g l a s s have been used, as f o r  i n s t a n c e , by Chantrenne and Lipmann the p y r u v a t e - f o r m a t e exchange  (1950)  i n t h e i r studies of  system o f E. c o l i .  Nachmansohn  and John ( 1 9 4 5 ) used s i l i c a i n s t e a d o f g l a s s t o e x t r a c t a c e t y l a s e from b r a i n . Lipmann  (1945)  D i f f e r e n t g r i n d i n g f l u i d s can be used.  used phosphate t o study t h e a c e t y l a t i o n o f  s u l p h a n i l a m i d e by l i v e r .  Cohen and Hayano ( 1 9 4 6 ) used R i n g e r ' s  s o l u t i o n f o r the enzymes c o n v e r t i n g mammalian  choline  liver.  c i t r u l l i n e t o arginine i n  Ames ( 1 9 4 7 ) i n h i s . s t u d i e s o f the s u c c i n i c  o x i d a s e system of r a t muscle, used i c e w a t e r , w h i l e  Elliott  and K a l n i t s k y ( 1 9 5 0 ) used s a l i n e t o i n v e s t i g a t e t h e o x i d a t i o n  -  22  -  o f a c e t a t e by r a b b i t - k i d n e y c o r t e x . used a l o o s e p e s t l e apparatus genates capable The  Ratner  and Pappas (1949)  t o o b t a i n a c t i v e l i v e r homo-  of converting c i t r u l l i n e to a r g i n i n e .  Booth-Green m i l l (1938) c o n s i s t s o f t h r e e  of r o l l e r b e a r i n g s w i t h a c e n t r a l t a p e r e d  shaft forced i n t o  the centre to cause zero c l e a r a n c e between the r o l l e r The  races are h e l d i n heavy h o s i n g .  bearings.  Paper-thin b a k e l i t e  spacers are p l a c e d between t h e r o l l e r s t o prevent wear.  races  excessive  The s h a f t i s d r i v e n at 6,000 r.p.m. a t which speed  y e a s t s are fragmented i n 10 minutes and t h e most r e s i s t a n t m i c r o o r g a n i s m s i n two h o u r s .  A heavy c e l l suspension i s  poured i n t o the t o p o f t h e m i l l and drops through t h e r o l l e r s , c o l l e c t s i n a pump and i s r e c i r c u l a t e d through to t h e t o p o f t h e machine.  a c o o l i n g bath  W i r t h and Nord (1940) used t h e  m i l l on F u s a r i a t o study a l c o h o l i c f e r m e n t a t i o n .  Still  (1941)  used a b a c t e r i a l paste d i l u t e d w i t h an equal volume o f water i n t h e i r i n v e s t i g a t i o n o f p y r u v i c dehydrogenase o f E. c o l i . The  m i l l has not been as w i d e l y used as t h e P o t t e r homogenizer  s i n c e i t i s much more c o m p l i c a t e d and e x p e n s i v e , and i t i s d o u b t f u l i f i t i s any more e f f e c t i v e .  I t s c h i e f advantage i s  i t s large capacity. Muys (1949) d e s c r i b e d an a u t o m a t i c b a c t e r i a l i n which c e l l s  c o u l d be crushed  mill  a n a e r o b i c a l l y i f necessary.  A rounded c y l i n d e r o f g l a s s i s made t o r e v o l v e i n a h o r i z o n t a l plane i n s i d e a t h i c k - w a l l e d g l a s s - t u b e . der i s arranged  The r e v o l v i n g c y l i n -  t o touch a t i t s end t h e rounded end o f the  t u b e , and i t i s a c r o s s t h i s p o i n t o f contact t h a t the b a c t e r i a  -  23  are f o r c e d by a g r a v i t y feed b e f o r e t h e y f a l l i n t o a r e c e i v e r . Hughes (1951) has d e s c r i b e d a p r e s s which has  the  s p e c i a l f e a t u r e of combining low t e m p e r a t u r e s and a s h o r t p e r i o d of m e c h a n i c a l t r e a t m e n t .  The  c e l l suspension,  together  w i t h a p p r o p r i a t e a b r a s i v e , i s • p l a c e d i n a c y l i n d e r hollowed i n a s t a i n l e s s s t e e l block p r e v i o u s l y cooled.  An a c c u r a t e l y  machined p i s t o n i s d r i v e n by means of a f l y p r e s s at a f o r c e of 12 to 15 t o n s per square i n c h , on to the c e l l s , and  the  l a t t e r are f o r c e d from the c y l i n d e r cut i n the b l o c k .  At tem-  p e r a t u r e s from -20°C t o -35°C, a b r a s i v e s c o u l d be  dispensed  w i t h , i c e c r y s t a l s being presumed t o perform t h i s f u n c t i o n . G h i r e t t i and Barron  (1954) have s t u d i e d the glucose  dehydro-  genase i n C o r y n e b a c t e r i u m e r e a t i n o v o r a n s by t h i s method; P o l l o c k (1953) i n v e s t i g a t e d p e n i c i l l i n a s e a d a p t a t i o n  by  B a c i l l u s cereus by c r u s h i n g the b a c t e r i a l spores i n the  cy-  l i n d e r ; B e a l i n g and Bacon (1953) d i s r u p t e d mold mycelium by t h i s method 4.  t o study the i n v e r t a s e i n the Shaking t e c h n i q u e s ;  system.  Shaking c e l l s w i t h sand  or o t h e r a b r a s i v e s has been used t o e f f e c t c e l l u l a r  fragmenta-  t i o n (Curran and Evans, 1942), and w h i l e the method i s e f f e c t i v e , the c h i e f d i f f i c u l t i e s i n v o l v e d are t h a t a g r e a t d e a l o f heat r e s u l t s from the a b r a d i n g o f the p a r t i c l e s , and the cedure i s f a i r l y l e n g t h y .  pro-  E i t h e r the equipment must be  equipped w i t h a c o o l i n g system or the procedure c a r r i e d  out  i n a c o l d room. M i c k l e (1948) designed  an a p p a r a t u s f o r the r a p i d  m e c h a n i c a l s h a k i n g o f b a c t e r i a l o r yeast c e l l s w i t h g l a s s  24  beads.  A description  (1954)•  o f t h i s apparatus i s g i v e n by Hugo  S t u d i e s on t h e c o n d i t i o n s f o r optimum b r e a k i n g by t h i s  method have been made f o r E. c o l i coccus aureus 5.  (Cooper,  ( F u r n e s s , 1 9 5 2 ) and S t a p h y l o -  1 9 5 3 ) .  Sonic d i s i n t e g r a t i o n ;  One o f t h e newer methods  of c e l l u l a r f r a g m e n t a t i o n i s by t h e use o f s o n i c and u l t r a sonic v i b r a t i o n s  which may be produced  n i c k e l rod o r by means o f c r y s t a l s .  e i t h e r by means o f a  Stumpf and Green  (1944)  exposed a 1 : 2 d i l u t i o n o f a washed c e n t r i f u g e paste o f P r o t e u s vulgaris  i n a 2 0 0 ml. volumetric f l a s k t o a three inch quartz  g e n e r a t o r o p e r a t i n g a t 1 , 0 0 0 v o l t s and 5 0 0 w a t t s f o r 2 0 minutes. They were s t u d y i n g t h e L-amino a c i d o x i d a s e i n t h i s  organism.  The o i l i n t h e bath was c o o l e d by c i r c u l a t i o n through an i c e bath which kept t h e f l a s k c o n t e n t s below 3 8 ° C .  Stumpf  i n a study o f t h e p y r u v i c o x i d a s e of P r o t e u s v u l g a r i s g r a t e d c e l l s by  exposure  i n a 5 0 m l . erlenmeyer  (1945)  disinte-  flask  which  was lowered i n t o t h e o i l bath t o t h e c r i t i c a l d i s t a n c e from t h e crystal.  The c r i t i c a l d i s t a n c e was found to be t h e p o i n t a t  which t h e cone o f f l u i d  i n t h e f l a s k was h i g h e s t .  bottomed f l a s k was found e s s e n t i a l  f o r proper  A thin  fragmentation.  A t h i n ( 2 5 mg. d r y wt./ml.) s u s p e n s i o n i s more e f f e c t i v e a heavy one. dissipated  than  A heavy s u s p e n s i o n causes t h e energy t o be  i n heat and l i t t l e  t h e s e c o n d i t i o n s a 1 0 minute, The v i b r a t i o n s  flat-  fragmentation takes place. i r r a d i a t i o n was found  were generated by a c r o s s - c u t q u a r t z  Under  sufficient. crystal  one t o f i v e i n c h e s i n diameter and ground t o 6 0 0 k c . The g e n e r a t o r r a n at 2 0 0 v o l t s and 7 0 0 w a t t s .  The f r e q u e n c y o f  25  the waves was found t o be o f l i t t l e tude ( i n t e n s i t y ) was c r i t i c a l .  importance but the a m p l i -  Pappenheimer and Hendee  (1949)  fragmented a s a l i n e s u s p e n s i o n o f Gorynebacterium d i p h t h e r i a e by a 3 0 minute t r e a t m e n t i n a 9 k c . o s c i l l a t o r manufactured by the Raytheon C o r p o r a t i o n t o study t h e s u c c i n o x i d a s e system. Paege and S c h l e n k ( 1 9 5 0 ) i r r a d i a t e d resuspended acetone t r e a t e d c e l l s o f E. c o l i f o r 2 0 minutes i n t h e same a p p a r a t u s t o achieve f r a g m e n t a t i o n and i s o l a t e c y t o s i n e n u c l e o s i d e deaminase. K a l l i o ( 1 9 5 1 ) p r e p a r e d an a c t i v e d i s u l p h y d r a s e from Pseudomonas m o r g a n i i by s u b j e c t i n g t h e p r e v i o u s l y d r i e d c e l l s t o s o n i c o s c i l l a t i o n i n 0 . 0 5 M phosphate b u f f e r .  Slade ( 1 9 5 3 ) o b t a i n e d an  a r g i n i n e d i h y d r o l a s e from S t r e p t o c o c c u s f a e c a l i s by t r e a t i n g a 3 p e r cent ( d r y wt.) s u s p e n s i o n i n water t o s o n i c  oscillations  i n a Raytheon 9 k c . o s c i l l a t o r f o r t h r e e h o u r s . 6.  Lysis:  T h i s t e c h n i q u e has not been t o o w i d e l y  used s i n c e d u r i n g l y s i s any s e n s i t i v e enzymes w i l l be destroyed.  Some of the e a r l i e r workers used the method q u i t e  s a t i s f a c t o r i l y b e f o r e o t h e r t e c h n i q u e s were a v a i l a b l e .  Stick-  l a n d ( 1 9 2 9 ) a l l o w e d E. c o l i c e l l s t o l y s e i n o r d e r t o study the d e c o m p o s i t i o n o f f o r m i c a c i d .  Due t o m i l d  proteolysis  d u r i n g l y s i n g , i n s o l u b l e enzymes w i l l be f r e e from p a r t i c u l a t e matter.  I n c i d e n t a l l y s i s may be r e s p o n s i b l e f o r t h e s u c c e s s  of some of the o t h e r t e c h n i q u e s f o r f r e e i n g enzyme a c t i v i t y . The  s i m p l e s t method o f l y s i s i s t o p l a c e t h e c e l l s i n d i s t i l l e d  water a f t e r washing i n an i s o t o n i c s o l u t i o n .  Harvey  (1949)  in-  v e s t i g a t e d t h e c a r b o h y d r a t e metabolism o f Trypanosoma h i p p i c u m by t h i s method; w h i c h , however, i s v a l i d o n l y f o r v e r y f r a g i l e  26  cells.  A u t o l y s i s by i n c u b a t i o n o f c e l l s i n b u f f e r  i s another s i m p l e method o f f r e e i n g enzymes.  solutions  Neuberg and  L u s t i g (1948) l y s e d d r i e d y e a s t s by i n c u b a t i n g i n diammonium phosphate  w i t h s h a k i n g a t 37°C.  Stephenson  l y s e d E.  (1928)  c o l i and l i b e r a t e d l a c t i c dehydrogenase by suspending t h e washed c e l l s i n M/2 phosphate  f o r f i v e t o s i x days.  Umbreit  and Gun-  s a l u s ( 1 9 4 5 ) a u t o l y s e d d r i e d c e l l s o f E. c o l i f o r two hours a t 37°C i n 0 . 1 5 M b o r a t e a t pH 8 . 2 .  B o v a r n i c k ( 1 9 4 1 ) l y s e d E. c o l i  communior f o r 1 6 days a f t e r acetone d r y i n g .  A more d r a s t i c  t r e a t m e n t makes use of crude enzymes found i n egg w h i t e and saliva.  I n order to e x t r a c t o x a l a c e t a t e decarboxylase Krampitz  and Werkman ( 1 9 4 1 ) l y s e d a 1 0 p e r cent acetone o f M i c r o c o c c u s l y s o d e i k t i c u s w i t h lysozyme  suspension  i n 1 / 1 0 volume o f  s a l i v a by i n c u b a t i n g f o r 6 0 minutes a t 36°C. ( 1 9 4 6 ) i n an i n v e s t i g a t i o n o f  cell  U t t e r e_t a l .  a c e t y l phosphate  oxidation,  l y s e d acetone p r e p a r a t i o n s o f t h e same organism by a d d i n g 3 m l . of a 1 : 2 0 d i l u t i o n o f egg w h i t e p e r gm. o f c e l l p r e p a r a t i o n . I n t h e i r s t u d i e s o f t h e p r o d u c t i o n o f p e r o x i d e by Avery and N e i l l  (1924)  pneumococci,  added 0 . 