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A study of the glucose oxidizing system of pseudomonas aeruginosa Stewart, J. E. (James Edward) 1954

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A Study of the Glucose Oxidizing System of Pseudomonas Aeruginosa by JAMES EDWARD STEWART A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Dairying We accept t h i s thesis as conforming to the Standard required from candidates for the degree of MASTER OF SCIENCE Members of the Department of Dairying THE UNIVERSITY OF BRITISH COLUMBIA May 1954 ABSTRACT C e l l , free extracts of glucose grown cultures of P. aeruginosa were prepared by exposure to sonic v i -bration. These sonicates were capable of o x i d i z i n g glucose-6-phosphate, ribose-5-phosphate, glucose, gluconic acid and gluconolactone. Treatment of the sonicate with (NH4)2S04 resulted i n the formation of precipitateswhich possessed the a b i l i t y to oxidize glucose, gluconolactone and gluconic a c i d . After (NH^JgSO^. treatment neither the superna-tant nor the p r e c i p i t a t e could oxidize the phosphory-l a t e d compounds. Since 30.0% (NH^JgSO^ or centrifuga-t i o n f o r one half hour at 25,000X g precipitated the enzymes they were considered to be insoluble. P r e c i p i t a t i o n with d i f f e r e n t concentrations of (NH4)2S04 and MI1SO4 f a i l e d to separate the glucose and gluconate enzymes from one another. Extraction with b i l e s a l t s s o l u b i l i z e d the gluconate enzyme, but pr e c i p i t a t e d the glucose enzyme along with a high proportion of the gluconate system. An increase i n concentration of the b i l e s a l t s destroyed the glucose enzyme while a reduction l e f t a large percentage i n so l u t i o n . The addition of glycine to the sonicate s o l u b i l i z e d the enzymes but did not aid i n t h e i r separation. When added before the sonic treatment, the enzymes became l a b i l e to protein p r e c i p i t a n t s . Etihyl alcohol, dioxane and acetone destroyed glucose oxidizing and gluconate oxidizing enzyme ac-t i v i t y while ethyl ether destroyed only the glucose system. The use of a growth substrate other than glucose resulted i n the formation of a reduced gluconic acid system, but the re s u l t s were not uniform i n that the gluconate enzyme frequently was very a c t i v e . The glucose system was sensitive to KCN and NaN3, and 8-hydroxy-quinoline but not to NaF. The 8-hydroxy-quinoline i n h i b i t i o n could be overcome by Mg**. Adenosinetriphosphate, f l a v i n e adenine dinucleotide, diphosphopyridinonucleotide, triphosphopyridinonucleo-t i d e , had no ef f e c t on the glucose oxidizing system. Methylene blue, b r i l l i a n t c r e s y l blue and pyocyanin had no a b i l i t y to act as a hydrogen acceptor. However 2.6 dichlorobenzenoneindophenol stimulated the reaction. When t h i s dye was added i n the presence of MgS04 a 300.0$ increase was noted. The product of the reaction was determined by paper chromatography to be gluconic a c i d . These data indicate that the glucose dehydrogenase d i f f e r s from any previously described glucose dehydro-genase and that some unknown hydrogen transport system apparently functions i n the transport of electrons to the cytochromes. The data support e a r l i e r conclusions that the reaction does not involve phosphorylation. ACKNOWIJinXjMENTS I wish, to express my sincere thanks to Dr. J . J . R. Campbell f o r h i s encourage-ment and assistance during the course of th i s work, and to Dean B. A. Eagles f o r his valuable suggestions and assistance throughout t h i s study. I also wish to thank the National Research Council f o r a grant to carry out part of th i s work. TABLE OF CPATENTS Page Introduction 1 Methods 10 B a c t e r i o l o g i c a l 10 Chemical 11 Experimental 18 I I s o l a t i o n and P u r i f i c a t i o n of the Enzyme 18 Table I 21 Table II 25 Table I I I 25 Table IV 27 Table V 29 Figure I 31 Table VI 33 II Properties of the Enzyme System 34 Table VII 35 Figure I I 36 Table VIII 37 Table IX 38 Discussion 40 Summary 45 Bibliography 48 1. INTRODUCTION Interest i n the study of aerobic metabolism r e -ceived an impetus when Dickens (1938 a, b) f i r s t demon-strated the metabolic scheme involving glucose-6-phosphate, 6-phosphogluconic and pentose phosphoric acids. This pathway was d i s t i n c t from the g l y c o l y t i c scheme and was the f i r s t p o s i t i v e evidence that an alternate pathway for carbohydrate metabolism existed. P r i o r to t h i s f i n d i n g , i t was almost u n i v e r s a l l y accepted that the oxidation and fermentation of glucose proceeded by a common pathway as f a r as the pyruvic acid. The oxidative steps i n the degradation of glucose were pictured as taking place a f t e r the formation of pyruvate and involved the c l a s s i -c a l Kreb*s t r i c a r b o x y l i c acid cycle. Under anaerobic conditions, pyruvate either acted as a hydrogen acceptor with the resultant formation of l a c t i c a c i d , or was cleaved to compounds, such as acetaldehyde, which would act as hydrogen acceptors. However, fundamental studies on t h i s problem were interrupted by the war and Dickens' i n i t i a l observations were not confirmed or extended during the next ten years. In fact Colowick and Kaplan (1951) stated that "The universal existence of the Embden-Meyerhof pathway i s so well accepted as to hardly require further documentation." Umbreit (1949) made a s i m i l a r 2. statement, "The concept of a f b a s i c metabolic ground plan' has suffered no severe blows i n the studies of the past year." At the time these statements were made considerable evidence was also available to indicate that glucose could be oxidized by a pathway which did not involve phosphorylated hexoses. However, these non-phosphorylated reactions were not considered to be important or even possible physiological reac-tions and so did not concern the majority of workers. A scheme of glucose oxidation based on the i n i t i a l observations of Dickens (1938,a,b) i s now commonly accepted. This pathway, which i s apparently very widely d i s t r i b u t e d , has been studied intensively by Horecker, Smyrniotis and aeegmiller (1951) using a p u r i f i e d yeast preparation. G-lucose was shown to be oxidized by way of glucose-6-phosphate and 6-phospho-gluconate. The l a t t e r compound was then oxidized with the formation of ribulose-5-phosphate (Cohen and Scott 1951, Horecker e t ' a l . 1951). In order to account f o r t h i s reaction, a hypothetical intermediate was postu-l a t e d (Horecker et a l . 1951). I t was shown that ribulose-5-phosphate was. i n equilibrium with ribose- . 5-phosphate. S i m i l a r l y , Seegmiller and Horecker (1952) obtained 3. an enzyme preparation from rabbit l i v e r which catalysed the quantitative oxidation of 6-phosphogluconate to pentose phosphate and carbon dioxide. The presence of ribulose-5-phosphate and ribose-5-phosphate was demonstrated. An enzyme preparation from rabbit bone marrow catalysed the formation of glucose-6-phosphate from 6-phosphogluconate by way of a pentose phosphate intermediate, thereby demonstrating a reversal of the dir e c t oxidative scheme. While Horecker et a l . have, at the present time, presented adequate proof of the d i r e c t oxidative scheme and have shown that i t i s present i n a vari e t y of t i s s u e , a number of other workers have added to the completeness of the picture by studies on other organ-isms or ti s s u e s . A v a r i a t i o n of t h i s oxidative scheme was discovered i n Pseudomonas saccharophila by Entner and Doudoroff (1952), This organism oxidizes glucose by way of glucose-6-phosphate and 6-phosphogluconate, but the next step i s the cleavage of the l a t t e r compound to y i e l d 3-phosphoglyceraldehyde and pyruvate. The reaction may be quite widely distributed and could be important i n the synthesis of hexoses. 