..,..‘.. 'u-l‘ Inhabit; ¥fl2.mnncé‘ 13-.'...;.. . U; _.. ..,:. .3 \. 5'“ . o y y 0" 3". a ct, v r 5' L i‘ b ' .fl .' '_ - also! a .Zs—a' A J 4. .. b * 'J ’ . ~ 'A . I '- l '~ ‘1 1 1. . l ”‘1 1.51;". J; .',: ~ 7" M‘W ‘5' S“~~“!\.‘\.‘.‘lm" ‘ [Jim rant. .. W -h‘ ‘ ' album 31 " -« l0“ 8: SOIS’ BOOK llllDEllY umm muons mm". mm! PARTIAL PURIFICATION AND CHARACTERIZATION OF A UNIQUE PROLINE DEHYDROGENASE FROM CLOSTRIDIUM SPOROGENES By Ronald Dale Cooper A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1976 \q ‘0’ ABSTRACT PARTIAL PURIFICATION AND CHARACTERIZATION OF A UNIQUE PROLINE DEHYDROGENASE FROM CLOSTRIDIUM SPOROGENES By Ronald Dale Cooper An NAD+—dependent dehydrogenase catalyzing the con- version of L-proline to Al-pyrroline 5—carboxylic acid (PSCA) was partially purified from Clostridium sporogenes. The enzyme is specific for L-proline and NAD+. It is not an oxidase, and it is not affected by oxygen. Apparent Km values measured at pH 8.0 for L-proline and NAD+ were 33 mM and 1.2 mM, respectively. However, these may be errone— ous due to the presence of an NADH—dependent PSCA reduc- tase in the extracts. The optimum temperature for the de- hydrogenase is approximately SOC, and the optimum assay pH is approximately 10. The conversion of L—proline to PSCA is strongly inhibited by low concentrations of L—glu- tamate. The presence of excess L-proline in the growth medium increases the levels of enzyme activity in cell extracts.. Cells grown with D-glucose have lowered proline dehydrogenase activity. The partially purified proline de— hydrogenase preparation contained a very low activity of glutamate dehydrogenase, which tended to copurify with the proline dehydrogenase. Also, the PSCA reductase mentioned above copurified with proline dehydrogenase. The activity of this enzyme is not affected by L-glutamate but is inhib- ited by L-proline at high concentrations. Studies with labeled proline and ornithine indicate that the proline de- hydrogenase may function in the conversion of proline to glutamate in g. sporogenes. DEDICATION I dedicate this thesis to my wife, Mary, for all her love, patience, and understanding during the comple— tion of my graduate education at Michigan State Univer- sity. ii an ACKNOWLEDGMENTS I wish to express my sincere gratitude to my major professor, Dr. R. N. Costilow, for his guidance and en- couragement throughout the course of this investigation and the preparation of this thesis. I would also like to express my sincere thanks to Dr. H. L. Sadoff and Dr. R. R. Brubaker for the use of their laboratory facilities and to Ms. Barbara Goelling for her technical assistance. iii H! I" f G $ t. '-, (I) '71 TABLE OF CONTENTS DEDICATION ........ ....... ........ ..... .... ..... ... ACKNOWLEDGMENTS ...... ............................. LIST OF TABLES .................................... LIST OF FIGURES ...... ........ . ......... .... ....... INTRODUCTION ...................................... LITERATURE REVIEW ............ ........... . ......... MATERIALS AND METHODS ....... O O O O I O O O O O O O O I O I O O O I O 0 Culture and Cultural Methods .... ..... ........... Enzyme Assays ................................... Preparation of Al—Pyrroline S-carboxylic Acid ... In Vitro Interconversion of Proline and Glutamate Fractionation of Cells Grown in Synthetic Media with Labeled Amino Acids ....... ................ RESULTS ...... ............ .... ...... ..... .......... Studies on the Role of Proline Dehydrogenase in Cells of Clostridium sporogenes .......... ..... . Effect of growth medium on proline dehy~ drogenase activity . ....... ................. Interconversion of proline and glutamate .... Partial Purification of Prolinc Dehydrogcnasc .. Production of cells and preparation of crude extract .............................. Dialyzed extract .. ....... . ......... ...... Streptomycin sulfate treatment .............. Ammonium sulfate precipitation .............. DEAE-cellulose chromatography ............... Sephadex G- 200 gel filtration .. ........... Analytical polyacrylamide disc gel electro- phoresis of the Sephadex G 200 fraction .... iv Page ii iii 10 10 10 13 1h 16 19 l9 19 21 C 26 26 27 27 28 29 33 (TABLE OF CONTENTS) Page Separation of proline dehydrogenase and glutamate dehydrogenase by polyacrylamide gel electrophoresis ........................ 