5 m l . o f s t e r i l e o x - b i l e t o 1 5  m l . o f a b a c t e r i a l suspension and i n c u b a t e d a n a e r o b i c a l l y a t 37°C f o r 12 h o u r s .  I n o r d e r t o o b t a i n f o r m i c dehydrogenase  S t i c k l a n d ( 1 9 2 9 ) l y s e d a washed a e r a t e d s u s p e n s i o n o f E. c o l i with 5 ml. of l i q u o r p a n c r e a t i c u s per 1 0 0 ml. of suspension i n a s o l u t i o n b u f f e r e d a t pH 7 . 6 .  Fluoride  ( 0 . 1 p e r c e n t ) was  sometimes added t o t h e l y s i n g s o l u t i o n t o i n h i b i t  growth.  Sher and M a l l e t t e ( 1 9 5 3 ) o b t a i n e d e x t r a c t s o f E. c o l i by l y s i n g the c e l l s w i t h the s p e c i f i c b a c t e r i o p h a g e .  I t was found  that  -  the  27  -  l y s i n e and a r g i n i n e d e c a r b o x y l a s e s i n t h e c e l l - f r e e e x -  t r a c t had f i v e t i m e s t h e a c t i v i t y o f t h e whole 7. (1942)  Free z i n g and t h a w i n g :  cells.  K o e p s e l l and Johnson  prepared p y r u v i c a c i d o x i d i z i n g enzymes from C l o s t r i d i u m  b u t y r i c u m by f r e e z i n g a S h a r p i e s p a s t e f o r 1 2 d a y s .  The  f r e e z i n g r u p t u r e d the c e l l s and on t h a w i n g t h e d e b r i s was r e moved by c e n t r i f u g i n g .  K o e p s e l l <et a l . ( 1 9 4 4 ) f r o z e a c e l l  p a s t e ' o f ' t h e same organisms f o r 1 4 days and ground thawing.  Nason e_t a l . ( 1 9 5 1 ) f r o z e Neurospora mats a t -18°C  f o r one t o t h r e e hours t o r u p t u r e t h e c e l l s . (1947)  after  f r o z e and thawed a p r e p a r a t i o n o f  E.  Wood and Gunsalus c o l i and subse-  quently l e t i t autolyze t o obtain tryptophanase.  Loomis  (1950)  s t a b i l i z e d m i t o c h o n d r i a l a c t i v i t y f o r o x i d a t i o n and phosphoryl a t i o n by f r e e z i n g a t -100°C and s t o r i n g i n d r y i c e w h i l e frozen.  B l a c k ( 1 9 5 1 ) p r e p a r e d an aldehyde dehydrogenase  still from  Baker's y e a s t by f r e e z i n g t h e c e l l s i n l i q u i d n i t r o g e n and e x t r a c t i n g a t pH 8 . 3 a t 4-5°C f o r f i v e  days.  METHODS Bacteriological;  The organism used throughout t h e  e x p e r i m e n t a l work was Pseudomonas a e r u g i n o s a ATCG 9 0 2 7 .  In  some comparative e x p e r i m e n t s Pseudomonas f l u o r e s c e n s A. 3 1 2 o b t a i n e d from Dr. R. Y. S t a n i e r was used.  Stock c u l t u r e s were  i n c u b a t e d a t 30°C f o r 4 8 hours and t h e n removed t o t h e r e f r i gerator f o r storage. once a month.  A f r e s h c u l t u r e was t a k e n from s t o c k  The stock medium had t h e f o l l o w i n g c o m p o s i t i o n :  28 Per cent Tryptone  1.0  K HP0  0.5  2  4  Glucose  0.1  Glycerol  0.5  Yeast e x t r a c t  0.1  Agar  0.5  Gelatin  2.0  pH  7.2  The medium used f o r s e c u r i n g  active resting  w i t h a l o w r a t e o f endogenous metabolism  cells  was t h a t o f Campbell  et a l . (1949) but w i t h g l u c o n a t e r e p l a c i n g g l u c o s e as t h e c a r bon source.  T h i s medium has t h e f o l l o w i n g  composition:  Per NH H P0 4  2  K HP0 2  cent  0.3  4  0.3  4  Sodium g l u c o n a t e  0.3  MgS0 4 .7H 0  0.1  Fe as FeCl3  0.5 p.p.m.  2  pH  7.0  For t h e growth o f l a r g e q u a n t i t i e s o f c e l l s t h e medium was d i s p e n s e d i n 100 m l . q u a n t i t i e s i n t o Roux f l a s k s .  Inoculum  was prepared  from a s t o c k c u l t u r e by t r a n s f e r r i n g t o a g l u c o s e  agar s l a n t .  At l e a s t t h r e e d a i l y t r a n s f e r s were made b e f o r e  the c u l t u r e was used f o r i n o c u l a t i n g a l a r g e r volume o f medium. N o r m a l l y one Roux f l a s k c o n t a i n i n g inoculated  t h e g l u c o n a t e medium was  and i n c u b a t e d at 30°C f o r 18 t o 24 h o u r s .  This  then  served as the source  medium.  28a -  of inoculum  f o r the l a r g e volume o f  Inoculum at the rate of 1 . 0 per cent was used. Production of active c e l l preparations;  were harvested i n a Sharpies  The c e l l s  c e n t r i f u g e and washed twice  M/30 pH 7 . 0 phosphate b u f f e r .  with  The r e s u l t i n g c e l l paste was  resuspended i n a s i m i l a r b u f f e r at a r a t e of, 200 m i l l i g r a m s per ml. of d i s t i l l e d water.  The c e l l  suspension was subjected t o  sonic o s c i l l a t i o n f o r 20 minutes i n a Raytheon 10 kc. o s c i l l a tor.  The r e s u l t a n t mixture  ( h e r e a f t e r c a l l e d the s o n i c a t e )  was placed i n c h i l l e d p l a s t i c  c e n t r i f u g e cups and c a r r i e d i n  an i c e bath to a S e r v a l l SS-1 c e n t r i f u g e maintained mately - 1 0 ° C .  The s o n i c a t e was then  at approxi-  centrifuged f o r five  minutes at 2 5 , 0 0 0 times g r a v i t y and the p a r t i c u l a t e matter discarded.  The supernatant  could be stored i n the deep freeze  u n t i l needed f o r manometric or f r a c t i o n a t i o n s t u d i e s . Chemical:  Metabolic gas exchanges were measured i n  the Warburg respirometer a c c o r d i n g to the standard  procedure  o u t l i n e d by Umbreit et, a l . ( 1 9 4 9 ) . Two-ketogluconic  a c i d was detected by paper chromato-  graphy with an ethanol-methanol-water (45"-45"10) solvent system  and 0.1N AgN03 i n 5N NH^OH as d e s c r i b e d by N o r r i s and  Campbell ( 1 9 4 9 ) .  Phosphate e s t e r s were looked f o r by chromato-  graphy with an i s o p r o p a n o l - 4 per cent ammonium sulphate ( 6 0 : 35)  solvent system and 4 per cent ammonium molybdate i n 60 per  cent p e r c h l o r i c a c i d A x e l r o d , 1951) violet  light.  (Hanes and Isherwood, 1949; Bandurski and  and observing the paper under a source o f u l t r a -  29 When c h e m i c a l a n a l y s i s o f t h e cup c o n t e n t s was des i r e d t h e r e a c t i o n was c a r r i e d out i n a 125 n i l . Warburg vessel containing the following  solutions: ml.  Glucose ( 2 5 uM/ml.) KH P0 2  4  (M/15,  2.0  pH 5 . 6 )  Water  15.0  10.0  Enzyme  1.0  Pyocyanine  ( 3 . 7 5 mg./ml.)  2.0  The r a t e o f o x i d a t i o n was f o l l o w e d w i t h a c o n v e n t i o n a l Warburg system c o n t a i n i n g one-tenth o f t h e above - c o n s t i t u e n t s and w i t h K0H i n t h e c e n t r e  well.  When t h e r e a c t i o n was complete, t h e  l a r g e cup was t a k e n out and t h e r e a c t i o n m i x t u r e was concent r a t e d t o 2 m l . by l y o p h i l i z a t i o n . used f o r chromatographic In t h e i n i t i a l  T h i s c o n c e n t r a t e was t h e n  purposes. s t a g e s o f p u r i f i c a t i o n p r o t e i n was  e s t i m a t e d by a m o d i f i c a t i o n o f t h e method o f H i l l e r (1948) w i t h c a s e i n as t h e s t a n d a r d .  ejb a l .  F i v e m l . of 10 p e r cent  t r i c h l o r o a c e t i c a c i d were added t o 0 . 5 m l . o f t h e s o n i c a t e and t h e r e s u l t i n g p r e c i p i t a t e s e p a r a t e d by c e n t r i f u g a t i o n a t 3,000 r.p.m.  The p r e c i p i t a t e was d i s s o l v e d i n f r e s h l y p r e -  pared 3 . 0 p e r cent NaOH and t h e n 0 . 6 m l . o f a 20 p e r cent CuS0 .5H20 s o l u t i o n was added f o r t h e b i u r e t c o l o u r r e a c t i o n . 4  The s o l u t i o n was r a p i d l y brought t o a f i n a l volume o f 2 5 m l . w i t h 3 p e r cent NaOH and shaken v i g o r o u s l y f o r one m i n u t e ; a l l o w e d t o stand f o r 10 m i n u t e s , and c e n t r i f u g e d a g a i n a t 3 , 0 0 0 r.p.m. f o r 10 m i n u t e s .  The s u p e r n a t a n t was t h e n read  -  30  at 560 m i l l i m i c r o n s i n a Beckman DU the p r o t e i n s o l u t i o n was was  spectrophotometer.  When  c o l o u r e d as when sodium g l y c o c h o l a t e  present the c o l o u r was  compensated f o r by u s i n g as a  blank a d u p l i c a t e sample which had r e c e i v e d a s i m i l a r  treatment  w i t h the e x c e p t i o n t h a t the copper s u l p h a t e  omitted.  stage was  A f t e r the b u l k of the n u c l e o p r o t e i n had been r e moved the p r o t e i n was of Warburg (1944)• present  determined by the 280/260 r a t i o method  T h i s a l s o gave the amount o f n u c l e o p r o t e i n  i n the sample.  The  Beckman DU  spectrophotometer  used f o r t h i s e s t i m a t i o n w i t h hydrogen lamp as the source.  A l l the ammonium s u l p h a t e  was  light  f r a c t i o n s are r e p o r t e d  as  the per cent of s a t u r a t i o n ; a s a t u r a t e d s o l u t i o n o f (NH/^^SO^ c o n t a i n i n g 70.6 The  gm.  of the s o l i d i n 100 ml. o f water at  f r a c t i o n s were o b t a i n e d by a d d i n g s o l i d (NH/^gSO^ s l o w l y  w i t h s t i r r i n g t o the p r o t e i n s o l u t i o n . the s o l u t i o n was The  0°C.  c e n t r i f u g e d at 25,000 x G f o r 10 m i n u t e s .  temperature was  denaturation.  A f t e r f i v e minutes  kept below 10°C  t o l e s s e n the danger of  A f t e r s e p a r a t i o n , the f r a c t i o n s were d i a l y z e d ,  w i t h s t i r r i n g , a g a i n s t i c e - c o l d d i s t i l l e d water f o r one For a l k a l i n e ammonium s u l p h a t e  f r a c t i o n a t i o n a saturated  s o l u t i o n of ammonium s u l p h a t e was and ammonium h y d r o x i d e  The  i n g t o the method of L i n d s t r o m s u l p h a t e i s d i s s o l v e d i n pH 5.2 per m l .  in distilled  water  d i l u t i o n of the  7.5.  protamine s u l p h a t e  t r a t i o n of 20 mg.  prepared  added u n t i l a 1:5  s o l u t i o n showed a pH o f  hour.  The  s o l u t i o n was  prepared  (1953) i n which the buffer with a f i n a l  accord-  protamine concen-  c o l d protamine s u l p h a t e  was  31  -  added s l o w l y beneath the s u r f a c e of a s t i r r e d p r o t e i n c o n t a i n i n g 10-15 mg.  of p r o t e i n per m l .  solution  The protamine  sul-  phate presumably removes n u c l e o p r o t e i n from s o l u t i o n on a mole for  mole b a s i s .  T h e r e f o r e , t h e r e should be no protamine  phate l e f t i n s o l u t i o n .  E x c e s s protamine s u l p h a t e a l s o  sulcarries  down some of the a c t i v e enzyme and t o p r e v e n t t h i s i t was added i n s m a l l p o r t i o n s , c e n t r i f u g e d a f t e r each a d d i t i o n and a p r o t e i n d e t e r m i n a t i o n c a r r i e d out on t h e s u p e r n a t a n t by t h e 2 8 0 / 2 6 0 r a t i o method.  T h i s was  continued u n t i l the n u c l e o -  p r o t e i n c o n t e n t was reduced t o about The b i l e  0:90).  s a l t , sodium g l y c o c h o l a t e , o b t a i n e d from  C i t y Chemical C o r p o r a t i o n , New s o n i c a t e and homogenized homogenate was  5 per cent ( r a t i o  Y o r k , was added d i r e c t l y t o the  i n a van P o t t e r homogenizer.  The  c e n t r i f u g e d f o r 10 minutes at 2 5 , 0 0 0 x G and  then d i a l y z e d against d i s t i l l e d water.  T h i s method i s a modi-  f i c a t i o n o f the procedure f o l l o w e d by W i l l i a m s and S r e e n i v a s a n (1953).  The t r i c a l c i u m phosphate f o r a d s o r p t i o n e x p e r i m e n t s was p r e p a r e d by t h e method of K e i l i n and H a r t r e e ( 1 9 3 8 ) , 1 5 0 m l . of CaCl2 s o l u t i o n (88.5 g.CaCl2.2H 0 per l i t r e ) was  diluted  2  to  about 1 6 0 0 cc. w i t h t a p w a t e r and shaken w i t h 1 5 0 m l . t r i -  sodium phosphate  s o l u t i o n (,L5 2 g. Na3P0 .12H 0 per r  4  2  litre).  