4. Ac t u a l l y there was s t i l l e a r l i e r evidence of an alternate pathway of glucose oxidation, for i n 1931, Harrison had i s o l a t e d a glucose dehydrogenase from l i v e r which could convert glucose to gluconic a c i d without the intermediate formation of any phosphory-lated compounds. This reaction was not regarded seriously by biochemists for several reasons. The f i r s t was that, since phosphorylation was not demon-strated, the reaction was considered to be either an a r t i f a c t or p h y s i o l o g i c a l l y unimportant. The second was that, since the reaction was studied as an isolated system, i t was not brought into a scheme of metabo-li s m and the gluconic acid was pictured as an end product rather than an intermediate. Strecker and Eorkes (195S) reported a further p u r i f i c a t i o n of t h i s enzyme. This glucose dehydrogenase oxidized beta-glucose p r e f e r e n t i a l l y w i t h delta-gluconolactone as the probable product. Diphosphopyridinonucleotide (DPN) was the prosthetic group of the system. K e i l i n and Hartree (1949 a, b,), working with the enzyme Notatin, discovered by Muller (1928) i n Aspergillus niger and P e h i c i l l i u m glaucum, established that f l a v i n e adenine dinucleotide (FAD) was the pros-t h e t i c group. The enzyme system was not affected by 5 cyanide and was glucose s p e c i f i c . Further work by Bentley and Neuberger (1949) established that Notatin was a dehydrogenase and that the product of glucose oxidation was delta gluconolactone• Evidence of s t i l l another system of glucose o x i -dation began to accumulate with the demonstration by Barron and Friedemann (1941) that the oxidation of glucose by Pseudomonas aeruginosa was not affected by sodium f l u o r i d e and therefore did not involve phospho-r y l a t i o n . Lockwood et a l . (1940) reported the forma-t i o n of large quantities of gluconate and 2-ketoglu-conate during the growth of Pseudomonas fluorescens i n a glucose medium. Lockwood and Stodola (1946) showed that with P. fluorescens NREL B-6, alphaketoglutaric a c i d was obtained as a major product. With the same organism Lockwood and Nelson (1946) obtained a prepa-r a t i o n which could oxidize pentoses to pentonic acids . However, these workers were interested only i n the com-mercial production of the organic acids and did not interpret t h e i r data as in d i c a t i n g a new metabolic pathway• Norris and Campbell (1949) found detectable amounts of both gluconic and 2-ketogluconic acids i n sixteen and twenty four hour cultures of P. aeruginosa when 6. glucose was the growth substrate. Since strong systems for the oxidation of these compounds were also demonstrated over t h i s period' of time, i t was concluded that the compounds must be continuously formed and oxidized and must therefore be part of the metabolic pathway for the complete oxidation of glucose. Campbell and Norris (1951) concluded that the conventional g l y c o l y t i c scheme was not present i n P. aeruginosa. This conclusion was based on the insen-s i t i v i t y of the system to sodiuig. f l u o r i d e , the lack of a c t i v i t y under anaerobic conditions, the absence of hexose phosphates, and the formation and u t i l i z a t i o n of gluconic and 2-ketogluconic acids. Using a dried c e l l preparation of P. aeruginosa, Stokes and Campbell (1951) found that glucose and gluconate were quantita-t i v e l y converted to 2-ket©gluconic acid i n d i c a t i n g that at least i n dried c e l l s t h i s was the only mechanism available for the oxidation of glucose. I t was also confirmed that gluconic acid was an intermediate i n the formation of 2-ketogluconate. Warburton, Eagles and Campbell (1951) determined pyruvic acid i n glucose cultures of P. aeruginosa at sixteen, twenty-eight and f o r t y hours and at the same time were able to demonstrate a strong system for i t s 7 oxidation. This led them to conclude that pyruvic a c i d was a normal intermediate i n glucose oxidation. Campbell and Stokes (1951), working with the same organism, found that while r e s t i n g c e l l s had no a b i l i t y to oxidize c i t -r a t e , c i s aconitate, i s o c i t r a t e , alpha-ketoglutarate, succinate or fumurate, dried c e l l s could oxidize the previously l i s t e d compounds as w e l l as malate, acetate, oxalacetate and pyruvate. They concluded that the or-ganism possessed a conventional t r i c a r b o x y l i c acid c y c l e . K l e i n and Doudoroff (1950) i n t h e i r studies of Pseudomonas putrefaciens found that the w i l d type had l i t t l e or no a b i l i t y to attack glucose and did not possess hexo-kinase and could u t i l i z e glucose quite r e a d i l y . This agrees quite w e l l with the data reported by Wood and" Schwerdt (1953, 1954), obtained with a c e l l free sonic extract of P. fluorescens. The l a t t e r workers were able to demonstrate that the sonic extract possessed the a b i l i t y to oxidize glucose-6-phosphate, 6-phosphogluconate, ribose-5-phosphate, glucose and gluconic a c i d . Using ammonium sulphate f r a c t i o n a t i o n they were able to separate the glucose-gluconic o x i d i z i n g system from the soluble enzyme system which oxidized the phosphorylated compounds. The glucose-gluconic system was not a flavoprotein oxidase and did not involve a DPN or TPN s p e c i f i c glucose 8. dehydrogenase, cytochrome c a r r i e r s apparently were involved and so t h i s glucose dehydrogenase d i f f e r s from Notatin or from that of Harrison. The soluble enzyme system f o r the phosphorylated compounds appears to be quite cl o s e l y related to the one reported by Entner and Doudoroff (1952) f o r P.  saccharophila. Wood and Schwerdt could not show the existence of hexokinase and so the presence of glucose-s-phosphate oxidizing system i s d i f f i c u l t to explain. However these data agree with the findings of K l e i n and Doudoroff. The l i t e r a t u r e reviewed offers abundant proof f o r the existence of a direc t pathway for the oxidation of glucose. Moreover, t h e Embden-Meyerhof scheme seems to play a minor role i n aerobic systems. Work with r e s t i n g and dried c e l l s has shown numerous routes and while these studies have been rewarding, the actual mechanism of act i o n w i l l not be d e f i n i t e l y shown or characterized u n t i l p u r i f i e d enzyme systems are ava i l a b l e . Very l i t t l e work has been done on the glucose oxidizing enzymes of bac t e r i a . Since the glucose o x i -dizing system of P. aeruginosa has been shown to be similar i n many respects to the glucose dehydrogenase 9. of Harrison (1931) and Muller»s Notatin (1928), i t was thought that the study of t h i s enzyme would be p r o f i t a b l e . Although oxidative enzymes i n general are i n -soluble, attempts were made to i s o l a t e the enzyme responsible f o r the i n i t i a l oxidation of glucose. The task immediately at hand was the i s o l a t i o n ^ p u r i f i c a t i o n and characterization of the non-phos-phorylative glucose oxidizing enzyme system of Pseudomonas aeruginosa ATCC 9027. 