35 Separation of proline dehydrogenase and glutamate dehydrogenase by DEAR—cellulose and hydroxylapatite chromatography ... ..... . 36 Characterization of Proline Dehydrogenase ....... A2 Enzyme stability ............................ A2 Relationship between enzyme concentration and measured activity ...................... A6 Effect of pH on enzyme activity ............. A6 Effect of temperature on enzyme activity .... 51 Response of enzyme activity to oxygen ....... Sl Affinity of proline dehydrogenase for L-pI‘Oline coo.ooooooooooooocoooooooo-oncoo-o 5’4 Affinity of proline dehydrogenase for NAD+ .. 5A Affinity of proline dehydrogenase for Al-pyrroline S-carboxylic acid .... ..... .... 59 Inhibition of proline dehydrogenase by L-glutamate ..... . ........ .......... ...... .. 62 DISCUSSION ........... ....... . ..... ........... ..... 65 LITERATURE CITED ........ . ............. . ........... 72 LIST OF TABLES Table Page 1. Effect of growth medium on proline dehy— drogenase activity ......................... 2O 2. Conversion of L-[U-th]-proline to L—[U—JhC]— glutamate by cells of g, sporOgenes ........ 22 3. Conversion of [U~th]— PSCA to L-[U-1“]— glutamate in the presence of various concentrations of hydrazine sulfate ........ 2A A. Interconversion of proline, glutamate, and ornithine during growth of g. sporogenes in a synthetic medium ...................... 25 5. Partial purification of proline dehydrogen~ ase ......OOOOOOOOOOOOOOOOO00......O...‘OIOO 32 6. Partial purification of proline dehydrogen— ase II ......C...........$‘..O.............. hs 7. Inhibition of proline dehydrogenase by L-glutamate 0................OOIDCCOOOOOCOOO 63 vi 10. ll. 12. 13. LIST OF FIGURES Interconversion of ornithine, proline and glutamate in microorganisms and animals . Sephadex G—200 gel filtration of DEAE— cellulose fraction ...................... Polyacrylamide disc gel electrophoresis of the Sephadex G—200 fraction .......... Separation of proline dehydrogenase from glutamate dehydrogenase by polyacrylamide disc gel electrophoresis ................ Elution of enzymes from DEAR—cellulose .... Elution of enzymes from hydroxylapatite ... Correlation of enzyme concentration and measured activity ....................... Effect of pH on proline dehydrogenase actiVity 0.6.6.00.0.00.........OOOOOOOOCO Effect of temperature on proline dehy— drogenase actiVity ......OOCOOOOOOOOCOIOO O f Lineweaver—Burk plot for L—proline ......... Lineweaver-Blll‘k plOt fOl‘ N4A13+ o o o o o o c c o o a o o Lineweaver—Burk plot for PSCA ............. Postulated relationships of arginine, orn— ithine, proline, and glutamate in C. fiporogenes O O O O C O O O O O O O O O O O ‘ I O O O O O O O O C P O . vii Page 30 3A 37 A0 A3 A7 A9 52 55 57 6O 69 -o ..- .1 Fab 2‘ Q ‘5- INTRODUCTION During studies of the conversion of ornithine to proline by Clostridium botulinum and Clostridium sporogenes, Costilow and Laycock (10) discovered an enzyme that appeared 1--pyrroline S—carboxylic to catalyze the interconversion of A acid (PSCA) and proline. Both NAD+—dependent proline dehy— drogenase and NADH—dependent PSCA reductase activities were demonstrated. The overall equilibrium of the interconversion was far in the direction of proline. Further studies showed that the true intermediate between ornithine and proline was 41-pyrroline 2-carboxylic acid rather than PSCA (23). There- fore, it was postulated that PSCA may be an intermediate in the interconversion of glutamate and proline in C. sporogenes as it is in many other microorganisms and animals. The purpose of this investigation was to study the enzyme described above, which will be referred to by the triv— ial name of proline dehydrogenase. Experiments were designed to: (a) determine the physiological role of the enzyme; (b) partially purify and determine optimal conditions for dehy— drogenase activity; and (c) study the inhibition of the enzyme by glutamate. LITERATURE REVIEW The Interconversion of Proline and Glutamic Acid The metabolic relationship between glutamic acid and proline has long been an area of interest. As early as 1912, Abderhalden observed that an alcohol—extracted casein hydrolysate, presumably free of proline and rich in glu— tamate, permitted the normal growth of dogs (38). Somewhat later, Womack and Rose (50) recovered ll‘tC—proline from rats fed