The m i x t u r e was brought t o pH 7.4 w i t h d i l u t e a c e t i c a c i d the in The  p r e c i p i t a t e washed w i t h l a r g e volumes of d i s t i l l e d a tall  and  water  j a r u n t i l the p r e c i p i t a t e was f r e e of c h l o r i d e  ions.  g e l was kept moist and a d r y weight d e t e r m i n a t i o n c a r r i e d  out a f t e r which more d i s t i l l e d water was added u n t i l t h e s u s -  32  p e n s i o n c o n t a i n e d 25 mg.  per m l .  For a d s o r p t i o n work, the  enzyme was d i a l y z e d a g a i n s t M/100 methane b u f f e r o f pH 7 . 0 .  tris  (hydroxymethyl)  V a r y i n g amounts of the  amino  tricalcium  phosphate were added t o the enzyme s o l u t i o n kept i n t e s t tubes i n an i c e b a t h .  A t y p i c a l p r o t o c o l i s g i v e n below. 1  2  3  4  5  Enzyme (ml.)  1.0  1.0  1.0  1.0  1.0'  Calcium phosphate s u s p e n s i o n (ml.)  0.0  0.05  0.10  0.25  0.50  Water ( m l . ) .  1.0  0.95  0.90  0.75  0.50  Tube  A s t i r r i n g rod was p l a c e d i n each tube and the g e l and enzyme were mixed f o r f i v e m i n u t e s . t u r e was  At the end o f t h i s time t h e mix-  c e n t r i f u g e d and t h e p r o t e i n c o n t e n t and enzyme  a c t i v i t y o f the supernatant d e t e r m i n e d .  E l u t i o n of the enzyme  from the g e l was a c c o m p l i s h e d by adding 1 . 0 7.0  m l . of M/50  phosphate b u f f e r t o the c e n t r i f u g e d g e l and  t h e i c e d m i x t u r e f o r 15 m i n u t e s .  pH  extracting  I f the enzyme was  not e l u t e d  by t h i s method, i n c r e a s i n g c o n c e n t r a t i o n s o f b u f f e r were used. The i n h i b i t o r s , whose f i n a l c o n c e n t r a t i o n i n the r e a c t i o n m i x t u r e s i s r e p o r t e d h e r e , were added d i r e c t l y t o the Warburg cup.  The  s u b s t r a t e was  t i p p e d i n w i t h i n 20  of the a d d i t i o n of the i n h i b i t o r . cups used f o r cyanide i n h i b i t i o n  The  minutes  centre w e l l s of the  c o n t a i n e d 0 . 2 m l . o f 4N NaCN  i n 10 per cent K0H ( E i s e n b e r g , 1953).  33 F i n a l Concentration of I n h i b i t o r s  The  Sodium f l u o r i d e  2.5  x 10"" M  Sodium a z i d e  3  x  10 3M  8-hydroxyquinoline  1  x  10~^M  Sodium I o d o a c e t a t e  1  x  10~3M  2:4  2  x  10-3M  Sodium a r s e n i t e  1  x 10 -3M  Potassium  1  x  dinitrophenol  cyanide  coenzymes were prepared  2  -  10~3M  a c c o r d i n g t o the Merck  (1952) and added d i r e c t l y t o t h e Warburg cup.  Index  T h e i r concen-  t r a t i o n s i n the r e a c t i o n m i x t u r e s are g i v e n below. F i n a l Amount s of Coenzymes Per Warburg V e s s e l 5.0  micromoles  F l a v i n adenine d i n u c l e o t i d e (FAD)  5.0  micromoles  Diphosphopyridine  5.0  micromoles  T r i p h o s p h o p y r i d i n e n u c l e o t i d e (TPN)  5.0  micromoles  Adenosine t r i p h o s p h a t e (ATP)  100  micromoles  Cytochrome  520  micromoles  2.5  x  R i b o f l a v i n phosphate  (FMN)  n u c l e o t i d e (DPN)  C  Magnesium ( M g S 0 4 . 7 H 0 ) 2  10-3M  The adenosine t r i p h o s p h a t e was n e u t r a l i z e d w i t h sodium  hydr-  oxide before u s e . C y s t e i n e or g l u t a t h i o n e was used i n a f i n a l c e n t r a t i o n o f 1 x 10~3M.  con-  The hydrogen a c c e p t o r s were added  i n the same manner as the i n h i b i t o r s .  The f i n a l  t i o n i n the r e a c t i o n m i x t u r e i s r e p o r t e d  here.  concentra-  34 F i n a l C o n c e n t r a t i o n o f Hydrogen  Acceptor  Methylene b l u e  1 x 10-4M  B r i l l i a n t c r e s y l blue  1 x 10"4M  2:6 d i c h l o r o p h e n o l i n d o p h e n o l  1 x 10~3M  Potassium  1 x 10~3M  ferricyanide  Pyocyanine  1 x 10~3M  EXPERIMENTAL Entner  and S t a n i e r (1951) have r e p o r t e d t h a t t h e  glucose o x i d i z i n g enzyme o f P_. f l u o r e s c e n s i s c o n s t i t u t i v e whereas t h e g l u c o n i c enzyme i s a d a p t i v e i n n a t u r e .  To check  on whether o r not the same s i t u a t i o n p r e v a i l e d i n P. aeruginosa a c u l t u r e was grown i n t h e medium of Campbell ejb a l . w i t h a c e t a t e r e p l a c i n g g l u c o s e as t h e carbon source.  The c e l l s  were h a r v e s t e d , washed, made t o a c o n c e n t r a t i o n of 20 t i m e s growth and t e s t e d f o r t h e i r a b i l i t y t o o x i d i z e glucose and gluconic acid.  As shown i n F i g u r e 1, a d a p t a t i o n t o gluconate  o c c u r r e d a f t e r one hour w h i l e g l u c o s e was o x i d i z e d  immediately.  However, i t can be seen t h a t a d a p t a t i o n t o g l u c o n a t e was a p r e r e q u i s i t e t o f u r t h e r glucose o x i d i z a t i o n thus  confirming  t h a t g l u c o n a t e i s an i n t e r m e d i a t e i n g l u c o s e o x i d a t i o n i n t h i s organism. I t was f e l t t h a t growth o f the c u l t u r e on g l u c o n a t e medium might induce t h e f o r m a t i o n of a h i g h e r c o n c e n t r a t i o n o f gluconate o x i d i z i n g enzyme. the case.  T h i s was indeed found t o be  The weight of c e l l s grown on g l u c o n i c a c i d was,  however, t h e same as t h a t on g l u c o s e .  35  A s e r i e s of experiments were c a r r i e d out i n an e f f o r t t o determine the optimum pH o f g l u c o n a t e by the whole c e l l s . c o n c e n t r a t i o n was  V e r o n a l b u f f e r o f t w i c e the  used as t h e r e was  of the s o l u t i o n , t o a t t a i n 14 ,,9 . 7„,/ 14.714  gms. gms.  oxidation ordinary  a tendency, on the  part  neutrality.  sodium a c e t a t e,)< j• sodxum veronal;  d i .s s-,„o l v e d ^„xn r  Bn  aA  0  c ™i <£50 n i l . n  5 m l . of t h i s stock s o l u t i o n i n a t o t a l volume o f 2 5 m l . of b u f f e r . A pH of 7 . 0 was The pH  c u l t u r e was,  found t o be the most s a t i s f a c t o r y ( F i g . 2 ) . t h e r e f o r e , grown i n the gluconate  medium at  7 . 0 .  Methods f o r d i s r u p t i n g c e l l s :  V a r i o u s methods were  employed i n o r d e r t o d i s r u p t the c e l l s and enzyme.  solubilize  the  C e l l s were ground i n a van P o t t e r homogenizer w i t h  and w i t h o u t  powdered g l a s s and e x t r a c t e d w i t h c o l d s a l i n e .  In  o t h e r e x p e r i m e n t s t h e y were ground e i t h e r w i t h sand or w i t h two  and o n e - h a l f t i m e s t h e i r weight of a b r a s i v e alumina  t o n Company) and  e x t r a c t e d as b e f o r e w i t h c o l d s a l i n e .  n e i t h e r case was  the supernatant  enzyme was  was  also t r i e d .  The  In  a c t i v e , i n d i c a t i n g t h a t the  not r e l e a s e d from the c e l l s .  looked very promising  (Nor-  A technique  which  because of the low t e m p e r a t u r e s used g l u c o n i c a c i d grown c e l l s  were  a f t e r 18 hours and washed once w i t h d i s t i l l e d w a t e r .  harvested They  were t h e n suspended i n d i s t i l l e d water at a c o n c e n t r a t i o n o f 2 0 0 mg./ml. s l o w l y added t o a s t e e l c y l i n d e r which had cooled i n the deep f r e e z e , and d r y i c e - a l c o h o l . A f t e r the  f i n a l l y p l a c e d i n a bath  been of  c e l l s were f r o z e n s o l i d a c o l d  -  FIGURE  3 6  -  1;  C o n s t i t u t i v e nature o f the g l u c o s e enzyme and t h e a d a p t i v e n a t u r e o f the g l u c o n a t e enzyme i n whole c e l l s h a r v e s t e d from the a c e t a t e medium. The r e a c t i o n was c a r r i e d out a t 3 0 . 5 ° C The Warburg cups c o n t a i n e d 1 . 0 m l . o f c e l l s u s p e n s i o n ; 1 . 5 m l . o f M / 1 5 phosphate b u f f e r of pH 7.0; 0.2 m l . o f s u b s t r a t e ( 5 uM); 0 . 1 5 m l . of 20 per cent K0H; water t o 3 . 1 5 m l . Endogenous • has been s u b t r a c t e d .  -  37  -  s t e e l p i s t o n was i n s e r t e d i n t o t h e c y l i n d e r and 1 2 , 0 0 0 pounds p r e s s u r e from a C a r v e r L a b o r a t o r y p r e s s was a p p l i e d .  Within  a minute or two t h e f r o z e n mass melted and the c e l l s were ruptured  i n the process.  at 4°C t h e p r e p a r a t i o n  was t e s t e d a t the r a t e o f 1 ml./cup  i n manometric s t u d i e s . wards g l u c o n i c  A f t e r c e n t r i f u g i n g a t 5 , 0 0 0 r.p.m.  Though t h e p r e p a r a t i o n  was a c t i v e t o -  a c i d , t h e r e were a few l i v e c e l l s p r e s e n t and  the r e a c t i o n was not completed even a f t e r t h r e e  hours.  The  r e s u l t s are a l s o i n c o n s i s t e n t and not r e p r o d u c a b l e - p r o b a b l y because whole uncrushed c e l l s adhered t o t h e gummy supernat a n t m a t e r i a l and r e s i s t e d c e n t r i f u g i n g . The  use o f a b a l l m i l l f o r c r u s h i n g  also investigated.  the c e l l s was  The washed c e l l s were d r i e d under vacuum  i n a d e s i c c a t o r and ground w i t h d r y washed alumina i n t h e ratio  3 / 7 . 5 i n a b a l l m i l l i n vacuo f o r 20 h o u r s .  The ground  c e l l s were e x t r a c t e d w i t h M/30 phosphate b u f f e r o f pH 7 . 2 . I n none of t h e s e c a s e s , however, was the s u p e r n a t a n t found t o be a c t i v e a g a i n s t  gluconic  a c i d showing t h a t t h e enzyme had  not been s o l u b i l i z e d by any o f these methods. The  object  o f t h e i n v e s t i g a t i o n was t o o b t a i n t h e  g l u c o n a t e o x i d i z i n g enzyme i n a s o l u b l e form, and from t h i s p o i n t o f v i e w , t h e o n l y s u c c e s s f u l method of d i s i n t e g r a t i o n of t h e c e l l s was t h a t o f s u b j e c t i n g the c e l l s t o s o n i c waves i n a Raytheon o s c i l l a t o r 200  mg. p e r m l .  uptake. M/30  f o r 2 0 minutes at a c o n c e n t r a t i o n o f  Higher concentrations  gave e r r a t i c  oxygen  The s u s p e n s i o n o f the c e l l s can be made e i t h e r i n  phosphate b u f f e r o f pH 7 . 0 o r i n d i s t i l l e d w a t e r .  The  -  38  -  FIGURE 2 :  Rate o f o x i d a t i o n of g l u c o n a t e by whole c e l l s as a f u n c t i o n o f pH.  The Warburg cups c o n t a i n e d 0 . 5 m l . o f c e l l s u s p e n s i o n ; 0 . 2 m l . gluconate ( 5 u M j ; 2 . 0 m l . o f M / 1 5 v e r o n a l b u f f e r ; 0 . 1 5 m l . o f 2 0 p e r cent K 0 H ; water t o 3 . 1 5 m l . Endogenous has been subtracted.  -  39  s o n i c a t e took up 2 atoms of oxygen p e r mole o f g l u c o s e and 1 atom f o r g l u c o n a t e , i n d i c a t i n g t h a t both g l u c o s e and g l u c o n a t e were o x i d i z e d  t o 2-ketogiuconate ( F i g . 3 ) »  Attempts t o f r a c t i o n a t e were conducted w i t h  the s o n i c a t e :  Experiments  a view t o s e p a r a t i n g t h e g l u c o s e o x i d i z i n g  enzyme from t h e g l u c o n i c  enzyme.  The a d d i t i o n  o f 2 0 per cent  s o l i d ammonium s u l p h a t e brought both the enzymes down i n the p r e c i p i t a t e and f u r t h e r not  possible.  