10. METHODS B a c t e r i o l o g i c a l Pseudomas aeruginosa ATCC 9027 was used through-out these studies. The organism was maintained on atock culture media consisting of tryptone 1.0%, dipotassium hydrogen phosphate 0.3%, glucose 0.1%, g l y c e r o l 0.3%, yeast extract 0.1%, agar 0.5%, g e l a t i n 2.0%, adjusted to pH 7.2. The stock cultures were stored at approxi-mately 10*0. The medium used f o r obtaining a good y i e l d of c e l l s was that of Campbell et a l . (1949 b). In general the medium was dispensed i n 200 ml. quantities i n 500 ml. capacity Florence f l a s k s . A f t e r inoculation these were shaken on a horizontal rotary shaker f o r approxi-mately 18 hours. The inoculum was prepared from a stock culture by t r a n s f e r r i n g at least three times, at 24 hour i n t e r v a l s , on glucose agar s l a n t s . The r e s u l t i n g growth was washed off with s t e r i l e water and added to a Florence f l a s k containing the previously described l i q u i d medium. Aft e r 18 to 24 hours incubation t h i s f l a s k was used as the source of the 1.0% inoculum for the large volume of medium. 11. The c e l l s were harvested by use of a Serval Angle centrifuge at 5,000 revolutions per minute with a Serval SS-1 centrifuge at 14,000 revolutions per minute, or with a Sharpies continuous centrifuge at approxi-mately 85,000 revolutions per minute. After harvesting, the c e l l s were washed once with d i s t i l l e d water and then resuspended i n d i s t i l l e d water at a concentration of 200 to 400 milligrams per m i l l i l i t r e . This c e l l suspension was subjected to sonic o s c i l l a t i o n f o r f i f t e e n to twenty minutes i n a Raytheon 10 K i l o c y c l e O s c i l l a t o r . The r e s u l t i n g mixture, c a l l e d a sonicate, was then centrifuged f o r ten minutes at 14,000 revolutions per minute and resulted i n the separation of a s l i g h t amount of sedimentable material, which was discarded. The centrifuged sonicate, which was a reddish colour, exhibited a marked Tyndall e f f e c t . Chemical Metabolic gas exchanges were measured i n the Warburg respirometer according to the standard procedures of Umbreit et a l . (1949). Gluconic acid was detected by paper chromatography with an ethanol-methanol-water (45:45:10) solvent system 12. and 0.1N AgN0 3 i n 5N NH4OH spray as described by Norris and Campbell (1949 a ) . The reaction was aarried out i n a 125 ml. capacity Warburg reaction vessel containing 50 micromoles of glucose (2 ml.), 15 ml. of a pH 7.3 M/15 phosphate buffer, 10 ml. of a sonicate from asparagine grown c e l l s (showing no gluconic acid oxidation), 1 ml. of 8XL0" 3 M MgS04, 2 ml. of sodium 2, 6, dichlorobenzenoneindophenol (0.75 mg. per ml.) and 10 ml. of d i s t i l l e d water. The rate of oxidation was followed with a conventional Warburg system con-t a i n i n g one tenth of the previous constituents. When the reaction was almost complete the large cup was cleared of protein by the use of d i l u t e sulphuric a c i d which lowered the pH to 2.0. After centrifugation, the pH was r a i s e d to 7.4 and the reaction mixture was concen-trated to four ml. i n vacuo at 25*c. This solution was then used f o r chromatographic purposes. Protein was determined i n the following manner: 5 ml. of 10.0% t r i c h l o r o acetic acid were added to 0.5 ml. of the protein s o l u t i o n , the r e s u l t i n g p r e c i p i t a t e was separated by centrifugation at 5,000 revolutions per minute. The p r e c i p i t a t e was dissolved i n f r e s h l y pre-pared 3.0% NaOH and 0.6 ml. of a 20.0% CuS^'SHgO soluti o n was added for the biuret colour reaction. The s o l u t i o n 13. was rapidly brought to a f i n a l volume of 25 ml. with 3.0$ NaOH and shaken vigourously f o r one minute; allowed to" stand for ten minutes, and centrifuged again at 5,000 revolutions per minute f o r 10 minutes. The supernatant was then read at 560 millimicrons i n a Beckman Model B. Spectrophotometer. This procedure i s a modification of the method of Robinson and Hogden (1940) and uses casein as a standard. A saturated solution of (NH4) 2 SO4 was prepared by adding 70.6 gm. to 100 ml. of d i s t i l l e d water. A l l the fractions reported are given as the per cent of saturation. The fra c t i o n s were obtained by adding (NH4)gS04 slowly with s t i r r i n g to the protein s o l u t i o n . A f t e r f i v e minutes the solution was centrifuged at 10,000 revolutions per minute for ten minutes. The temperature was kept below 10*C to lessen the danger of denaturation. A f t e r separation, the fr a c t i o n s were dialysed, with s t i r r i n g , against ice cold d i s t i l l e d water f o r one hour, using a cellophane sac. The alumina c-alpha was prepared according to the method of Hawk, Oser and Summerson (1949). The alumina wa's used i n amounts equal to the s o l i d s i n solution i n the sonicate, i . e . , i f the sonicate had 200 mg. of c e l l material per ml. then 200 mg. of Alumina were added per 14. ml. of s o l u t i o n . The alumina was shaken on a rotary shaker with the preparation and then centrifuged to remove i t from s o l u t i o n . E l u t i o n was c a r r i e d out "by shaking the e l u t i n g agent with the alumina and again removing the alumina by centrifugation. The IRC-50 r e s i n was conditioned by allowing i t to remain f o r IE hours i n 10.0% HC1, washing free of acid with d i s t i l l e d water and then suspending i n pH 7.2 M/15 phosphate buffer. The solution was adjusted to pH 7.2 with NaOH u n t i l the pH remained constant, and was then l e f t for 12 hours at less than 10«G. The pH was tested a f t e r 12 hours and the r e s i n was then washed with a large excess of pH 7.2 M/15 phosphate buffer. The pH was tested again and i f the r e s i n was at the desired pH i t was ready for use. This procedure i s a modification of the one used by H i r s , Mooie and S t e i n (1953). The solvents ethyl alcohol, ethyl ether, and acetone were cooled to -18*0 before use. The dioxane was used at room temperature. The solvents were added slowly with s t i r r i n g to a sonicate whose temperature was below 10'U. The p r e c i p i t a t e s were removed by centrifugation at 10,000 revolutions per minute for 10 minutes. The pr e c i p i t a t e s were resuspended to t h e i r o r i g i n a l volume i n d i s t i l l e d 15. water or buffer pH 7.3. The traces of solvent were removed from the pre c i p i t a t e s by d i a l y s i s as before or by aeration. The solvents were removed from the super-natant by separating the layers, by d i a l y s i s or by aeration. The sonicate was made <3>.0235M with regard to MnSC-4, according to the method of Kuby and Lardy (1953) and was allowed to stand at approximately 10*C for 38 hours. The mixture containing the pre c i p i t a t e was divided into four parts and centrifuged at 14,000 revolutions per minute. The four samples were c e n t r i -fuged f o r 5, 10, 20 and 30 minutes resp e c t i v e l y . Another