with 1” C—glutamic acid, indicating a possible metabolic pathway between these two amino acids. The intermediates in the pathway of the conversion of proline to glutamic acid were first worked out in mamma— lian systems. In 19AA, Stetten and Schocnheimer (36) iso— lated significant amounts of 15N-glutamate from rats fed with 15N-proline. Other evidence from their work indicated that proline was oxidized to a pyrroline carboxylic acid which was presumably futher oxidized to glutamate. Vogel and Davis (A7) subsequently showed that the product of oxidized proline was most likely A} —pyrroline S-carboxylic acid (PSCA), which was in spontaneous equilibrium with glutamic X—semialdehyde (GSA), as determined in vitro with chemically synthesized PSCA. Strecker (A1), a major contributor to the study of the interconversion of glutamate and proline, showed that with partially purified rat liver and calf liver enzymes and pure 'chemically synthesized PSCA, the enzymatic conversion of proline ... .O-' '->‘ 0 VP. I No.1. L. is to glutamate proceeds via two distinct one-way steps: (a) the oxygen—dependent oxidation of proline, catalyzed by a "proline oxidase," and (b) the spontaneous conversion of the PBCA formed to GSA and the subsequent oxidation of this compound to glutamate, catalyzed by an NAD(P)+-dependent "PSCA dehydrogenase." This pathway of proline catabolism may be a universal one in animals, having already been identi- fied in guinea pig kidney, rabbit liver and kidney, human liver, brain and kidney, and rat brain (A2). There is no evi- dence as yet for any other pathway of proline catabolism in animals. The biosynthesis of proline in animals has been shown to proceed from L—glutamate through the same intermediates (PSCA 3 GSA), using a distinctly different system of enzymes catalyzing essentially irreversible reactions. One enzyme (not yet isolated) catalyzes the conversion of L—glutamate to GSA, and another enzyme, an NAD(P)H—dependent PSCA reductase, reduces PSCA to proline (25). I In microorganisms, most of the early work on the in- terconversion of proline and glutamate was concerned with the biosynthesis of proline from glutamate in Escherichia coli (Ah) and Neurospora (6). Vogel and Davis (A7) first demon- strated that the pathway from glutamate proceeds via the same intermediates as those in animal systems by showing that chemically synthesized GSA and/or PSCA could satisfy the pro— line requirements of E, ggli proline auxotrophs. The present accepted pathway of prOline biosynthesis in E. coli includes an as yet unisolated phosphorylated intermediate between glu- tamate and GSA, as proposed by Baich (2). The catabolism of proline in microorganisms has been studied extensively in recent years. Early work by Bernheim (5) and Stone and Hoberman (39) showed that L-proline could be utilized as a single carbon source in E. coli in the pres- ence of oxygen. Frank and Rybicki (15) later demonstrated , that the degradation of L-proline to glutamate in E. 3311 proceeds, as is the case in animals, via the same intermedi- ates as those for the biosynthesis of L—proline from glutam- ate (PSCA ¢ GSA). .In a follow—up study, Frank and Ranhand (16) showed that the enzymes involved in L—proline catabolism were not reversible by demonstrating that mutant strains of E. 331; BA, blocked in the enzymes for proline biosynthesis from L-glutamate, could still convert L-proline to glutamate. This system of separate enzymes for the biosynthesis and ca- tabolism of L-proline is comparable to the mechanism of in— terconversion of proline and glutamate in animals. Little information is available concerning the meta- bolic relationship of proline and glutamate in plants. Very recently, a NAD+—dependent proline dehydrogenase and a NAD(P)H~ dependent PSCA reductase have been described in Chlorella pyrenoidosa (21) and in cotyledons from the pumpkin, Cucur- bita moschata (31). The proline dehydrogenase described in these organisms is not an oxidase and can function normally in the absence of oxygen, unlike the proline oxidizing en— zymes described thus far in animals and microorganisms. Also, .AK —: r; .ru ~ 5..