f r a c t i o n a t i o n o f t h e p r e c i p i t a t e was  Alumina CV and c a l c i u m phosphate adsorbed the  enzymes, but e l u t i o n c o u l d not be e f f e c t e d bants. bed  S i m i l a r l y , A m b e r l i t e i o n exchange r e s i n I R C - 5 0 a d s o r -  both enzymes, but i t was not p o s s i b l e  them.  from these a d s o r -  These r e s u l t s i n d i c a t e d  to elute e i t h e r of  t h a t the enzymes were  w i t h p a r t i c u l a t e m a t t e r and t h a t  associated  f r a c t i o n a t i o n c o u l d be e f f -  e c t e d o n l y a f t e r t h e enzymes had been c o n v e r t e d t o a  soluble  form. S o l u b i l i z a t i o n o f the enzyme:  Attempts were made  t o b r i n g the enzymes i n t o s o l u t i o n by t h e use o f s u r f a c e a c t i v e agents.  Sodium g l y c o c h o l a t e was found t o e f f e c t  this.  One gm. o f t h e s a l t was added t o 1 0 m l . o f the s o n i c a t e which was homogenized i n a P o t t e r centrifuged  homogenizer f o r one minute and  at 2 5 , 0 0 0 x G f o r 3 0 minutes i n the c o l d .  s u p e r n a t a n t was s e p a r a t e d from t h e r e s i d u e ,  The  but no enzymic  a c t i v i t y was found i n e i t h e r o f t h e f r a c t i o n s .  The presence  of 0 . 2 ml. o f 2 : 6 d i c h l o r o p h e n o l indophenol a c t i v a t e d the supernatant towards g l u c o n i c  a c i d and t o some e x t e n t t o  g l u c o s e , w h i l e the same added t o t h e p r e c i p i t a t e  activated  40  FIGURE 3:  -  A c t i v i t y of s o n i c d i s i n t e g r a t e d c e l l g l u c o s e and g l u c o n a t e .  s u s p e n s i o n on  The r e a c t i o n was c a r r i e d out a t 30.5°C. Each cup c o n t a i n e d ; 1.0 m l . o f s o n i c e d c e l l s u s p e n s i o n ; 1.5 ml. of M/15 phosphate b u f f e r of pH 6 . 0 ; 0.2 ml. o f s u b s t r a t e (5 uM); 0.15 m l . of 20 per cent K0H; water t o 3.15 m l .  41  m a i n l y the o x i d a t i o n o f g l u c o s e .  -  The dye presumably  p l e t e s the hydrogen t r a n s p o r t system.  com-  N e i t h e r methylene  nor f e r r i c y a - n i d e a c t e d as hydrogen a c c e p t o r s  blue  but pyocyanine  was found t o g i v e a f a s t e r r a t e t h a n even i n d o p h e n o l .  Indo-  phenol was d e c o l o r i z e d i n the presence of g l u c o n i c a c i d and was not r e o x i d i z e d by oxygen whereas pyocyanine was r e o x i d i z e d and q u a n t i t a t i v e oxygen uptake r e s u l t e d .  I t was found t h a t a  c o r r e l a t i o n between t h e r a t e of r e d u c t i o n o f 2 : 6 d i c h l o r o phenol i n d o p h e n o l and the r a t e o f oxygen uptake i n t h e p r e s ence of pyocyanine c o u l d be e s t a b l i s h e d .  2 : 6 dichlorophenol  i n d o p h e n o l has a maximum a b s o r p t i o n a t 6 0 0 mu and the r a t e o f r e a c t i o n can be f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y by measuring the r e d u c t i o n i n o p t i c a l d e n s i t y a t t h i s wavelength and conv e r t e d t o oxygen u p t a k e .  The r e d u c t i o n was found t o be over  i n e i g h t minutes whereas 1 atom o f oxygen was t a k e n up i n 9 0 minutes w i t h g l u c o n i c a c i d as s u b s t r a t e .  The c o r r e s p o n d i n g  amount o f oxygen uptake can be c a l c u l a t e d from t h e r e d u c t i o n of 2 : 6 d i c h l o r o p h e n o l i n d o p h e n o l : Amount o f dye used  =  o . 2 6 uM  . 2 6 uM dye can t r a n s f e r . 2 6 uM (H2) = . 2 6 u Atoms O2 i n e i g h t m i n u t e s . •  , . I n 9 0 minutes t h i s c o r r e s p o n d s t o an uptake of 2.9  u Atoms o f oxygen.  For t h e dye r e d u c t i o n 0 . 2 5 m l . o f t h e enzyme was used  while  f o r t h e manometric experiment 0 . 5 0 m l . o f the enzyme was used.  42  ._. . I n 9 0 minutes w i t h 0 . 5 m l . enzyme d i c h l o r o p h e n o l 5*8  i n d o p h e n o l the c o r r e s p o n d i n g uptake would be u Atoms of oxygen. I n the same p e r i o d o f time pyocyanine  reduces  5 micro  atoms of oxygen. D i c h l o r o p h e n o l i n d o p h e n o l i s t h u s a more e f f i c i e n t hydrogen a c c e p t o r t h a n p y o c y a n i n e , but s i n c e the former i s not a u t o o x i d i z a b l e , pyocyanine a c c e p t o r i n manometric  had t o be used as the hydrogen  experiments.  Ammonium s u l p h a t e f r a c t i o n a t i o n ; which had been  11  c h i l l e d t o 5°C.  The enzyme e x t r a c t  s o l u b i l i z e d " by g l y c o c h o l a t e t r e a t m e n t S o l i d ammonium s u l p h a t e was  was  then added s l o w l y  w i t h constant s t i r r i n g t o b r i n g t h e m i x t u r e t o 2 5 per cent o f saturation.  The  r e s u l t i n g p r e c i p i t a t e was  f u g a t i o n and resuspended  removed by  centri-  i n the o r i g i n a l volume of M / 3 0 phos-  phate b u f f e r , pH 6 . 0 . I n the presence  o f pyocyanine  the r e -  suspended p r e c i p i t a t e o x i d i z e d g l u c o s e at a good r a t e w h i l e i t s a c t i o n on g l u c o n a t e was weak.  The  s u p e r n a t a n t , on the o t h e r  hand, o x i d i z e d g l u c o n a t e at a good r a t e and had o n l y v e r y weak a c t i v i t y towards g l u c o s e ( F i g . 4 ) .  I t would, t h e r e f o r e ,  appear t h a t t h i s step has l a r g e l y s e p a r a t e d the two  enzymes.  From the curves i t can be seen t h a t a l t h o u g h g l u c o n i c a c i d i s q u a n t i t a t i v e l y converted t o 2 - k e t o g l u c o n i c a c i d ,  glucose  o x i d a t i o n stops a f t e r a p p r o x i m a t e l y 1 atom of oxygen has been t a k e n up.  T h i s can be e x p l a i n e d on the assumption  thattwo  enzymes are n e c e s s a r y f o r the c o n v e r s i o n of g l u c o s e t o  -  4 3  -  g l u c o n i c a c i d , one o f which i s m i s s i n g .  T h i s l e n d s weight that glucono-A-  to t h e f i n d i n g o f B r o d i e and Lipmann ( 1 9 5 4 ) l a c t o n e i s t h e p r i m a r y o x i d a t i o n product  o f glucose  enzymatically hydrolyzed t o gluconic a c i d .  and i t i s  The h y d r o l y z i n g  enzyme i s p r o b a b l y absent i n t h e 2 5 p e r cent ammonium  sulphate  fraction. S o l i d ammonium s u l p h a t e was a g a i n added t o t h e chilled  supernatant  s l o w l y , w i t h constant  per cent o f s a t u r a t i o n was reached. t a t e was separated  stirring, until 9 0  The r e s u l t i n g  precipi-  by c e n t r i f u g i n g ; d i s s o l v e d i n t h e o r i g i n a l  volume o f M / 3 0 phosphate b u f f e r o f pH 6 . 0 and t e s t e d f o r i t s a c t i v i t y against gluconic a c i d . of t h e o r i g i n a l s o l u t i o n . did  I t contained a l l the a c t i v i t y  The presence o f ammonium  not a f f e c t t h e measurement o f enzymic Nucleoprotein removal:  sulphate  activity,  The 2 5 - 9 0  f r a c t i o n was  d i a l y z e d a g a i n s t c o l d d i s t i l l e d water f o r one hour w i t h cons t a n t , vigorous mechanical  stirring.  The d i a l y z e d p r o t e i n was  t r e a t e d w i t h protamine s u l p h a t e t o remove n u c l e o p r o t e i n s by the method o f L i n d s t r o m necessary  (1953).  The d i a l y s i s was found t o be  f o r o b t a i n i n g a heavy p r e c i p i t a t e .  The most f a v o u r -  a b l e c o n d i t i o n s f o r the p r e c i p i t a t i o n were a pH o f 6 . 0 and a p r o t e i n c o n c e n t r a t i o n of about 1 0 mg. p e r m l . protamine s u l p h a t e a c i d content  Sufficient  s o l u t i o n was added t o b r i n g t h e n u c l e i c  o f t h e supernatant  t o about 5 p e r c e n t .  The  p r e c i p i t a t e was c e n t r i f u g e d o f f at 2 5 , 0 0 0 x G f o r 2 0 minutes and d i s c a r d e d .  The supernatant  which was c l e a r and n e a r l y  c o l o u r l e s s , was d i a l y z e d a g a i n s t M / 1 0 0 t r i s  (hydroxymethyl)  44 -  FIGURE 4:  A c t i v i t y o f c e l l e x t r a c t s s o l u b i l i z e d w i t h sodium g l y c o c h o l a t e a g a i n s t glucose and g l u c o n a t e . The r e a c t i o n was c a r r i e d out at 3 0 . 5 ° C Each cup c o n t a i n e d ; 0 . 5 m l . o f c e l l e x t r a c t ; 1 . 5 m l . o f M / 1 5 phosphate b u f f e r o f pH 6.0; 0 . 2 m l . o f p y o c y a n i n e • ( 1 0 - 3 1 0 } 0 . 2 m l . o f s u b s t r a t e ( 5 uM); 0 . 1 5 m l , of 20 per cent K0H; water t o 3 . 1 5 m l . Endogenous has been s u b t r a c t e d . A = sodium g l y c o c h o l a t e t r e a t e d e x t r a c t f B = sodium g l y c o c h o l a t e t r e a t e d e x t r a c t + C = s u p e r n a t a n t from 2 5 p e r cent ammonium p r e c i p i t a t i o n of s o l u b i l i z e d e x t r a c t D = supernatant from 2 5 p e r cent ammonium p r e c i p i t a t i o n of s o l u b i l i z e d extract  glucose. gluconate. sulphate 4- g l u c o s e . sulphate + gluconate.  45  aminomethane b u f f e r pH 7 . 0 t o p r e c i p i t a t e any n u c l e o p r o t e i n which might remain d i s s o l v e d , due t o the presence o f t r a c e s o f ammonium s u l p h a t e .  I f a p r e c i p i t a t e formed, i t t o o was c e n t r i -  fuged o f f and d i s c a r d e d .  The supernatant  retained the a c t i v i t y  of t h e o r i g i n a l s o l u t i o n a g a i n s t g l u c o n i c a c i d ( F i g . 5 ) . F i n a l f r a c t i o n a t i o n w i t h ammonium s u l p h a t e :  The  s o l u t i o n was now d i v i d e d i n t o f o u r p o r t i o n s by f r a c t i o n a l c i p i t a t i o n w i t h ammonium s u l p h a t e . 25-42,  42-59  pre-  The f r a c t i o n s were 0 - 2 5 ,  and 5 9 - 9 0 p e r cent o f s a t u r a t i o n .  The a c t i v i t y  of these v a r i o u s f r a c t i o n s (made up t o t h e o r i g i n a l volume) a g a i n s t g l u c o n i c a c i d was determined m a n o m e t r i c a l l y . a c t i v i t y was found t o be i n the 0 - 2 5 and 2 5 - 4 2 Saturation of Ammonium S u l p h a t e  A c t i v i t y o f Enzyme (Oxygen uptake i n u l p e r hr./ml.)  The  fractions:  Total Volume  Total Activity  ml.  2159  105.9  30 ml.  3177  42-59  0  30 ml.  0  59-90  0  30 ml.  0  0-25  75.3  25-42  30  Another experiment u s i n g 0 - 2 0 , 2 0 - 3 5 and 3 5 - 4 2 s a t u r a t i o n s w i t h ammonium s u l p h a t e concentrated  r e s u l t e d i n t h e major a c t i v i t y  i n the 2 0 - 3 5 f r a c t i o n .  a c t i v i t y against  glucose:  being  T h i s f r a c t i o n had no  -  Saturation of Ammonium S u l p h a t e  46  -  A c t i v i t y o f Enzyme (Oxygen uptake i n u l p e r hr./ml.)  Total Volume  Total Activity  ml.  0-20  26.4  30  792  20-35  138.0  30 ml.  4140  35-42  2.8  30 ml.  84  42-90  0  30 m l .  0  T h i s l a s t named f r a c t i o n was d i s s o l v e d i n the o r i g i n a l  volume  of M/30 phosphate b u f f e r and was f u r t h e r f r a c t i o n a t e d w i t h saturated  a l k a l i n e ammonium s u l p h a t e  o f pH 7 . 5 .  The g l u c o n i c  dehydrogenase was found t o be i n t h e 2 0 - 3 0 c u t . Saturation of Ammonium S u l p h a t e  A c t i v i t y o f Enzyme (Oxygen uptake i n u l per h r . / m l . l  0-20  Total Volume  0•  20  Tot'al Activity  ml.  0  20-30  272.0  20 ml.  5240  30-40  4.4  20 m l .  88  40-50  0  20 ml.  0  P u r i f i c a t i o n by c a l c i u m phosphate g e l :  The f r a c -  t i o n which was brought down between 2 0 - 3 0 p e r cent s a t u r a t i o n of a l k a l i n e ammonium sulphate b u f f e r o f pH 7 . 