experiment was undertaken i n which the concen-t r a t i o n of MnSO^ was added to two separate volumes of the sonicate bringing the concentration of M11SO4 to one ha l f and one tenth of the 0.0235M o r i g i n a l l y used. These two samples were then centrifuged at 14,000 revolutions per minute fo r f i v e minutes. The MnSO^ was removed by d i a l y s i s and the various f r a c t i o n s were tested f o r a c t i v i t y . The protamine sulphate s o l u t i o n was prepared ac-cording to the method of Lindstrom (1953), i n which the protamine sulphate i s dissolved i n pH 5.0 buffer with a f i n a l concentration of 20 mg. per ml. The cold protamine sulphate was added slowly beneath the surface of a s t i r r e d 16. protein s o l u t i o n . The r e s u l t i n g p r e c i p i t a t e was r e -moved by centrifugation. The protamine sulphate presumably removes nucleoprotein from solution on a mole for mole basis. Therefore there should be no protamine sulphate l e f t i n solu t i o n . The b i l e s a l t s , sodium taurocholate and sodium glycocholate, were added d i r e c t l y to a sonicate and homogenized i n a Van Potter homogenizer. The homoge-nates were centrifuged for t en minutes at 14,000 revolutions per minute and then dialysed against d i s -t i l l e d water. This method i s a modification of the procedure followed by Williams and Sreenivasan (1953). The i n h i b i t o r s , whose f i n a l concentration i n the reaction mixtures i s reported here, were added d i r e c t l y to the Warburg cup. Substrate was tipped i n w i t h i n twenty minutes of the addition of the i n h i b i t o r . The centre wells of the cups used for cyanide i n h i b i t i o n contained 0.2 ml. of 4N NaCN i n 10.0% KOH (Eisenberg (1953) ). F i n a l Concentration of Inhibitors Sodium f l u o r i d e Sodium azide 8-hydroxy quinoline Sodium iodoacetate 2.4 dinitrophenol sodium arsenite Potassium cyanide Pyocyanin 1 X 10-4 M 1 X 10-2 M 2 X l O " 4 M 1 X 10-2 M 1.5 X 10" 4 M 3 X 10-4 M 17. The co-enzymes were prepared according to the Merck Index (1952) and added d i r e c t l y to the Warburg cup. Their f i n a l concentrations i n the reaction mixtures are reported here. F i n a l Amounts of Co-enzymes Ri b o f l a v i n phosphate Flavine adenine dinucleotide (FAD) Triphosphopyridinonucleotide (TPNJ Diphosphopyridinonucleotide (DPN) Adenosinetriphosphate (ATP) Cytochrome - C Magnesium sulphate (MgSO^ .) The hydrogen ion acceptors 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 concentration i n the reaction mixture i s reported here. F i n a l Concentration of Hydrogen Acceptors Methylene blue B r i l l i a n t c r e s y l blue 2.6 dichlorobenzenoneindophenol Potassium ferricyanide Pyocyanin 5.0 micromoles 5.0 micromoles 5.0 micromoles 5.0 micromoles 100 micrograms 520 micrograms 2.5 X 10~5 M 1 X 10"4 M 1 X 10 "J M 1 X IO"*3 M 1 X 10-3 M 1 X 10~4 M 18. EXPERIMENTAL I I s o l a t i o n and P u r i f i c a t i o n of the Enzyme System The i s o l a t i o n and the p u r i f i c a t i o n of the glucose o x i d i z i n g system were to be the f i r s t steps i n the procedure. Therefore a method of rupturing the c e l l membranes with the release of the system i n an int a c t form was required. C e l l free extracts were obtained by the exposure of a c e l l suspension containing approximately 31 mg. of protein per ml. to sonic v i -bration generated by a 10 K i l o c y c l e 60 cycle Raytheon o s c i l l a t o r . The exposure time which gave the greatest l i b e r a t i o n of active enzyme was f i f t e e n minutes. Physiological s a l i n e , M/30 phosphate buffers pH6 and pH7, T r i s buffer pH7.5 and pH8.0, and d i s t i l l e d water were tested i n an ef f o r t to determine the o p t i -mum medium for suspending the c e l l s f o r exposure to sonic v i b r a t i o n . D i s t i l l e d water proved to be the best medium. D i a l y s i s of the sonicate against d i s t i l l e d water reduced the endogenous r e s p i r a t i o n by seventy-five per cent with only a s l i g h t decrease i n the rate of glucose oxidation. 19 Ammonium Sulphate Fractionation The sonicate prepared from glucose grown c e l l s was active against glucose, gluconic a c i d , d e l t a -gluconolactone, and to a lesser degree against glucose-6-phosphate and ribose-5-phosphate. When (HH^JgSO^ (30.0% of saturation) was added, the pre-c i p i t a t e so formed was capable of oxidizing glucose, gluconic acid and delta-gluconolactone. The glucose-6-phosphate, ribose-5-phosphate and endogenous systems remained i n s o l u t i o n . An attempt was then made to separate the glucose enzyme from the gluconic acid enzyme by a more refined f r a c t i o n a t i o n of the system with (NH^gSO^.. Fractions of the sonicate were obtained by adding 2.0% of satu-r a t i o n (HH4)2S04 and increasing i t by 2.0% increments. The r e s u l t i n g p r e c i p i t a t e s showed equal a c t i v i t y with regard to glucose and gluconic acid and so no separa-t i o n was achieved. The glucose and gluconic acid enzyme system was concluded to be insoluble as determined by i t s p r e c i p i -t a t i o n by (NH4)2S04 (30,0% of saturation). Since the two enzymes were inseparable with regard to (NB^JgSfl^ "treat-ment i t was decided to attempt a separation by the use of adsorbents. 20. Alumina G-Alpha Alumina C-alpha was used i n the hope that i t would either adsorb the enzyme s e l e c t i v e l y or take out the other constituents of the preparation pre-f e r e n t i a l l y . However, a f t e r treatment the suspen-sion was found to have retained reduced but equal a c t i v i t y toward glucose and gluconic a c i d . E l u t i o n of the adsorbent released the endogenous a c t i v i t y and the portion of the glucose and gluconate ox i d i z i n g system which had been adsorbed. Since the endogenous system was adsorbed, i t was thought that i t might have interfered with the adsorption of the enzymes i n question. Therefore an ammonium sulphate (25.0$ of saturation) f r a c t i o n of a sonicate was used f o r t h e adsorption procedure. The r e s u l t s remained the same i n that the glucose and gluconate enzymes were s t i l l adsorbed and eluted i n the same f r a c t i o n s , as shown by Table I . 81. TABLE I THE RESPIRATORY ACTIVITY OF SONICATE FRACTIONS TREATED WITH ALUMINA-C -ALPHA OXYGEN UPTAKE IN 1.5 HOURS FRACTION Endogenous micro-l i t r e s Glucose • micro-l i t r e s Gluconic acid * m i c r o l i t r e s O r i g i n a l Sonicate 72 101 39 i'raction not Adsorbed on Alumina 12 82 31 Fractio n Eluted from Alumina 65 94 38 25% (NH4)2 SO4 Fraction 0 98 50 Frac t i o n not Adsorbed on Alumina 0 46 27 Fractio n Eluted from Alumina 0 83 41 * The values reported i n these columns refer to net oxygen uptake. The Warburg cups contained 1.5 ml. pH 7.4 M/15 TRIS buffer; 1.0 ml. enzyme preparation; 0.8 ml. substrate con-ta i n i n g 5 micromoles of substrate; 0.3 ml. d i s t i l l e d water; 0.15 ml. of 80.0% KOH i n centre well. F i n a l volume i n the main chamber equalled 3.0 ml. 22 The samples treated with (NPL^gSC^ were dialysed for 1.0 hours with s t i r r i n g against d i s t i l l e d water. LRC-50 Resin The r e s i n was prepared as described e a r l i e r and packed i n a column 10 inches high and 2 centimetres i n diameter. The sonicate was passed through t h i s column at a slow rate and tested f o r a c t i v i t y a f t e r each passage. Af t e r the f i r s t passage the endogenous a c t i v i t y was reduced by h a l f , but the glucose and gluconic acid a c t i v i t y remained the same. The second, t h i r d and fourth passages reduced the glucose-gluconate system but evidently eluted the endogenous system. The e l u -t i o n with 10 ml. of M/30 phosphate buffer pH 7.2 gave a high endogenous with s l i g h t glucose and gluconate a c t i v i t y a f t e r the f i r s t washing. The next four eluents gave a low endogenous and only s l i g h t l y higher glucose and gluconate oxidation rates. The s i x t h eluent which was M/5 phosphate pH 7.2 gave no a c t i v i t y . The r e s i n did not give any separation except with the endogenous system which can be r e a d i l y separated from the glucose-gluconate system by (NH4.)2S04 fEactionation. The data are summarized i n Table I I . 