‘ F. ~x EU ..1 some evidence is presented by Rena and Splittstoesser (32) that proline dehydrogenase and PSCA reductase activity may occur on the same protein molecule in pumpkins. The only other NAD+-dependent proline dehydrogenase reported in the literature is the one found in Clostridium sporogenes by Costilow and Laycock (10). Further investiga— tion of this enzyme is the subject of this thesis. Regulation of Proline-Glutamic Acid Interconversion in Microorggnisms Since most of the work concerning the investigation of the regulation of proline—glutamic acid interconversion has been done with bacteria and yeast, only these systems will be discussed. Synthesis of L-proline from L—glutamate in E, £211 has been shown to be regulated by feedback inhibition. Baich (2) and Baich and Pierson (3) demonstrated that L-proline in- hibits as well as represses the conversion of L-glutamate to GSA. The reduction of PSCA to L—proline is neither inhibited nor repressed in this organism, since the bacterium will ex~ crete L-proline when supplied with exogenous P5CA (3). Berg and Rossi (A) showed that E. 3313 will also excrete L—proline when the conversion of L—glutamate to GSA is blocked and PSCA is formed by an alternate route (from Nuacetylglutamate). Kuo and Stocker (17) obtained the same results with Salmonella typhimurium. The inducibility by L—proline (and not PSCA) of both enzymes of the catabolic pathway from L—proline to glutamate has been demonstrated in a number of bacteria, all growing aerobically (12, 15, 2A). In all of these instances, catabo— lite repression of proline oxidase and PSCA dehydrogenase by D-glucose was also observed. Prival, Brenchley, and Magasanik (7, 27. 28) presented evidence that in Klebsiella aerqgenes, proline oxidase is repressed by glucose only when the supply of nitrogen is abundant. In the presence of limiting nitro- gen, glucose does not repress the enzyme, presumably due to increased activities of glutamine synthetase under these con— ditions. Adenosine 3',5'—monophosphate (cyclic AMP) does not appear to play a role in this relief of catabolite repression. Laishley and Bernlohr (18) reported that the enzymes involved in arginine breakdown (arginase, ornithine JLtrans- aminase, and PSCA dehydrogenase) could be coincidentally in~ duced by arginine or ornithine and could be repressed by glu- cose in Bacillus licheniformis. Also, proline oxidase and PSCA dehydrogenase were induced by L—proline, and the dehy- drogenase was found to be under catabolite repression control. They suggest that.P5CA could be a common in XEXQ inducer of arginine and proline catabolism in this organism. Lundgren and Ogur (20) showed that a Saccharomyces glutamate auxotroph blocked in the tricarboxylic acid cycle possessed a catabolic pathway to glutamate from proline, ar- ginine, and glutamine, and grew on any of these amino acids in a minimal medium. The mutant did not, however, grow on these amino acids in a medium containing a full complement of common V». p». AN» u~U HR. amino acids minus glutamate. Their investigation showed that the PSCA dehydrogenase (and not the proline oxidase) in this organism is significantly inhibited by a wide variety of amino acids, including glutamate. They suggested that there may be a non—specific regulatory mechanism in yeast in which the size of the amino acid pools controls glutamate formation from proline and arginine. Relationship of Ornithine to Proline and Glutamic Acid Metabolism The recognized biosynthetic and catabolic routes for the interconversions of ornithine, proline, and glutamate are shown in Fig. l (l, 29, 30, 33, 3?). In E. EQEE, the major pathway of ornithine synthesis from glutamate proceeds via acetylated intermediates, while acetylation is not required in Neurospora crassa, Torulopis utilis, or animal tissues (A6). Little information is available on the interconversion of these amino acids in plants. The conversion of ornithine to glutamate X—semialde- 'hyde is catalyzed by ornithine J-transaminase with pyridoxal phosphate required for the transamination. In E. EQEE, the equilibrium of this reaction is far in the direction of the semialdehyde, presumably due to the tendency of the semial- dehyde to cyclize (A6). The glutamic U-semialdehyde is nor— mally oxidized to glutamate, but may be reduced to proline as an alternate method of synthesizing proline in E, gal; (33, A6). ’,G1utamate / // k/ N-Acetylglutamate l l {r N-Acetylglutamic _*G1utamic U-semialdehyde ————— U-semialdehyde ,/ I l I .c 1' 1 4r N—Acetylornithine A -Pyrroline S-carboxylic acid + 1+ Ornithine g/ >Proline l v Citrulline \ \ \\ ‘\ fit Arginine Fig. 1.