0 o v e r n i g h t  was d i a l y z e d a g a i n s t M/100 " t r i s "  as a d s o r p t i o n  by c a l c i u m  i s best e f f e c t e d i n a s o l u t i o n o f low i o n i c  phosphate  concentration.  The i n v e s t i g a t i o n s were o n l y o f a p r e l i m i n a r y n a t u r e t o e s t a b l i s h c o n d i t i o n s f o r c a l c i u m phosphate a d s o r p t i o n .  To 1 . 0 m l .  of the enzyme v a r i o u s amounts o f t r i c a l c i u m phosphate s u s p e n s i o n c o n t a i n i n g 2 5 mg. s o l i d p e r m l . were added:  -  60r  47  -  Gluconic acid o  —  —  o  —  50 ©  x o a. o 30 CO  a: 6IUC086  20  -fe  gb—sb—nfo—r£rr  TIME IN MINUTES  140  FIGURE 5 : A c t i v i t y o f protamine t r e a t e d enzyme s o l u t i o n on glucose and g l u c o n a t e . Each cup c o n t a i n e d ; 0 . 5 m l . o f enzyme s o l u t i o n ; 1 . 5 m l . o f M/15 phosphate b u f f e r o f pH 6.0; 0.2 m l . o f pyo cyanine (10-3M); 0.2 m l . o f s u b s t r a t e ( 5 uM); 0 . 1 5 m l . o f 20 p e r cent K0H; water t o 3 . 1 5 n i l .  48  -  -  Enzyme (ml.)  1.0  1.0  1.0  1.0  1.0  C a l c i u m phosphate (ml.)  0  0.05  0.10  0.25  0.50  Water (ml.)  1.0  0.95  0.90  0.75  0.50  The m i x t u r e s were kept i n an i c e bath w i t h s t i r r i n g rod i n each and s t i r r e d f o r f i v e m i n u t e s a f t e r which t h e y were c e n t r i f u g e d and enzymic a c t i v i t y and p r o t e i n determined on t h e supernatant. M/100  For p r o t e i n d e t e r m i n a t i o n by t h e 280/260 method,  " t r i s " b u f f e r shaken w i t h the same amount o f t r i c a l c i u m  phosphate was used as t h e b l a n k . I t was found t h a t 0 . 2 5 m l . o f c a l c i u m  phosphate  s u s p e n s i o n d i d not t a k e up any s i g n i f i c a n t amount o f enzyme but t h a t 0 . 5 m l . adsorbed a l l o f i t . Calcium Phosphate Suspension  Total Volume  Protein Concen.in Supernatant  Enzyme A c t i v i t y p e r mg. P r o t e i n (ul oxygen t a k e n up per hour  Total Activity  0  ml.  2.0 ml.  1.82 mg./ml.  420  1528.8  0.05  ml.  2.0 m l .  1.82 mg./ml.  420  1528.8  0.1  ml.  2.0 m l .  1.54 mg./ml.  420  1293.6  0.25  ml.  2.0 m l .  0 . 9 3 mg./ml.  337  628.8  0.5  ml.  2.0 m l .  0 . 5 2 mg./ml.  0  0  A second s e r i e s o f a d s o r p t i o n e x p e r i m e n t s showed t h a t 0 . 3 5 m l . was an a p p r o p r i a t e amount o f t r i c a l c i u m phosphate i n t h a t a l a r g e p r o p o r t i o n o f the enzyme was adsorbed.  49  Calcium Pho sphate Suspension  Total Volume  0  ml.  2 . 0 ml.  0.3  ml.  2 . 0  0.35  ml.  0.4  ml.  -  Protein Enzyme A c t i v i t y Concen.in p e r mg. P r o t e i n Supernatant, ' (ul oxygen t a k e n up p e r hour)  ml.  1 . 8 2 mg./ml.  Total Activity  1528.8  4 2 0  mg./ml.  3 1 5  467.0  2 . 0 ml.  0 . 7 5 mg./ml.  2 1  3 1 . 6  2 . 0 ml.  0 . 6 7 mg./ml.  0  Next the . 3 5 f r a c t i o n  0 . 9 0  of calcium  0  phosphate was mixed w i t h 2 . 0  m l . o f M / 3 0 phosphate b u f f e r o f pH 7 . 0 , a g i t a t e d f o r 1 5 minutes i n t h e c o l d and c e n t r i f u g e d .  The g e l was then mixed w i t h M / 1 0  b u f f e r o f t h e same pH and c e n t r i f u g e d  i n the same manner.  The  p r o c e s s was r e p e a t e d w i t h M/5 and M / l phosphate b u f f e r . Concen. o f Buffer  Protein Concen. In E lute  Enzyme A c t i v i t y p e r mg. P r o t e i n ( u l Oxygen Up^~ take per h r . ) ~  Total Volume  Total Activity  M/30  0 . 3 5 mg./ml.  0  2 . 0 ml.  0  M/10  0 . 5 3 mg./ml.  0  2 . 0 ml.  0  M/5  0 . 7 1  mg./ml.  6 3 2  2 . 0  ml.  897.6  M/l  0 . 7 7  mg./ml.  6 3 2  2 . 0  ml.  .973.2  From t h e t a b l e i t can be seen t h a t M/5 phosphate b u f f e r o f pH 7.0  i s able t o s e l e c t i v e l y  phosphate. but  S i x t y p e r cent o f t h e o r i g i n a l p r o t e i n was recovered  t h e r e was o n l y a l o s s o f 1 0 p e r cent o f t h e a c t i v i t y . The  the  e l u t e t h e enzyme from t h e c a l c i u m  various  following table:  stages o f p u r i f i c a t i o n  are summarized i n  Stage o f Purification Sonic  extract  Glycocholate solution 25-90(NH ) S0 4  2  Protamine SO^ treated extract 4  2  4  21-30 Calcium elut e  2  Total Activity /hr.  118  560.0  2.1  1176.0  960  293.0  32.8  9610.4  444  138.0  64.O  8832.0  2.78  300  83.4  107.8  8989.5  30  2.25 .  300  67.5  133.3  8997.5  20  1.28  540  25.6  421.5  10790.4  20  0.71  485  15.4  631.5  9724.1  10  56.00  10  29.30  20  6.93  30  Activity /hr./ml.  4  fraction Alk.(NH ) S0  Activity /hr./mg. Protein  Protein per ml.  4  fraction  20-35(NH ) S0  Total Protein  Volume ml.  4  fraction phosphate  There i s thus a 300 f o l d  purification  51  -  P r o p e r t i e s o f the Enzyme The  S t a b i l i t y of the enzyme: s t o r e d at -10°C  i n M/10  c o n d i t i o n s i t was the  enzyme was  " t r i s " b u f f e r , pH 7.0.  s t a b l e f o r s e v e r a l weeks.  routinely  Under t h e s e  At 4°C > under  same c o n d i t i o n s , the enzyme could be kept f o r t h r e e  f o u r days w i t h o u t any alyzed against  appreciable  l o s s of a c t i v i t y .  d i s t i l l e d w a t e r , t h e r e was  to  When d i -  a gradual  l o s s of  a c t i v i t y a f t e r e i g h t t o 10 h o u r s , accompanied by p r e c i p i t a t i o n . D i a l y s i s against  n e u t r a l phosphate or " t r i s " b u f f e r s f o r as  l o n g as 24 hours i n the  c o l d produced no l o s s of  A c t i v i t y of e l e c t r o n o f methylene b l u e , 2 : 6  acceptors:  dichlorophenol  activity.  I n the  i n d o p h e n o l or pyocyanine,  g l u c o n i c dehydrogenase c a t a l y z e d the o x i d a t i o n of acid.  F e r r i c y a n i d e appeared t o p o i s o n the  c r e s y l b l u e was  inactive.  Reaction  slow w h i l e t h a t w i t h i n d o p h e n o l blue manometric work s i n c e the dye  system and  was  c o u l d not be used f o r oxygen.  Oxygen t a k e n up/hr. ul.  blue  dichlorophenol  brilliant  w i t h methylene b l u e  26.0  Pyocyanine 2:6  gluconic  i s not r e o x i d i z e d by  Hydrogen A c c e p t o r Methylene  presence  78.0 indophenol  0*  B r i l l i a n t c r e s y l blue  0  Fe r r i c y a n i d e  0  *Dye reduced i n e i g h t minutes as measured i n photometer.  spectro-  52  Reaction  w i t h pyocyanine was rapid, and t h e r a t e  with increasing concentrations concentration  increased  o f pyocyanine u n t i l an optimum  o f 680V o f pyocyanine p e r 3 m l . was r e a c h e d .  Beyond t h a t t h e r e was no i n c r e a s e i n r a t e ( F i g . 6 ) . Cofactors:  No c o f a c t o r s o t h e r t h a n the hydrogen  acceptor  were found t o be n e c e s s a r y f o r the a c t i v i t y o f t h e  enzyme.  The a d d i t i o n o f DPN, TPN, ATP, FMN, FAD, cytochrome  c, Mg, Mn o r Fe++ d i d not i n c r e a s e t h e a c t i v i t y . was not reduced as measured by i n c r e a s e the r e a c t i o n m i x t u r e at 340 mu.  DPN o r TPN  i n o p t i c a l density of  The r e a c t i o n m i x t u r e  contained  0.1 m l . o f t h e p u r i f i e d enzyme, 0.2 m l . o f g l u c o n i c a c i d , 0.1 m l . o f .005M DPN o r TPN, 1.5 m l . o f pH 5.6 b u f f e r and d i s t i l l e d water t o make up t o 3.0 m l . Treatment  w i t h c h a r c o a l t o remove  any bound DPN ( T a y l o r et_ a l . 1948) d i d not reduce t h e enzymic activity. the  The enzyme s o l u t i o n i n t h i s case was t r e a t e d i n  c o l d w i t h 1.3 mg. o f n o r i t p e r m l . and a f t e r g e n t l e a g i t a -  t i o n f o r t h r e e minutes t h e n o r i t t i o n and f i l t r a t i o n .  was removed by c e n t r i f u g a -  The absence o f m e t a l requirement f o r  the enzyme was c o n f i r m e d by d i a l y z i n g a g a i n s t versene t o the method o f Racker ( 1 9 5 3 ) .  according  The s o l u t i o n was d i a l y z e d  a g a i n s t 1000 volumes o f 0.6 p e r cent versene i n 0 . 0 2 M o f pH 7.4 for 20 hours and then a g a i n s t  phosphate  1000 volumes of 0.6  per cent versene i n 0.9 p e r cent KC1 f o r another 20 h o u r s . E f f e c t of i n h i b i t o r s :  Iodoacetate,  sodium  azide,  2:4 d i n i t r o p h e n o l , sodium a r s e n i t e and sodium f l u o r i d e d i d not i n h i b i t t h e a c t i o n o f the enzyme.  The absence o f f l u o r i d e  i n h i b i t i o n i n d i c a t e s that phosphorylation  o f s u b s t r a t e i s not  53  FIGURE 6:  E f f e c t o f pyocyanine c o n c e n t r a t i o n on v e l o c i t y o f o x i d a t i o n by g l u c o n i c dehydrogenase. Oxygen uptake i s shown u s i n g 5 micromoles o f subs t r a t e and v a r y i n g c o n c e n t r a t i o n s o f p y o c y a n i n e . The r e a c t i o n  was  carried  out a t 30.5°C and pH  5.6.  -  involved.  54  Cyanide, glutathione  -  and c y s t e i n e i n c r e a s e d t h e  r a t e of o x i d a t i o n ( F i g . ? ) • Substrate ketogluconic  specificity;  G l u c o s e , glucuronic a c i d , 2-  a c i d , f r u c t o s e , g l u c o s e - 6 - p h o s p h a t e , 6-phospho-  g l u c o n a t e and mannose, were not o x i d i z e d by the The  enzyme system.  p u r i f i e d enzyme a p p e a r s , t h e r e f o r e , t o be q u i t e  for gluconic  acid.  P r o d u c t o f t h e enzymic a c t i v i t y :  The p r o d u c t ' o f  the o x i d a t i o n o f g l u c o n i c a c i d has been r e p o r t e d ketogluconic  specific  acid.  t o be 2-  I n t h i s r e a c t i o n no CC>2 e v o l u t i o n t a k e s  place. C6 12°7 H  +  2°2  Data f o r t h i s were o b t a i n e d Dixon ( 1 9 4 3 ) .  C H 6  1 0  0  7  +-H 0 2  by the manometric t e c h n i q u e of  Manometric e x p e r i m e n t s were conducted w i t h ( l )  enzyme and s u b s t r a t e  and w i t h KOH i n t h e c e n t r e  w i t h no KOH but w i t h 0.3 ml. o f 3N H C 1 i n the  w e l l ; (2) side-arm.  At  the end o f t h e r e a c t i o n (where | uM o f oxygen p e r uM o f g l u conate was t a k e n u p ) , t h e HC1 was t i p p e d i n t o t h e r e a c t i o n chamber i n the t h i r d dioxide evolution.  cup.  There was no evidence o f carbon  There was a l s o no d i f f e r e n c e between t h e  apparent oxygen uptake i n t h e cup c o n t a i n i n g KOH and t h a t where KOH had been o m i t t e d ,  showing t h a t no CO2 was e v o l v e d  during the o x i d a t i o n . For i d e n t i f i c a t i o n o f t h e product t h e chromatographic procedure d e s c r i b e d ammoniacal-silver  under "Methods" was f o l l o w e d .  n i t r a t e reagent t h e p r o d u c t from  With gluconic  a c i d o x i d a t i o n gave a spot i d e n t i c a l i n appearance w i t h 2-  55  I  ••  i i  20  40 60 MlNUTES  FIGURE 7: Influence of reducing  agents on the o x i d a t i o n o f  gluconate.  Each cup c o n t a i n e d ; 0 . 5 m l . o f p u r i f i e d enzyme; 1 . 5 m l . o f M/15 phosphate b u f f e r o f pH 5 . 6 ; 0 . 2 m l . o f g l u conate ( 5 uM); 0 . 3 m l . o f KCN (lO~3Mj w i t h AN KCN i n 20 p e r cent KOH i n c e n t r e w e l l ; ( o r 0 . 3 m l . o f g l u t a t h i o n e or cysteine 10-3M); 0 . 2 m l . o f p y o c y a n i n e . A = gluconate B = gluconate  alone + KCN ( o r g l u t a t h i o n e o r c y s t e i n e ) .  -  ketogluconic a c i d .  -  Rf v a l u e s f o r the  The  t h e r s u b s t a n t i a t e d the  56  chromatogram f u r -  finding.  H e i g h t of s o l v e n t f r o n t -  19.25  ins.  H e i g h t o f glucose  =  10.00  ins.  0 . 5 2  H e i g h t of g l u c o n i c a c i d spot =  5.75  ins.  0 . 