23. TABLE I I ACTIVITY OF SONICATE TREATED WITH I.R. C-50 OXYGEN UPTAKE IN 1.5 HOURS STEP Endogenous micro-l i t r e s Glucose + micro-l i t r e s Gluconic Acid * m i c r o l i t r e s O r i g i n a l Sonicate 84 90 43 Af t e r 1st Passage through Column 45 86 52 After 2nd Passage Through Column 61 45 27 Aft e r 3rd Passage Through Column 58 33 n 14 After 4th Passage Through Column 69 32 20 E l u t i o n with M/30 PO4 Buffer (10ml. fractions) Endogenous Glucose* Gluconic Acid Fraction 1 41 24 13 F r a c t i o n 2 14 16 7 Fraction 3 8 8 4 Fraction 4 6 6 6 Fraction 5 <7 "§ S E l u t i o n with M/5 PO4 Buffer (10 ml.) 0 0 0 The values reported i n these columns refer to net oxygen uptake The V/arburg cups contained 1.5 ml. pH 7.2 M/15 phosphate buffer; 1.0 ml. enzyme preparation; 0.2 ml. 24. substrate (5.0 microiaoles) ; 0.3 ml. d i s t i l l e d water; 0.15 ml. of 20.0% KOH i n the centre w e l l . P r e c i p i t a t i o n by Solvents When a sonicate was treated with 2.0% acetone, ethyl alcohol or dioxane, the oxidative a b i l i t y of both the p r e c i p i t a t e and the supernatant toward glucose and gluconic a c i d was destroyed. E t h y l ether i n the same concentration proved to be an exception. The a c t i v i t y of the supernatant was destroyed but the pre-c i p i t a t e had the a b i l i t y to oxidize gluconic acid alone. This loss of a b i l i t y was not due to a separation of the protein part of the enzyme from i t s co-factors since a recombination of the p r e c i p i t a t e with the supernatant did not r e s u l t i n a recovery of a c t i v i t y . (Table I I I ) . 25. TABLE I I I EFFECT OF SOLVENT PRECIPITATION ON THE RESPIRATORY ACTIVITY OF SONICATE Solvent Oxygen Uptake Added Endogenous Glucose Gluconic (2% by volume) micro- micro- Acid l i t r e s l i t r e s m i c r o l i t r e s A 0 0 0 Acetone B 0 0 0 A 0 0 0 Et h y l Alcohol B 0 0 0 A 0 0 0 uioxane B 0 0 0 A 0 0 0 Ethyl Ether B 0 0 50 A = Supernatant B s Ppt. resuspended i n o r i g i n a l volume Ppt. and Superna- Endogenous Glucose Gluconic tant Recombined Ac i d Acetone 0 0 0 E t h y l Alcohol 0 0 0 Dioxane 0 0 0 E t h y l Ether 0 0 45 The Warburg cups contained 1.5 ml. pH 7.3 M/15 phosphate buffer; 1 ml. enzyme preparation; 0.2 ml. substrate (5.0 micromoles) ; 0.3 ml. d i s t i l l e d water; 0.15 ml, of 20.0% KOH i n centre w e l l . 26. P r e c i p i t a t i o n by Manganous Sulphate Manganous sulphate treatment of a protein solu-t i o n has been shown to remove nucleoproteins. An attempt was made to remove i n t e r f e r i n g nucleoproteins from solu t i o n i n order to aid i n dissolv i n g the glucose enzyme. I t was thought that the removal of a l l extraneous material would greatly aid i n the sepa-r a t i o n of a pure enzyme. The sonicate was treated with MnS04 as described previously. Upon resuspen-sion the pre c i p i t a t e proved to have the greatest amount of the glucose-gluconate oxidizing enzymes. It was found that varying concentrations of the MnS04, and varying times of centrifugation removed correspon-ding amounts of both enzymes but did not ef f e c t a separation as can be seen from Table IV. 27. TABLE IV EFFECT OF MANGANOUS SULPHATE ON RESPIRATORY ACTIVITY OF SONICATE 0.0235M. MnS04 Time of Net ; Oxygen Uptake Centrifugation (14,000 B.P.M.) Glucose m i c r o l i t r e s Gluconic Acid m i c r o l i t r e s B A B 5 minutes 35 112 15 56 10 minutes 15 112 0 56 20 minutes 10 112 0 56 30 minutes 0 112 0 56 A B Supernatant B = Ppt. Resuspended i n o r i g i n a l volume 5 Min. Centrifugation (14,000 B P M ) Concentration of Glucose m i c r o l i t r e s Gluconic Acid m i c r o l i t r e s M11SO4 A B A B 0.0235M. 35 112 15 56 0.01175M. 45 112 20 56 0.00235M. 55 112 25 56 A B: = Supernatant = Ppt. Resuspended i n o r i g i n a l volume The Warburg cups contained 1.5 mi. pH 7.3 m/15 phosphate buffer; 1.0 ml. enzyme preparation; 0.2 ml. substrate (5.0 micromoles); 0.3 ml. d i s t i l l e d water; 0.15 ml. of 20.0% KOH. A l l enzyme fractions were dialysed as before. 28. B i l e Salt Extraction When glycocholate was added to a sonicate i n the proportion of 0.5 gnu per 10.0 ml. of the sonicate, the r e s u l t i n g supernatant oxidized only gluconic acid i n the presence of 2.6 dichlorobenzenoneindophenol as the hydrogen acceptor. The p r e c i p i t a t e oxidized gluconic acid more r a p i d l y than i t did glucose and the addition of the dye was again necessary for the oxidation. Combination of the dialysed p r e c i p i t a t e and the dialysed supernatant did not restore the a c t i v i t y on glucose to i t s o r i g i n a l r a t e . Taurocholate used i n the same concentration as the glycocholate gave s i m i l a r r e s u l t s . The p r e c i p i -tate contained both enzymes and the supernatant only the gluconic enzyme. An increase i n the concentration of b i l e s a l t s caused a destruction of both enzymes. The data are summarized i n Table "V. 29. TABLE V EXTRACTION OF THE SONICATE WITH BILE SALTS B i l e S a l t Used as Extractant Oxygen Uptake  Glucose Gluconic Acid micro-l i t r e s micro-l i t r e s 1. Glycocholate A = Supernatant A +2, 6, dichlorobenze-noneindophenol P r e c i p i t a t e 0 0 35 0 56 50 2. Taurocholate A = Supernatant 0 0 A if 2, 6, dichlorobenze-noneindophenol 0 56 Prec i p i t a t e 35 50 The Warburg cups contained 1.5 ml. pH 7.3 M/15 phosphate buffer; 1.0 ml. of enzyme preparation; 0.2 ml. substrate (5.0 micromoles); 0.3 ml. d i s t i l l e d water ex-cept when the 2.6 dichlorobenzenoneindophenol was added, i n 0.2 ml. volumes i n which case the water was reduced to 0.1 ml; 0.15 ml. of 20.0% K0H i n the centre w e l l . A l l enzyme f r a c t i o n s were dialysed as before. Use of Carbon Source Unrelated to Glucose Since the glucose ox i d i z i n g enzyme has been reported 30. to be co n s t i t u t i v e while the gluconic acid enzyme was adaptive it should be possible to grow the organism on a medium which would not stimulate the formation of the gluconic acid oxidizing enzyme system (Entner and Stanier (1951) ). In some cases, when grown i n 0.3% asparagine alone, 0.2% asparagine plus 0.2% g l y c e r o l , and 0.2% asparagine plus 0.2% succinate, in t a c t c e l l s proved to be adaptive to a l l substrates except glucose. Acetate grown c e l l s had a constitutive though s l i g h t l y reduced system f o r the oxidation of gluconic a c i d . (See F i g . I.) The sonicates of the c e l l s grown on the various carbon sources had only a very weak system for the oxidation of gluconic a c i d . However the systems were not dependable, i n that the gluconic acid enzyme would frequently appear quite strongly, though not to the same degree as i n the glucose grown c e l l l s . This would appear to be a promising approach however and should be investigated i n greater d e t a i l . 