—~Interconversions of ornithine, proline, and glutamate in microorganisms and animals. Dashed arrows represent bio— synthetic and solid arrows catabolic pathways. Species of Clostridium catabolize ornithine by two pathways, both different from those described above. Dyer and Costilow (13, 1A) and Tsuda and Friedman (A5) have re— ported that Clostridium sticklandii oxidized L-ornithine as a single substrate to ammonia, alanine, acetate, and carbon dioxide by a coupled oxidation-reduction with proline as the electron acceptor. Costilow and Laycock (8, 9, 10) also showed that both E. botulinum and E. fipgiggenes con- verted L-ornithine to L-proline by a single protein, orn— ithine cyclase (deaminating). The intermediates in this pathway are &éketo-JLaminovaleric acid in spontaneous equi— librium with AE-pyrroline 2-carboxylic acid (23). They (10) also found significant levels of a presumably rever— sible enzyme that catalyzes the interconversion of PSCA and L-proline which was not involved in the conversion of orni— thine to proline. . . . . .. ... L. I. Hi ‘ . ~ . ... ... . . ... . . PL C- . . .n .us .7“ v»: ... n. c an. #3 n . u- as ..a n! .ulu .5. Fa- C.‘ L. QK‘ MATERIALS AND METHODS Culture and Cultural Methods The organism used in this study was a putrefactive anaerobe, Clostridium sporogenes (NCA Clostridium PA 3679, ATCC 7955). Two basic kinds of media were used for the study of the proline dehydrogenase enzyme. The "standard trypticase" medium consisted of A.O% trypticase, 2 ppm thiamine hydro— chloride, and 0.05% sodium thioglycollate as a reducing agent. The other medium was a synthetic one containing salts, vitamins, and 12 amino acids adapted from the medium of Per- kins and Tsuji (26). Both kinds of media were adjusted to pH 7.A before autoclaving (15 psi for 20 min). Growth cultures were routinely started by inoculating a stock suspension of spores (kept at AC) into a 10 ml tube of standard trypticase medium. This suspension was heat shocked for 10 min in a 60C water bath and incubated in an anaerobic jar under a hydrogen atmosphere. Tube cultures pre— pared in this manner were used as 2% inocula into the appro- priate media. All cultures were incubated between 15 and 18 h. Enzyme Assays Three assay procedures for proline dehydrogenase were used in this study. One procedure was essentially that de— scribed by Costilow and Laycock (10) which measures the amount of Al—pyrroline 5-carboxy1ic acid (P5CA) formed from proline 10 11 during a 15 min incubation in the presence of g-aminobenzal- dehyde. The colored reaction product, a P5CA-g—aminobenza1- dehyde complex, is measured by absorbance at AA3 nm, with a mM extinction coefficient of 2.71 mM—1 cm"1 (25). Previously reported data demonstrated that the proline dehydrogenase can readily be detected by trapping off the PSCA product by this method (10). Reaction mixtures of 0.5 ml total volume contained 25 mM Tris~HC1, pH 8.0, 0.3 M L—proline, 2 mM NAD+, 20 mM sodium pyruvate, 0.01 mg rabbit lactic dehydrogenase (65 units/mg), and enzyme. The mixture without enzyme was saturated with g-aminobenzaldehyde and placed into 13 x 100 mm tubes. After equilibration for 5 min in a ACC water bath, enzyme was added, and the mixture incubated for 15 min and stopped with 0.5 ml 10% trichloroacetic acid (TCA). The TCA~ insoluble material was centrifuged out, and the absorbancy of the supernatant solution was read at AA3 nm against pre— acidified controls. In the second procedure, the formation of the colored reaction product of P5CA and g—aminobenzaldchyde was measured by monitoring the rate of increase in absorbency at AA3 nm. Reaction mixtures of 1.0 ml total volume contained 0.1 M Tris—HCl (saturated with g—aminobenzaldehyde), pH 8.0, 0.A5 M ' L—proline, 10 mM NAD+, and enzyme. The mixture without enzyme was equilibrated to 37C in a cuvette using a Haake constant temperature regulator. After addition of enzyme, the reaction was monitored immediately by measuring the increase in absor~ bancy at AA3 nm with time. 12 The third proline dehydrogenase assay procedure was conducted by measuring the rate of proline-dependentreduction of NAD+ as the increase in absorbancy at 3A0 nm with time. The reaction mixture for this assay