2 9  H e i g h t of 2 - k e t o g l u c o n i c a c i d spot =  7 . 0 0 ins.  O.36  H e i g h t of o x i d a t i o n product  7 . 0 0 ins.  O.36  Since  spot  =  5 - k e t o g l u c o n i c a c i d cannot be o x i d i z e d by  e i t h e r whole c e l l s or c e l l e x t r a c t s of P_. a e r u g i n o s a e a r l i e r work ( N o r r i s and  Campbell, 1 9 4 9 )  not i n c l u d e d as a standard  since  conclusively elimin-  ated i t as a p o s s i b l e p r o d u c t of the r e a c t i o n s b e i n g i t was  and  studied,  i n t h e s e chromatograms.  With H a n e s reagent no phosphate compounds c o u l d  be  1  detected  on a paper chromatogram. M i c h a e l i s constant:  E x p e r i m e n t s were conducted w i t h  the p u r i f i e d enzyme to determine the  c o n c e n t r a t i o n of sub-  s t r a t e r e q u i r e d t o s a t u r a t e the enzyme. s o l u t i o n c o n t a i n i n g O . 3 6 4 mg. i n the Warburg re s p i r o m e t e r v a r y i n g from 2 . 5 t o 1 5 . 0 uM. case was The  p l o t t e d a g a i n s t the  0 . 2 m l . of the  of p r o t e i n was  enzyme  allowed to react  w i t h amounts o f g l u c o n i c a c i d The  r a t e of r e a c t i o n i n each  substrate concentration  s u b s t r a t e c o n c e n t r a t i o n e x p r e s s e d i n moles of  (Fig. 8 ) .  substrate  per l i t r e  at h a l f the l i m i t i n g v e l o c i t y gave the M i c h a e l i s  constant  (Km).  57  7 Or  60* -  50 -  40 30  20  10  0.1  0.2  0.3  0.4  0.5  0.6  FIGURE 8: M i c h a e l i s constant o f g l u c o n i c dehydrogenase. Oxygen uptake i s shown u s i n g v a r y i n g c o n c e n t r a t i o n s o f g l u c o n a t e . Each cup c o n t a i n e d ; 0 . 2 m l . o f p u r i f i e d enzyme; 1.5 m l . of M/15 phosphate b u f f e r pH 5.6; 0 . 2 m l . o f pyoc y a n i n e ; g l u c o n a t e ; 0 . 1 5 m l . o f 20 p e r cent KOH; water t o 3.15 m l .  -  58  -  = 64 uM 0  Maximum v e l o c i t y Substrate concentration Corresponding to 3 2 uM 0 Km  =  2  P  e r  hour  ) )  P r hour )  -  0.9  ml. = 2 . 2 5 uM  e  2  2.25 x 1000 M = .00075 M 3 x 1000 x 1000  Properties of the Enzyme System Influence of pH:  Using the crude sonicate as the  source of enzyme, experiments were conducted over the ranges of pH 3 « 2 - 7 . 5 i n veronal buffer. be 5 . 6 ( F i g . 9 ) «  The optimum pH was found to  While the oxidation proceeds there i s a  strong tendency f o r the reaction mixture to become basic and so the veronal buffer had to be prepared with double the concentration of the reagents.  If any p r e c i p i t a t i o n occurred,  the solution was warmed to above 30°C and then used f o r the experiment. When the p u r i f i e d enzyme plus pyocyanine were used in place of the sonicate for these experiments, there was the suggestion of a second maximum at pH 4 « 6 ( F i g . 1 0 ) . Since decarboxylation was possible at t h i s low pH, manometric experiments f o r carbon dioxide determination by Dixon's method were repeated.  However, no carbon dioxide was evolved.  The  product was chromatographically analyzed as before, but 2 ketogluconic acid was the only product.  It may be concluded  from these experiments that at this pH i t was the hydrogen acceptor and not the enzyme which was influenced. As i n the case of many other enzymes, the gluconic  59  -  FIGURE 9; Rate of oxidation of gluconate by soniced c e l l s as a function of pH. Each cup contained; 0.5 ml. of sonicate; 2.0 ml. of M/15 veronal buffer; 0.2 ml. of gluconate (5 uM); 0.15 ml. of 20 per cent KOH; water to 3.15 ml.  60  dehydrogenase isolated from the c e l l has a pH optimum ( 5 . 6 ) different from that of the whole c e l l s ( 7 . 0 ) .  Although this  has been used i n arguing that working with isolated enzymes is making use of an a r t i f i c i a l environment, the advantages accruing from the study of an isolated enzyme are many-fold. Phosphorylation:  The lack of i n h i b i t i o n of the  enzyme by sodium fluoride indicated that phosphorylation of the  substrate was not involved i n i t s oxidation.  Another  proof of this was the fact that neither ATP nor AMP the  enzyme.  activated  However, Narrod and Wood (1954) reported that  P. fluorescens phosphorylates gluconate prior to oxidation and that the phosphorylated intermediate i s 6-phosphogluconate. In view of this observation and the fact that an enzyme capable of dehydrogenation of gluconate had never been i s o l a t e d , a series of experiments were conducted to determine whether or not  P. aeruginosa phosphorylated gluconate prior to oxidation. (a) Production of acid;  The production of  acid i s the method followed by Colowick and Kalckar (1943) to detect phosphorylation. In this procedure, the substrate i s reacted on by the enzyme i n the presence of ATP.  The  transfer of phosphate from ATP results i n the l i b e r a t i o n of 1 mole of acid per mole of phosphate.  In the presence of  bicarbonate, the rate of phosphate transfer can be followed manometrically by observing the evolution of carbon dioxide. The phosphate transfer i s thus proportional to a c i d i f i c a t i o n and i s measured manometrically as carbon dioxide l i b e r a t e d  -  i  61  -  . o.  FIGURE 10:  Rate of oxidation of gluconate by p u r i f i e d gluconic dehydrogenase as a function of pH.  Each cup contained; 0.2 ml. of enzyme solution; 2.0 ml. of M/15 veronal buffer; 0.2 ml. of gluconate (5 uM); 0.15 ml. of 20 per cent KOH; water to 3.15 ml.  62 from the bicarbonate. be concluded  If no carbon dioxide i s evolved i t can  that phosphorylation i s absent i n the system.  This experiment was carried out with extracts of P. aeruginosa and P. fluorescens under aerobic and anaerobic conditions. Warburg flasks f i t t e d with gas vents were used and the following solutions added; 0.5 ml. of the sonic extract, 50 uM ATP; .005 M iodoacetate (to i n h i b i t oxidative processes and to permit accumulation  of primary esters formed); M/18 sodium  fluoride (to i n h i b i t decomposition  of the phosphate esters)  and 0.05 M sodium bicarbonate to react with the acid formed during phosphate transfer thus releasing C02« was used as the substrate.  Gluconic acid  The C 0 - a i r mixture was c i r c u l a t e d 2  through the cups f o r 15 minutes and the stopcocks  closed o f f .  After gluconic acid was tipped i n , there was no carbon dioxide evolution, showing absence of phosphorylation.  The experi-  ment was repeated using nitrogen instead of a i r , but s t i l l phosphorylation was not shown.  To eliminate the p o s s i b i l i t y  that phosphorylation i s not evident because of the high pH used i n the experiments, another anaerobic experiment was run at pH 6.0 ( i n the presence of 6.6 x 10~*M sodium bicarbonate). S t i l l no CO2 was evolved. The above experiments were repeated with a-sonic extract of P. fluorescens A. 312 under i d e n t i c a l conditions. Here though aerobically no phosphorylation could be detected, anaerobically, carbon dioxide was evolved (156 u l of CO2 P r Q  5 uM of gluconate) showing phosphorylation.  I t i s thus clear  that there i s no phosphorylation i n the system i n P. aeruginosa  -  63  -  while i t i s present i n P. fluorescens.  The anaerobic experi-  ment i s much more useful for i t stops the reaction prior to the oxidative step and favors the accumulation  of a phosphory-  lated intermediate. (b) Reduction phosphorylated  of TPN;  to phosphogluconic  If gluconic acid i s  acid, phosphogluconic  dehy-  drogenase should be able to oxidize i t i n the presence of  TPN.  The measurement of TPN reduction should then indicate whether the i n i t i a l phosphorylation has taken place. Phosphogluconic  dehydrogenase was i s o l a t e d from  Brewer's yeast by the method of Horecker and Smyrniotis The enzyme was  (1951)*  f i r s t tested for i t s a c t i v i t y against phospho-  gluconic acid and when i t was  found active t r i e d against the  sonic extracts of P. aeruginosa 9027 and P. fluorescens A. The cuvettes contained 0.1 ml. sonic extract, 0.1 ml. ( 3 . 0 uM per ml.), 0.5 ml. phosphogluconic  2  TPN  dehydrogenase, 0.04  ml. gluconic acid (50 uM per ml.), 0.05 ml. of ATP ml.), 0.02 ml. of MgCl  312.  (200 uM per  (0.1 M) and water to 3.0 ml. were  added and the reduction of TPN,  i f any, measured at 340 mu  every minute for 10 minutes, ( F i g . 1 1 ) .  As the figure shows,  TPN reduction occurred only with P. fluorescens, thus showing that only i n this organism was  6-phosphogluconate being formed.  (c) Formation of high energy bond;  Although  one might expect that ATP would be needed to i n i t i a t e the reaction with gluconic acid, nevertheless, i t would be usual for the o v e r a l l oxidation to r e s u l t in the accumulation energy bonds.  Lehninger  (1951)  of high  calculated that for every atom  64  -  of oxygen taken up by a b i o l o g i c a l reaction, three high energy bonds are produced.  It has already been shown that no ATP  could be detected chromatographic a l l y as the product of gluconate oxidation.' Another method i s to substitute the oxidation of glucose or gluconate f o r ATP where the l a t t e r i s necessary for i n i t i a t i n g  a reaction.  Such a reaction i s the  formation of phosphogluconic acid from glucose. Glucose + ATP Hexokinase^ Glucose-6-Phosphate Glucose-6-Phosphate 6-Phosphogluconic  + TPN Glucose 6P0^ acid -f TPNH f H  + ADP  dehydrogenase^  +  ATP i s necessary f o r the i n i t i a l phosphorylation of glucose and the presence of ATP may be followed by measuring the reduction of TPN at 340 mu by the method of Romberg (1950). The components of the test werej glucose (5 uM) 0.2 ml., MgCl (15 uM) 0.1 ml., l y o p h i l i z e d hexokinase (10 mg. per ml.) ml., sonicate of P. aeruginosa 0.05 ml., AMP  2  0.1  (50 uM per ml.)  0.1 ml., and water to a f i n a l volume of 3.0 ml.  The a c t i v i t y  of the enzymes was tested with 5 uM ATP i n place of the Pseudomonas sonicate.  The blank adsorption c e l l contained  water i n the place of TPN. glueose-6-phosphate action.  I n i t i a l readings were obtained and  dehydrogenase  was added to s t a r t the re-  Readings were taken every minute, ( F i g . 12).  As the  figure shows, the oxidation of glucose by P. aeruginosa extract does not produce ATP which indicates that the reaction i s different from most which have previously been studied.  65  P. f luorescens  ^  2  o  4 6 MINUTES  FIGURE 11:  Gluconate phosphorylation by P. fluorescens as measured by TPN reduction i n the presence of excess phosphogluconic dehydrogenase. Extracts of P. aeruginosa 9027 gave no reduction of TPN under these conditions. The cuvette contained; 0.1 ml. of sonic extract; 0.2 ml. of gluconate (0.5 uM); MgCl2 (0.15 uM) 0.1 ml.; 0.5 ml. of phosphogluconic dehydrogenase; 0.1 ml. of TPN (2 mg. per ml); 0.05 ml. of ATP (10 uM); water to a f i n a l volume of 3.0 ml. Measurement of O.D. at 340 mu.  66  Alternative Methods of Enzyme P u r i f i c a t i o n , (1) (1954)  Use of Cutscum as s o l u b i l i z e r ;  Cotzias e_t a l . ,  have reported the use of isooctylphe.noxypolyethoxy-  ethanol.for s o l u b i l i z i n g enzymes.  This reagent i s an active  ingredient of the detergent Cutscum.  Experiments  were con-  ducted to replace sodium glycocholate by Cutscum since the l a t t e r does not leave ,any colour i n solution.  