100 too / 7 y ( 20 30 «K> MINUTES 50 FIG. I. The cons t i t u t i v e nature of the glucose enzyme (A) and the adaptive nature of the gluconic enzyme (B)• These curves demonstrate t y p i c a l glucose and gluconate oxidation by whole c e l l s when asparagine, asparagine * succinate or asparagine * gl y c e r o l was used as the car-bon source for growth. Sonicates of these c e l l s pos-sessed normal a c t i v i t y towards glucose but n e g l i g i b l e a c t i v i t y towards gluconate. Net oxygen uptake i s shown, using 5 micromoles of substrate. 32. Glycine and A n i l i n e Treatment The method of orewther et a l . (1953) was followed i n which glycine was added to a sonicate i n order to s o l u b i l i z e the enzymes. According to these authors glycine and a n i l i n e increase the s o l u b i l i t y of proteins so that they can withstand higher concentrations of protein p r e c i p i t a n t s . I t was found that a n i l i n e , added to a f i n a l con-centration of 0.01M., increased the s o l u b i l i t y but inhib i t e d the enzyme ac t i o n . When glycine was added to the sonicate at a f i n a l concentration of 0.1 M. i t did not i n h i b i t the enzyme action, and moreover greatly increased the s o l u b i l i t y . P r e c i p i t a t i o n with (NH^Jg SOA and protamine sulphate did not separate the glucose enzyme from the gluconic acid enzyme however. Sonic v i b r a t i o n i n the presence of 0.1M. glycine ( f i n a l concentration) resulted i n an active preparation. However (NILdJgSO^ and protamine sulphate deactivated both the pr e c i p i t a t e and the supernatant. TABLE VI THE EFFECT OF GLYCINE AND ANILINE ON THE SONICATE Oxygen Uptake i n 1.5 Hours Step Glucose Gluconic Acid m i c r o l i t r e s m i c r o l i t r e s O r i g i n a l Sonicate 112 56 Made 0.3M with Glycine 112 56 Made O.IM with A n i l i n e 80 35 O r i g i n a l Sonicate A 15 7 B 112 56 O.IM with A 112 56 glycine B 10 0 O.IM with A 75 35 an i l i n e B 10 0 30.0% (NH 4) 2S04 Fra c t i o n of Origin a l Sonicate 112 56 O.IM.Glycine Sonicate 112 56 Supernatant of 30.0% (NH4)2S04 Fr a c t i o n of Origin a l Sonicate 0 0 O.IM.Glycine Sonicate 45 25 Supernatant of 6.0% Protamine Sulphate Origi n a l Sonicate 70 30 O.LM.Glycine Sonicate 85 40 Sonicate prepared i n Presence of O.IM.Glycine 112 56 20.0% (NH4)2S04 Fr a c t i o n 15 0 Supernatant of 20.0% (NH4)2S04 Fract i o n 0 0 Supernatant of 6.0% Protamine Sulnhate 0 0 A = Supernatant B = Pr e c i p i t a t e The Warburg cups contained 1.5 ml. pH 7.3/&/15 phosphate 34 buffer; 1.0 ml. enzyme preparation; 0.2 ml. substrate (5.0 micromoles); 0.3 ml. d i s t i l l e d water; 0.15 ml. of 20.0% KOH i n the centre w e l l . A l l (NH4.)2S04 treated samples were dialysed as before. II Properties of the Enzyme System The attempts to i s o l a t e and purify the enzyme had met with very l i t t l e success, therefore i t was decided to study the enzyme system as i t existed i n the o r i g i n a l dialyzed sonicate without the benefit • of any p u r i f i c a t i o n procedure. I n h i b i t i o n Studies The ef f e c t of sodium iodoacetate, sodium f l u o r i d e , sodium arsenite, 2.4 dinitrophenol, sodium azide, potassium cyanide and pyocyanin, on a sonicate of asparagine-succinate grown c e l l s was studied. As shown i n Table III only sodium azide and potas-sium cyanide showed marked i n h i b i t i o n . These two i n h i b i t o r s depressed the r e s p i r a t i o n of the glucose oxidizing enzyme almost completely. When 8-hydroxy-quinoline was used on a 25% (NH4)2S04 f r a c t i o n of an asparagine grown sonicate, the i n h i b i t i o n could be overcome by the addition of magnesium. 35. TABLE VII THE EFFECT OF VARIOUS INHIBITORS ON G-LUCOSE OXIDATION  INHIBITOR 1o INHIBITION Sodium Iodoacetate 0 Sodium Arsenate 0 Sodium Fluoride 0 Sodium Azide 60 Potassium Cyanide 100 2.4 Dinitrophenol 0 Pyocyanin 0 8 OH Quinoline 80 8 OH Quinoline 0 * 2.5 X 10" 3 M. MgS0 4 FIG. II pH Curve The Warburg cups contained 1 ml..enzyme preparation, 0.2 ml. glucose (5.0 micromoles), 0.3 ml. d i s t i l l e d water and 1.5 ml. M/15 phosphate buffer. The pH was raised i n increments of 0.5 pH units be-tween pH 5.5 and pH 7.0, and 0.2 pH units between pH 7.0 and pH 8.0 Net oxygen uptake i n 1.5 hours. 37. Hydrogen Ion Acceptors B r i l l i a n t c r e s y l blue, methylene blue, 2.6 dichloro-benzenoneindophenol, ferricyanide and pyocyanin were tested as hydrogen ion acceptors. The ferri c y a n i d e proved to be Inhibitory, while only the 2.6 dich l o r o -benzoneindophenol was stimulatory as shown i n Table VTII. TABLE VIII THE EFFECT OF HYDROGEN ACCEPTORS ON GLUCOSE OXIDATION HYDROGEN ACCEPTOR B r i l l i a n t Cresylblue Methylene Blue 2.6 Dichlorobenzenoneindophenol Ferricyanide Pyocyanin EFFECT None None 100% Stimulation 40% I n h i b i t i o n None Coenzymes ATP, DPN, TPN, FAD, r i b o f l a v i n phosphate and cyto-chrome^ had no e f f e c t on the glucose oxidizing system. Magnesium sulphate i n a f i n a l concentration of 2.5 X 10"3 stimulated the oxidationo f glucose by over t h i r t y per cent when used alone while when used i n 38. conjunction with 2.6 dichlorohenzenoneindophenol the system was stimulated by approximately 300.0% (Table IX). TABLE IX THE EFFECT OF VARIOUS COENZYMES ON GLUCOSE OXIDATION COENZYME ATP FAD DPN TPN R i b o f l a v i n Phosphate Cytochrome C MgS0 4 2.6 Dichlorohenzenoneindophenol MgS0 4 * 2.6 Dichlorohenzenone-indophenol EFFECT None None None None None None 33% Stimulation 100% Stimulation 286% Stimulation The Reaction Product Gluconic a c i d was determined as the product of the reaction by paper chromatography with the previously des-cribed ethanol-methanol-water solvent system. An asparagine grown sonicate which showed only glucose oxidation was used. The reaction was allowed to continue 39. u n t i l i t was almost complete. The contents of the large Warburg cup, a f t e r being treated as described i n the methods, was chromatographed. Two spots were ob-served which corresponded to glucose and gluconic acid standards. The glucose spot had an Rf of 0.55 and the gluconic acid spot had an Rf of 0.40. 