Cutscum was  added slowly to the cold sonicate' to a t o t a l concentration of 5 per cent by volume, and homogenized i n a van Potter homoP r e c i p i t a t i o n with 3 0 per cent ammonium sulphate  genizer.  showed enzymic a c t i v i t y i n the supernatant.  However, the  detergent had to be dialyzed away completely before treatment with ammonium sulphate i n order to avoid the formation of a d i f f i c u l t l y separable scum.  The a c t i v i t y of the treated pre-  paration was the same as with sodium glycocholate. (2)  Use of the ultracentrifuge;  The addition of  a foreign agent such as sodium glycocholate or Cutscum produces complexes with the l i p i d s present i n the sonicate. These complexes were found to i n t e r f e r e i n electrophoretic analysis. enzyme  It was,  therefore, decided to t r y to separate the  mechanically from the c e l l surface without the use  of Cutscum and through the use of the u l t r a c e n t r i f u g e  0  The washed c e l l s as usual, were subjected to sonic o s c i l l a t i o n for 2 0 minutes and centrifuged f o r 1 0 minutes at 25,000  for  x G.  The supernatant was  centrifuged at  6 0 minutes i n an ultra-centrifuge at 4 ° C .  105,000  x G  A clear l i q u i d  -  67 -  A T P  .25 >•  •  /  to-20 z ui  /  O  O—— Q  Q  Q  j .15 < o £ .10 o .05  / - /  c  / P. a e r u g i n o s a + A M P  \  2  6 8 TIME IN MINUTES 4  10  FIGURE 12;  P r o d u c t i o n of h i g h energy phosphate d u r i n g g l u c o s e o x i d a t i o n by P. a e r u g i n o s a 9027 as measured by TPN r e d u c t i o n i n the presence of hexokinase and glucose-6-phosphate dehydrogenase. The f i r s t c u v e t t e c o n t a i n e d 0.2 m l . of g l u c o s e (5 uM)j 0.1 m l . of MgCl2 (15 uM); 0.1 ml. of hexokinase (10 mg. p e r ml.); 0.1 m l . o f glucose-6-phosphate dehydrogenase (3 mg. per m l . ) ; 0.1 ml. of TPN (2 mg. per m l . ) ; 0.1 ml. of ATP (10 uM); 1.5 m l . of M/15 phosphate b u f f e r pH 7.0 and water t o 3.0 m l . In t h e second c u v e t t e 0.1 m l . of s o n i c e x t r a c t of P. a e r u g i n o s a and 0.1 m l . o f AMP (10 uM) r e p l a c e d ATP.  68 separated from a reddish-brown precipitate which may be cytochromes.  The supernatant by i t s e l f had only poor a c t i v i t y  against gluconic  acid but was stimulated  presence of pyocyanine.  three-fold i n the  The precipitate suspended i n d i s -  t i l l e d water, also had some a c t i v i t y against  gluconic  acid.  Ammonium sulphate to 30 per cent of saturation was added to the supernatant and the precipitate formed was c e n t r i fuged o f f . against  Both the precipitate and supernatant had a c t i v i t y  gluconic  solubilized.  acid showing that part of the enzyme had been  The supernatant was dialyzed and treated with  protamine sulphate as usual and the nucleoproteins off.  centrifuged  The solution was l y o p h i l i z e d and dissolved i n the minimum  amount of M/10 phosphate buffer of pH 6.8 after which i t was dialyzed against one-third  the same buffer overnight and made up to  the o r i g i n a l volume.  The solution was clear and  colourless. The  f i n a l product was subjected to analysis i n the  electrophoretic  apparatus.  The following are the relevant  data f o r the analysis: Current  10 m.a.  Duration  2 hours, photographs taken each hour.  Temperature  1°C  Slit  40°  Filter  Red  Position  3 cm.  Buffer  0.1 ionic strength pH 6..83  of phosphate buffer of  Coulombs c a r r i e d  69  by l e f t  -  electrode:  754.00 c a t h o d e 778.00 anode The cause The  p h o t o g r a p h shows two p e a k s ,  of i t s h i g h m o b i l i t y , a p p e a r s t o be p r o t a m i n e  sulphate.  o t h e r may be t h e enzyme. F u r t h e r work t o c o r r o b o r a t e  be  one o f w h i c h b e -  carried  out because  these  finding  could not  of l a c k of f a c i l i t i e s *  DISCUSSION In has of  the p a s t  been a b a r r i e r metabolic  genase  preventing  pathways.  necessary  e n e r g y was u t i l i z e d  The p r e p a r a t i o n  Barron  (1954)  forits activity or produced  have p o i n t e d  m e t a b o l i s m must be made w i t h Some o f t h e s e different  ity  t h e e l u c i d a t i o n o f t h e mechanism  techniques  intermediates;  o f t h e enzyme.  erroneous  and t o f i n d  during  dehydro-  As  Ghiretti  o f t h e pathways o f  as many t e c h n i q u e s  as p o s s i b l e .  a r e d e t e c t i o n i n t h e medium o f t h e d e t e c t i o n o f t h e enzyme i n c e l l inhibitors  "Use o f o n l y one of t h e s e  on t h e a c t i v -  methods may g i v e  answers as shown i n t h e c o n t r a d i c t i n g c o n c l u s i o n s  by a number o f i n v e s t i g a t o r s who u s e d  approach".  out whether  oxidation.  out a study  e x t r a c t s and t h e u s e o f s p e c i f i c  reached  of g l u c o n i c  f o r t h e s u b s t r a t e has made i t p o s s i b l e t o s t u d y t h e  cofactors  free  o f o x i d a t i v e enzymes  i n a s o l u b l e f o r m i n a s t a t e o f p u r i t y where i t i s  specific  and  the i n s o l u b i l i t y  only a single  70 Earlier of  work  phosphorylation  aeruginosa absence lation  during  at least  i s involved This  activate  t h e enzyme.  during of  from  phosphate.  i s also that  no energy  When  the product  has  already  that  gluconic  reported  no e n e r g y  acid  of  growth  of  glucose,  firmed  acid  gluconic  from  was  this  de-  of  i s this  f o r gluconic  t o be p r e s e n t  the p u r i f i e d  i n  enzyme  during  with  No i n c r e a s e o f AMP  of gluconic  no e v i d e n c e  t h e same  occurs  equimolecular  the o x i d a t i o n  acid  This  was  of ATP.  the oxidation  acids.  inorganic  (Campbell  that  i n which  i n activity  and  by the f a c t with  the  o f ATP i s p r o d u c e d  laboratory  or 2-ketogluconic  by t h e e x p e r i m e n t  as  and t h e  by e x t r a c t s  experiments  acid.  there  i s produced  o f P. a e r u g i n o s a  from  of o x i d a t i o n  from  as e v i d e n c e d  by t h e  ionization  system  i n the presence  chromatographically  acid  no p h o s p h o r y l a t i o n  i n the form  of gluconic  was n o t i c e d  phosphory-  and K a l c k a r  and Schwerdt  evidence  analyzed  1954)  that  absent  no  The  9027»  P. a e r u g i n o s a  enzyme  t h e enzyme  b y Wood  b y P.  of phosphogluconic  of gluconic  i s , therefore,  the oxidation  been  of C o l o w i c k  lack  ATP does n o t  of increased  confirmation  reported  There purified  The a b s e n c e  a  stage.  that  that  The a d d i t i o n a l p h o s p h o r y l a t e d  P. f l u o r e s c e n s obtained  of glucose  of gluconic  TPN i n t h e p r e s e n c e  a r e added  oxidation  indicated  indicates  by the f a c t  i n the oxidation  organism. acid  by f l u o r i d e  by the technique  hydrogenase involved  the oxidation  i s confirmed  t o reduce  laboratory  i n the oxidation  enzyme.  failure  this  a s f a r as t h e 2 - k e t o g l u c o n i c  of i n h i b i t i o n  determined  from  It  e_t  a l .  of amount amounts  was  con-  of glucose  by  71  P.  a e r u g i n o s a d i d not  of  g l u c o s e by  by  P.  hexokinase.  This  aeruginosa i s different  according atom  provide energy  of  to Hunter,  oxygen  Ochoa,  i s taken  up  f o r the  finding  from  that  Lehninger with  the  phosphorylation  shows by  and  that  oxidation  other tissues other workers,  production  3  of  where, 1  moles  of  ATP. The the  production  oxidation  nucleotides, (1946)  of  the  molecules  substrate  flavoproteins  illustrates  3  of  this  and  with  of  high  i s associated the  the  with  cytochrome  following  energy  during  the  pyridine  system.  Lipmann  diagram:  0 1..2 Cytochromes  PO:  Flavoprot eins  P0T7  Pyridine nucleotides  PO;  v  Adenylic acid  v o  1  t  2H fx.  Metabolite In  the  reduced  case by  of the  mononucleotide  gluconic enzyme or  therefore,  does  nucleotide  system  a rapid that  hydrogen  this  pigment  nor  flavin  not  dehydrogenase  seem  or the  was  the  adenine t o be  DPN  system  in a  system similar  was  activated  through  flavoproteins.  functions  TPN  dinucleotide.  linked  acceptor i n the  or  by  The the  i t i s  manner  in  flavin  system,  pyridine  Pyocyanine and  not  acted  possible growing  as  -  72  -  cells. Friedheim  found t h a t pyocyanine c a t a l y s i s  (1931)  does n o t have an i n d i s c r i m i n a t e e f f e c t i n a l l o x i d a t i o n s , but o n l y i n the o x i d a t i o n of c e r t a i n s u b s t a n c e s c l o s e l y connected w i t h the b a c t e r i a l body l i k e g l u c o s e , acid.  a s p a r a g i n e and p y r u v i c  I t i s , therefore, l o g i c a l that gluconic acid oxidation  can be s i m i l a r l y a f f e c t e d .  Pyocyanine has been found t o i n -  c r e a s e the r e s p i r a t i o n of l i v i n g c e l l s t o a great degree and the r e v e r s i b i l i t y of i t s o x i d a t i o n and r e d u c t i o n i s r e s p o n sible for this.  Friedheim  and M i c h a e l i s  (1931)  have found  t h a t a t pH's l e s s than 6 , i t behaves e n t i r e l y as a r e v e r s i b l e dye  of q u i n o n o i d  s t r u c t u r e and t h a t the s l o p e  of the t i t r a t i o n  curve i n d i c a t e s t h a t 1 m o l e c u l e of the dye combines w i t h 2 hydrogen atoms s i m u l t a n e o u s l y .  This g i v e s an i n d i c a t i o n of t h e  k i n e t i c s of g l u c o n a t e o x i d a t i o n which i s most e f f i c i e n t a t pH optimum of and A s n i s  Most dehydrogenases have a h i g h pH optima  5«6«  and B r o d i e  (1953)  the o x i d a t i o n - r e d u c t i o n s  e x p l a i n t h i s by t h e f a c t t h a t i n  of t h e type i n which hydrogen i o n s  participate; Substrate-H  2  t DPN*  ^  *  a high hydrogen i o n c o n c e n t r a t i o n to the l e f t .  substrate  f DPNH f H+  would f a v o u r  the e q u i l i b r i u m  Where, on the o t h e r hand, 2 hydrogen atoms can  be accepted s i m u l t a n e o u s l y ,  l o w pH cannot a f f e c t the o x i d a t i o n  a d v e r s e l y w h i l e , a t t h e same t i m e , t h e r e i s no c o m p e t i t i o n the hydrogen a c c e p t o r  from t h e other  enzyme systems.  for  This  appears t o be what i s t a k i n g p l a c e i n g l u c o n a t e o x i d a t i o n  with  -  P.  73  aeruginosa. P y o c y a n i n e has been f o u n d t o c a t a l y z e  uptake  of the  s y s t e m as w e l l as  of h i g h energy is  similar  phosphate  to methylene  pyridine nucleotides Michaelis  (1935)  of b r i n g i n g about  compounds. blue  i n the  i n the  cozymase  from y e a s t c e l l s  esters.  These  amount of that  synthesis  esters  synthesis pyocyanine  and  blue, it  haemol-  addition  increased phosphates  went h a n d i n h a n d ;  by o m i t t i n g m e t h y l e n e  occur e i t h e r .  the  R u n n s t r o m and  of i n o r g a n i c  two e f f e c t s  i n o r g a n i c phosphorus  phosphate  thesized  d i d not  instance  and m e t h y l e n e  when r e s p i r a t i o n was s u p p r e s s e d phosphorylation  first  had two e f f e c t s ;  o x i d a t i o n and m e d i a t e d i n t h e to phosphate  the  i n a s y s t e m c o n s i s t i n g of  yzed b l o o d , hexosemonophosphate of  oxygen  In t h i s respect  second i n s t a n c e .  found t h a t  the  On t h e  contrary,  increased with time,  c o u l d , i n such a system,  o r b r o k e n down a c c o r d i n g t o w h e t h e r  blue, the  showing  e i t h e r be  or n o t  respira-  t i o n took p l a c e .  When m e t h y l e n e  cyanine,  r e s p i r a t i o n was c o u p l e d w i t h p h o s p h a t e  however,  synthesis  and t h e Since  not  a d d i t i o n of  it  o c c u r when g l u c o n i c  pyocyanine,  it  aeruginosa  the  transport  cytochrome  system.  other  shown t h a t  acid is  is possible  p e r i m e n t a l l y to act with  has been  b l u e was r e p l a c e d w i t h  cozymase  that  i n the  components  of the  capacity,  ester  p h o s p h o r y l a t i o n does  intact  presence  cells  a c t i n g as t h e  Though c y t o c h r o m e  pyo-  necessary.  o x i d i z e d i n the  system i s  in this  was n o t  syn-  c has n o t it  system l i k e  is  of  of  P.  electron  been f o u n d  possible  cytochrome  that a,  it  exalong acts  74  -  as a link between the substrate and molecular oxygen. It has been generally accepted that a bivalent oxidation i n homogeneous solution proceeds i n two successive univalent steps.  Since carbon i s quadrivalent, the product  of a univalent oxidation i s unstable as i t has a free r a d i c a l with one of i t s atomic valences unoccupied.  This i n s t a b i l i t y  implies a r e l a t i v e l y high energy content, to overcome which the substrate i s activated by the enzyme. Most of the b i o l o g i c a l oxidations are linked through the pyridinoprotein system which accepts 1 H ion and 2 electrons at a time and passes them on to the flavoproteins.  The flavo-  proteins have the capacity to pass on ,2 hydrogen ions and 2 electrons at a time to oxygen. oxide i s formed.  Under these conditions, per-  In aerobic microorganisms catalase i s  present i n the system to destroy peroxide. In the gluconic dehydrogenase  system both pyridino-  protein and flavoprotein seem to be absent as hydrogen acceptors and their place may be taken by pyocyanine or 2 , 6 - d i chlorophenol indophenol, both of which accept 2 hydrogen atoms at a time.  Ferricyanide, methylene blue or b r i l l i a n t  cresyl  blue, which can accept only 1 hydrogen atom at a time, were not effective i n this system. Dickens and Mcllwain (193&) determined the a c t i v i t y of the phenazines and some nonphenazine dyestuffs as carriers in the hexosemonophosphate system.  They found that the  phenazine derivatives (e.g., pyocyanine) were more active than  -  75  -  nonphenazine  derivatives like b r i l l i a n t  ylene blue.  M e t h y l C a p r i b l u e , though  p o t e n t i a l as t h e a c t i v e phenazine  c r e s y l b l u e and methi n the same range o f  d e r i v a t i v e s was found t o be  1  inactive.  They, t h e r e f o r e , c o n c l u d e d t h a t s t r u c t u r e i s more  important than  i n determining the a c t i v i t y of c a r r i e r s for  t h e i r system and t h e p e c u l i a r a c t i v i t y o f phenazines system may be due t o t h e i r a b i l i t y t o form  i n the  semiquinones.  Dickens and M c l l l w a i n a l s o found a p o i s o n i n g e f f e c t on t h e system by b r i l l i a n t  c r e s y l b l u e and methylene  b l u e when p r e s -  ent i n h i g h c o n c e n t r a t i o n s . The a c t i v i t y o f t h e hydrogen a c c e p t o r i n g l u c o n i c dehydrogenase seems t o f o l l o w t h i s p a t t e r n : Hydrogen A c c e p t o r Structure v  E^, pH 7 A c t i v i t y  2, 6 - d i c h l o r o p h e n o l i n d o p h e n o l Semiquinone  + 0 . 2 1 7  Good  Pyocyanine ( 4 - k e t o - N - m e t h y l phenazine)  Semiquinone  - 0 . 0 1 1  Good  Methylene  blue  Non-semiquinone  + 0 . 0 1 1  Feeble  Brilliant  c r e s y l blue  Non-semiquinone  + 0 . 0 4 5  N i l  Non-semiquinone  + 0 . 3 6 0  Nil  Ferricyanide 2,  6 - d i c h l o r o p h e n o l i n d o p h e n o l can a c c e p t 2 hydrogen atoms and  be  reduced. CI 0= (  y = N-<(  ^>-OH - ± % H0<^  ^ - NH-<f CI  ^-0H  75a Pyocyanine can accept 2 hydrogen atoms and an electron to form a semiquinone r a d i c a l . quinone.  It i s an example of a cationic semi-  Friedheim and Michaelis (1931) have shown that i n  ranges of pH greater than 6.0 i t behaved,.e'ntirely as a reversible dye of a quinonoid structure.  The slope of the t i t r a t i o n  curve indicated that 1 molecule of the dye combined with 2 hydrogen atoms simultaneously.  At pH ranges greater than 6.0  the t i t r a t i o n curves showed a different shape which was  inter-  preted by the assumption that the 2 H atoms were accepted i n two separate steps.  With gluconic dehydrogenase there i s a  definite drop i n a c t i v i t y at pH 6.0 even i n crude c e l l preparations which seems to indicate the p a r t i c i p a t i o n of pyocyanine i n the l i v i n g c e l l s also.  Weil-Malherbe (1937) found  a dehydrogenase i n animal tissues which oxidized 1 ( — ) o < hydroxyglutaric acid to °\-ketoglutaric  acid.  The properties  of t h i s enzyme are comparable to those of the gluconic dehydrogenase.  Its action did not depend on any coenzyme.  A carrier  was necessary f o r the reaction with molecular oxygen; pyocyanine was active whereas cytochrome c was i n a c t i v e .  Brilliant  cresyl blue and methylene blue were much less e f f i c i e n t than pyocyanine.  The structure of methylene blue indicates that  only 1 hydrogen atom can be accepted at a time while apparently this dehydrogenase and gluconic dehydrogenase both require simultaneous transfer of 2 hydrogen atoms.  -  76  -  The action of cyanide in stimulating the enzyme may be either i n forming a cyanohydrin derivative with the or i n reducing the potential of the system.  product  According to the  f i r s t alternative, 2-ketogluconic acid, formed by oxidation of gluconic acid may  be acting as an i n h i b i t o r  of the reaction and  cyanide removes i t by forming the cyanohydrin ever, semicarbazide  derivative.  How-  did not stimulate the enzyme nor did i n -  creasing concentrations of 2-ketogluconic acid i n h i b i t the a c t i v i t y of the enzyme.  The second alternative seems to be  the more acceptable one, since both glutathione and cysteine activated the enzyme to the same degree as cyanide. The nine-fold increase in t o t a l a c t i v i t y during purif i c a t i o n may  be due to the removal either of toxic factors or  competing reactions.  However, the s t a r t l i n g increase i n t o t a l  a c t i v i t y occurred at the s o l u b i l i z i n g  step and so probably  represents an increase in enzyme surface. Energy for synthesis i n b i o l o g i c a l materials may  be  available commonly i n the form of oxonium ion, sulfonium ion, quaternary nitrogen, thioester or high energy phosphate bond. Of these, the high energy phosphate bond i s probably the most common and has been studied i n d e t a i l .  77  The question of whether high energy bonds are formed during the oxidation of a l l substrates has been a highly cont r o v e r s i a l one.  It i s now  gen from reduced DPN  accepted that the transfer of hydro-  or TPN to oxygen i s associated with the  production of 3 high energy bonds and the oxidation of 3 phoswith the production of 1 molecule  phoglyceraldehyde  energy phosphate bond.  of high  Ochoa ( 1 9 4 7 ) working with pigeon breast  muscle found that ATP-ase present i n the tissue destroyed the A-TP which was being produced.  Even the addition of sodium  fluoride which i n h i b i t s ATP-ase could only bring the destruction down to 4 5 - 5 0 per cent.  To correct for t h i s , he studied  the oxidation of 3 phosphoglyceraldehyde DPN  in the presence of  and pyruvate and i n the absence of oxygen, a reaction which  was known to give 1 high energy bond per pair of hydrogens transferred.  The oxidation of 3 phosphoglyceraldehyde  i n the  muscle served as a means of measuring the amount of high energy bonds destroyed by the ATP-ase.  Next, pyruvate was  oxidized by  muscle and the values corrected for the amount of ATP in the reaction.  found that 3 moles of ATP were formed  It was  per atom of oxygen taken 2CH3C00H  +  2  J02  destroyed  up. ^  S  3C0  2  +  2H20  f  15  Ochoa also tentatively concluded that DPN-  ph. and  linked oxidations were not coupled with phosphorylation.  TPNHe  found that massive concentrations of DPNH were r e a d i l y oxidized by preparations of known a c t i v i t y towards substrates of the Kreb's cycle, but no phosphorylation occurred.  Secondly,  o  -  78  the oxidation of i s o c i t r a t e i n s i m i l a r systems poisoned by. arsenite, to abolish further oxidation of «C-ketoglutarate, likewise showed no phosphorylation. Lehninger findings.  (1951)  questioned the v a l i d i t y of Ochoa's  I t was found that i n his f i r s t experiment high con-  centrations of DPNH uncoupled phosphorylation in his second, arsenite uncoupled i t . Friedkin and Lehninger  (1948)  from oxidation;  Using a l a b e l l e d system  found that there was as much  high energy phosphate formed during the oxidation of 1 mole of DPNH as of 1 mole of malate.  They also found that i n the  case of mitochondria there was a difference between the a c t i v i t y of 'internal' and 'external' cytochrome c and DPN. Their experiments indicated that permeability and the inner structure of the mitochondria are of great importance i n determining  the P/0 r a t i o .  Thus 'internal' DPN i s more heat-  stable than externally added DPN; maximal P/0 r a t i o s were obtained  only at the optimum osmotic concentration;  'internal'  cytochrome c i s available for the oxidation of DPNH; and the rate of oxidation of DPNH i s three to f i v e times as fast when the mitochondria are f i r s t exposed to hypotonic conditions. DPN  or cytochrome c has also to be attached  before phosphorylation  can take place.  to the c e l l  surface  He found that the  oxidation of reduced DPN or TPN gives r i s e to 3 high energy bonds per mole. Slater  (1950)  has presented data i n d i c a t i n g that  the P/0 r a t i o s during**-ketoglutarate  oxidation by heart  79  muscle preparations  are the same when using either oxygen or  cytochrome c as the ultimate electron acceptor.  He  blocked  cytochrome c with cyanide and got the same amount of high energy bonds produced.  He,  therefore, concluded that no phos-  phorylation occurs between cytochrome c and oxygen. (1951) i s prone to argue this contention  Lehninger  since he feels that  the methods which Slater used are inadequate for measuring the very small exchanges which might occur i n the region of cytochrome c to oxygen which makes up a very substantial portion of the "E.M.F. span". The r e s u l t s obtained with gluconic dehydrogenase seem to corroborate  Slater's findings.  Though flavoproteins  and pyridinoproteins do not take part i n the oxidation by this enzyme, the lack of a c t i v i t y i n the absence of an added hydrogen acceptor chromes may  when the enzyme i s s o l u b i l i z e d shows that cytobe involved i n tfie oxidation.  Since no phosphory-  l a t i o n i s involved during the span of oxidation, i t seems reasonable to conclude that no phosphorylation  occurs i n this case  also between cytochrome c and oxygen. The oxidation of gluconic acid i n some mammalian tissues also seems to follow t h i s pattern. head (1954) have reported  Salmony and White-  that gluconic acid i s oxidized by  kidney s l i c e s , but they could not demonstrate the product of the reaction.  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