40 DISCUSSION Several related non-phosphorylative glucose oxidizing systems have previously been described i n the l i t e r a t u r e . I t should be noted however, that although the reaction which they catalyse i s s i m i l a r , the nature of the enzyme systems i s quite d i f f e r e n t . Since the enzymes are normally species s p e c i f i c t h e i r differences i n chemical composition w i l l be manifested by variations i n such physical constants as s o l u b i l i t y , s t a b i l i t y and pH optimum. This means that the i s o l a t i o n and p u r i f i c a t i o n of each enzyme system has to be es-tablished independently. I t i s for t h i s reason that a detailed study of the glucose oxidizing system of P.  aeruginosa was e s s e n t i a l . Manometric experiments have revealed that sonic extracts were able to oxidize glucose, gluconate, glucose-6-phosphate, ribose-5-phosphate and d e l t a -gluconolactone. Centrifugation or (NEL^JgSO^ f r a c t i o n a -t i o n precipitated the glucose, gluconate, and delta-glu-conolactone oxidizing systems i n an active form. However, the system f o r oxidizing the phosphorylated compounds was l o s t a f t e r treatment with (NH^JgSO^. Attempts to separate the glucose enzyme from the gluconic acid enzyme were not successful. The f a c t 41. that both enzymes were thrown down by half hour c e n t r i -fugation at 25,000 g or by 30.0% (NH 4) 2S0 4 showed that they were insoluble. An attempt to fractionate by the addition of d i f f e r e n t percentages of (NE^JgSO,! re-sulted i n a series of precipitates which had equal amounts of the two enzymes. The adsorption procedures using IRC-50 and Alumina-c-alpha also resulted i n fracti o n s containing equivalent amounts of the two enzymes. Treatment by MnS0 4 was i n e f f e c t i v e i n that i t r e -moved corresponding amounts of the two enzymes from the so l u t i o n . The use of various solvents for f r a c t i o n a t i o n denatured the enzymes. H0wever ethyl ether was an ex-ception i n that i t did not destroy the gluconic a c i d enzyme. Extraction of the sonicate with b i l e s a l t s resulted i n a solut i o n showing strong a c t i v i t y toward gluconic acid when 2.6 dichlorobenzenoneindophenol was present. The pr e c i p i t a t e possessed a strong system for the oxida-t i o n of both glucose and gluconic aci d . Reduction of the concentration of b i l e s a l t l e f t the glucose enzyme i n so l u t i o n while an increase i n concentration destroyed the glucose a c t i v i t y i n both f r a c t i o n s . When added before the sonic treatment, glycine aided i n s o l u b i l i z i n g both the glucose and gluconic a c i d enzymes 42. but did not a i d i n t h e i r separation. Moreover the s o l u b i l i z e d glucose and gluconic a c i d enzymes became l a b i l e to such precipitants as protamine sulphate and (NSiJgSC^. Growth on a carbon source other than glucose may almost e n t i r e l y eliminate the gluconic acid enzyme. However, t h i s method does not give uniform r e s u l t s . The glucose enzyme i s always constitutive but the g l u -conic acid enzyme frequently appears very strongly, a l -though not to the same degree as when glucose i s the carbon source. Goulthard et a l . (1942) were able to pu r i f y Notatin by acetone extraction of a tannic acid p r e c i p i t a t e of the mould culture f i l t r a t e . Strecker and Korkes (1952) were able to remove extraneous material from an aqueous ex-t r a c t of l i v e r homogenate with (NH^JgSC^. Both l i v e r glucose dehydrogenase and Notatin catalyse the oxidation of glucose to gluconic a c i d . These two enzymes, apparently possessing the same function as the glucose ox i d i z i n g enzyme of P. aeruginosa and therefore isodynamic are adequate proof of the differences i n the physical con-stants of three enzymes classed as being isodynamic. The glucose oxidizing enzyme of P. aeruginosa i s i n h i b i t e d by 1.5 X 10" 4 potassium cyanide and 1 X 1 0 " % 43. sodium azide thereby suggesting the function of cyto-chrome c a r r i e r s , and distinguishing i t from the f l a v o -protein glucose oxidase. The lack of i n h i b i t i o n by sodium f l u o r i d e and E.4 dinitrophenol would indicate the lack of phosphorylation or the absence of the g l y -c o l y t i c scheme. Since DPN, TPN, FAD, ATP, cytochrome-c or r i b o f l a v i n phosphate caused no stimulation these factors were regarded as not being coenzymes of t h i s system. This i s i n contrast to the l i v e r dehydrogenase which requires either DPN or TPN and Notatin which r e -quires FAD as the prosthetic groups. 2.6 dichlorohenzenoneindophenol stimulated the glucose oxidation while methylene blue, pyocyanin and b r i l l i a n t c r e s y l blue did not a f f e c t the rate of oxida-t i o n . This may indicate a role of cytochrome b or a similar component i n the oxidation (Wood and Schwerdt (1953) ). A requirement f o r magnesium was shown when the i n h i -b i t i o n by IXLO-^M 8-hydroxyquinoline was overcome by addition of 2.5 X lO^M MgSO^. The reaction product of glucose oxidation was found to be gluconic acid when determined under conditions which would s p l i t a lactone to the a c i d . Strecker and Korkes (1952) found that the product of the reaction catalysed by the l i v e r dehydrogenase of Harrison (1931) 44. was gluconolactone. Bentley and Neuberger (1949) showed that the enzymatic product of Notatin was delta-gluconolac-tone. The reaction product of glucose oxidation by P. aeruginosa could well be gluconolactone, since the sonicate can oxidize gluconolactone as r e a d i l y as g l u -conic a c i d . Brodie and Lipmann(1954) have reported that an enzyme from the p a r t i c u l a t e f r a c t i o n of Azotobacter  v i n e l a n d i i oxidized glucose and quantitatively accumu-lated d e l t a gluconolactone. A second enzyme from the supernatant of A. v i n e l a n d i i and baker's yeast r a p i d l y hydrolysed the lactone to the corresponding a c i d . This mechanism activated by Mg** i s s i m i l a r to the glucose oxidizing system of P. aeruginosa. From these r e s u l t s i t may be concluded that the glucose oxidizing enzyme of P. aeruginosa i s insoluble and i s cyanide and azide s e n s i t i v e . I t requires Mg as a co-factor and can use 2.6 dichlorobenzenoneindophenol as a hydrogen acceptor. Its mechanism of action i s probably the removal of hydrogen from glucose to form gluconolactone which may then undergo enzymatic or non-enzymatic hydrolysis to gluconic a c i d . 45 SQMMAHY 1. A c e l l free extract of P. aeruginosa was obtained by exposure to sonic v i b r a t i o n . This sonicate possessed two systems, one d i r e c t l y o xidizing glucose, gluconate and gluconolactone, the other an independent system oxidizing glucose-6-phos-phate and ribose-5-phosphate. 2. The glucose, gluconate and gluconolactone o x i -dizing system could be separated from the soluble proteins by centrifugation or INH4)2S04 p r e c i p i t a -t i o n but the enzymes could not be separated from each other. 3. pH 7.5 was found to be the optimum pH f o r t he g l u -cose system. 4. The enzymes were precipitated by MnSC-4 i n amounts proportional to the concentration of the s a l t . The r e l a t i v e amounts of each of the enzymes i n the pre-c i p i t a t e remained constant, with no separation of enzymes being achieved i n the process. 5. The r e s i n IRC-50 adsorbed the enzymes equally. I t was not possible to elute them separately. 6. Alumina-c-alpha absorbed the glucose and gluconate enzymes completely and i t was not possible to elute them separately. 46 7. E t h y l alcohol, dioxane and acetone destroyed glucose and gluconate enzymes. Eth y l ether des-troyed only the glucose enzyme. 8. The addition of glycine to the sonicate s o l u -b i l i z e d the enzymes but did not a i d i n t h e i r .separation. -Addition of glycine before sonic treatment caused the enzymes to become l a b i l e to protein p r e c i p i t a n t s . 9. Extraction of the sonicate with b i l e s a l t s l e f t a precipitate with equal a c t i v i t y toward glucose and gluconic a c i d . The supernatant possessed a portion of the gluconic acid enzyme i n a soluble form. An increase i n the bile, s a l t concentration destroyed the glucose a c t i v i t y . 10. Growth on a carbon source other than glucose r e -sulted i n the formation of a reduced gluconic ac i d system. However, the r e s u l t s were not u n i -form i n that the gluconate enzyme frequently was very a c t i v e . 11. Methylene blue, pyocyanin, and b r i l l i a n t c r e s y l blue had no a b i l i t y to act as hydrogen acceptors. 12. 2.6 dichlorobenzenoneindophenol acted as a hydrogen acceptor. 13. Ferricyanide i n h i b i t e d glucose oxidation. 14. A.T.P., F.A.D., D.P.N., T.P.N., cytochrome-c and 47. r i b o f l a v i n phosphate had no ef f e c t on glucose oxidation. 15. KCN, NaN3 and 8-hydroxy quinoline i n h i b i t e d g l u -cose oxidation. 16. MgSC-4 overcame the i n h i b i t i o n caused by 8-hydroxy-quinoline. 17. MgSC-4 stimulated glucose oxidation. 18. Sodium f l u o r i d e , 2.4 dinitrophenol, sodium iodo-acetate, and sodium arsenite caused no i n h i b i t i o n . 48. BIBLIOGRAPHY 1. Barron, E.S.G., and Friedemann, T.E., Studies of b i o l o g i c a l oxidations: XIV: Oxidations by micro-organisms which do not ferment glucose. J . B i o l . Chem., 137, 593 (1941) 2. Bentley, R., and Neuberger, A., Mechanism of the action of Notatin. Biochem. J . , 45, 584 (1949) 3. Brodie, A.F., and Lipmann, F., The enzymatic formation and hydrolysis of D-glucono-delta-lactone. Proc. Soc. Am. Bact., 54th meeting, 107 (1954) 4. Campbell, J.J.R., and Nor r i s , F.C., The interme-diate metabolism of Pseudomonas aeruginesa: IV: The absence of an Embden-^eyerhof system as evidenced by phosphorous d i s t r i b u t i o n . Can. J . Research, C 28, 203 (1950) 5. Campbell, J.J.R., N o r r i s , F.C., and Norris, M.E., The intermediate metabolism of Pseudomonas  aeruginesa: I I : Limitations of simultaneous adap-t i o n as applied to the i d e n t i f i c a t i o n of acetic acid, an intermediate i n glucose oxidation. Can. J . Research, C 27 t 165 (1949b) 6. Campbell, J.J.R., and Stokes, F.N., Tri c a r b o x y l i c acid cycle i n Pseudomonas aeruginosa. J". B i o l . Chem., 190, 853 (1951) 49 7. Cohen, S.S., and Scott, D.B.M., Formation of pentose phosphate from 6-phosphogluconate. Science, I I I , (1950) 8. Colowick, S.P., and Kaplan, N.O., Carbohydrate metabolism. Annual Rev. Biochem., 20, 513 (1951) 9. Coulthard, C.E., Michaelis, R., Short, W.F., Sykes, G., Skrimshire, G.E.H., Standfast, A.F.B., Brinkinshaw, J.H., and R a i s t r i c k , H., Notatin: An a n t i b a c t e r i a l glucose aer©dehydrogenase from P e n i c i l l i u m notatum westling. Nature, 150, 634 (1942) 10. Crewther, W.G., and Lennox, F.G., Enzymes of Aspergillus oryzae: Fractionation and prepara-t i o n of c r y s t a l s r i c h i n protease. A u s t r a l i a n J . B i o l . S c i . , 6, 428 (1953) 11. Dickens, F., Oxidation of phosphohexonate and pentose phosphoric acids by yeast enzymes. Biochem. J . , _32, 1626 (1938a) 12. Dickens, F., Yeast fermentation of pentose phos-phoric acids. Biochem. J . , 32, 1645 (1938b) 13. Eisenberg, M.A., The t r i c a r b o x y l i c cycle i n Rhodospirillum rubrum. 0". B i o l . Chem., 205, 815 (1953) 14. Entner, N., and Doudoroff, M., Glucose and gluconic acid oxidation of Pseudomonas sacchorophila. J . B i o l . Chem., 196, 853 (1952) 50. 15. Entner, N., and Stanier, R.T., Studies of the oxidation of glucose by Pseudomonas fluorescens. J . Bact., 62, 181 (1951) 16. Harrison, D.C., Glucose dehydrogenase: A new oxidizing enzyme from animal t i s s u e s . Biochem. J . , 25, 1016 (1931) 17. Hawk, P. B., Oser, B.L., and Summerson, W.H., P r a c t i c a l P h ysiological Chemistry, P.286, Twelfth e d i t i o n Philadelphia Blakiston Company, Toronto, (1949) 13. H i r s , C.H.W., Moore, s . , and Stein, W.H., A chroma-tographic investigation of pancreatic ribonu-clease. J . B i o l . Chem., 200, 493 (1953) 19. Horecker, B.L., Smyrniotis, P.Z., and Seegmiller J.E., The enzymatic conversion of 6-phosphogluconate to ribulose-5-phosphate and ribose-5-phosphate. J . B i o l . Chem., 193, 383 (1951) 20. K e i l i n , D., and Hartree, E.E., Properties of glucose oxidase (Notatin). Biochem. J . , 42, 221 (1948a) 21. K e i l i n , D., and Hartree, E.F., Use of glucose o x i -dase (Notatin) f o r the determination of glucose i n b i o l o g i c a l material find f o r the study of glucose-producing systems by manometric methods. Biochem. J . , 42, 230 (1948b) 22. K l e i n , P.S., and Doudoroff, M., The mutation of 51. Pseudomonas putrefaciens to glucose u t i l i z a t i o n and i t s enzymatic basis. J . Bact., 5_9, 739 (1950) 23. Kuby, S.A., and Lardy, H.A., P u r i f i c a t i o n and ki n e t i c s of beta-D-glactosidase from Escherichia  c o l i , s t r a i n K-12. J . Am. Chem. S o c , 75, 890 (1953) 24. Lindstrom, E.S., the alpha-ketoglutaric oxidase system of Azotobacter. J . Bact., 65_, 565 (1953) 25. Lockwood, L.B., and Nelson, G.E.N., The oxidation of pentoses by Pseudomonas. J . Bact., 52, 581 (1946) 26. Lockwood, L.B., and stodola, E.H. , Preliminary studies on the production of alpha-ketoglutaric acid by Pseudomonas fluorescens. J . B i o l . Chem., 164, 81 (1946) 27. Lockwood, L. B., Tabenkin, B., and Ward, G.E., The production of gluconic acid from glucose by species of Pseudomonas and Phytomonas. J , Bact. 42, 51 (1940) 28. Muller, D., Studies uber eines enzyme glykoseoxy-dase. I Biochem. Zeitshr., 199, 136 (1928) 29. Norris, F.C., and Campbell, J.J.R., The intermediate metabolism of Pseudomonas aeruginosa: I I I : The application of paper chromatography to the i d e n t i -f i c a t i o n of gluconic and 2-ketogluconic acids, i n t e r -52. mediate i n glucose oxidation. Can J . Research., C 27, 253 (1949) 30. Robinson, H.W., and H 0gden, C.G., the Biuret Re-action i n the determination, of serum proteins: I I : Measurements made with Duboscq colorimeter com-pared with values obtained by the Kjeldahl procedure. J . B i o l . Chem., 135, 707 (1940) 31. Seegmiller, J.E., and H orecker, B.L., Metabolism of 6-phosphogluconic acid i n l i v e r and bone marrow. J". B i o l . Chem., 194, 261 (1952) 32. stokes, F.N., and Campbell, J.J.R., Oxidation of glucose and gluconic acid by dried c e l l s of Pseudomonas aeruginosa. Arch. Biochem., 30, No. 1 (1951) 33. Strecker, H.H., and Korkes, S., Glucose dehydro-genase. J . B i o l , ohem., 196, 769 (1952) 34. Umbreit, W.W., Metabolism of microorganisms. Annual Rev. Mi c r o b i o l . , I l l , 81 (1949) 35. Umbreit, W.W., Bu r r i s , R.H., and st a u f f e r , Manometric Techniques and Tissue Metabolism, Bur-gess Publishing Company, Minneapolis (1949) 36. Warburton, R.H., Eagles, B.A., and Campbell, J.J.R., The intermediate metabolism of Pseudomonas aeruginosa: V: I d e n t i f i c a t i o n of pyruvate as intermediate i n glucose oxidation. Can. 3". Botan., 2_9, 143 (1951) 53 37. Williams, and Sreenivasan, A., Preparation of soluble choline dehydrogenase from l i v e r mito-chondria. J . B i o l . Chem., 205, 899 (1953) 38. Wood, W.A., and Schwerdt, R.F., Carbohydrate oxidation by Pseudomonas fluorescens: I : Mecha-nisms of glucose and gluconate oxidation. J . B i o l . Chem., 201, 501 (1953) 39. Wood, W.A., and schwerdt, R.F., Carbohydrate oxidation by Pseudomonas fluorescens: I I : Mechanisms of hexose phosphate degradation. J . B i o l . Chem., 206, 625 (1954) 

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