was exactly the same as that for the second procedure (above), but without g-amino- benzaldehyde. Enzyme was added to start the reaction after 5 min equilibration at 370, and the rate of absorbancy was monitored immediately. This third assay procedure was not used for the less purified enzyme fractions due to a signifi- cant amount of background reduction of NAD+ with these pre— parations. To assay for P5CA reductase, the PSCA—dependent oxi- dation of NADH was measured by monitoring the loss of absor- bancy at 3A0 nm with time. Reaction mixtures of 1.0 ml to- tal volume contained 0.1 M Tris—HCl, pH 8.0, 0.1 mM NADH, 1.3 mM P5CA, and enzyme. After 5 min equilibration at 370 in a cuvette, enzyme was added to start the reaction which was monitored immediately. Glutamate dehydrogenase assays were conducted by measuring theci—ketoglutarate-dependent oxidation of NADH as the rate of loss of absorbancy at 3A0 nm by the procedure of Winnacker and Barker (A9). A 1.0 m1 reaction mixture contained 0.15 M Tris—H01, pH 8.0, 0.18 M ammonium chlor- ide, 0.1 mM NADH, 5 mM:{—ketoglutarate, and enzyme. After 5 min equilibration in a cuvette at 37C, NADH was added to start the reaction. For all of these enzyme assays, a unit of enzyme is 1a. «\n and '- vs. 13 defined as that amount of enzyme necessary to convert l pmole of substrate to l pmole of product in one minute. And specific activity is defined as units of enzyme per mg of protein. For the reactions monitored at 3A0 nm, NADH was taken to have a millimolar extinction coeffi— cient of 6.2 mM”1 cm—l. All reactions were read on a Gil— ford multisample recording spectrophotometer. Protein con— centrations were estimated by the method of Lowry, 33. EE. (19), using bovine serum albumin as a standard, or by read- ing absorbancy at 260 nm and 280 nm and using a nomograph. Preparation of'Al—Pyrroline 5—carboxylic Acid Two methods were used to prepare PSCA. In one method it was formed by the transamination of L—ornithine, using par- tially purified rat liver ornithine detransaminase according to the method of Strecker (A0). Livers extracted from adult rats were partially homogenized in 0.25 M sucrose, washed in 0.05 M potassium phosphate, homogenized again in a Waring blendor, precipitated with ammonium sulfate, and dialyzed. To generate the PSCA, the reaction mixture adapted from -Strecker contained A0 mM L-ornithine (L-[U—th]~ornithine th]—P50A was needed), 8 mMI£~ketoglutarate, pH 7.1, when [U- 50 mM potassium phosphate, pH 7.1, and the partially puri— fied ornithine JLtransaminase, in a total volume of 10 ml. The reaction was run with shaking for 35 min at 37C and stopped with an equal volume of 10% TCA. The PSCA prepared by this method was separated from ..." .hv u . N. .7 u C» L. s u D n PF. ... . .r k ‘Lvd' . U- r:— -. >.¢ .l . "-". hr. 5. I ‘1. ( ... 1A the other amino acids in the final reaction mixture by the method of Costilow and Laycock (9). P5CA was also prepared by another method described by Strecker (A2), using a rat liver mitochondrial prepara— tion and cytochrome c with L—proline as substrate. In Vitro Interconversion of Proline and Glutamate To attempt to show the 13 vitro conversion of L—pro— line to glutamate, reaction mixtures of 0.6 ml total volume were combined in 13 x 100 mm tubes to contain 2) mM potassium phosphate, pH 7.5, 15 mM ATP, 1 mM magnesium chloride, 15 mM NAD+, 130 mM L-[U—th]-proline (specific activity = 0.0032 pCiflumole), and either whole cells or crude extract of E. sporogenes grown in a synthetic medium. Likewise, to attempt to show the £3 vitro conversion of L—glutamate to proline, similar reaction mixtures of 0.6 ml total volume were com- bined in 13 x 100 mm tubes to contain 25 mM potassium phos- phate, pH 7.5, 15 mM ATP, 1 nM magnesium chloride, 15 mM NADH, 30 mM L-[U-th]—glutamate (specific activity = 0.02 pCi/pmole), and whole cells or crude extracts of cells grown in synthetic media. In both cases, the reaction mixtures were equili- brated in a water bath to AOC for 5 min. Whole cells or crude extract was added to start the reactions, which were stopped after 1 h with an equal volume of 10% TCA. A cold TCA extraction was carried out in an ice bath for 30 min, after which the TCA insoluble material was centrifuged out .n. .In '- .7. .,.l-. '- .—. no . ...-.. u.“ . ~ . n Cd . . .. ... .—_ .u. F. v“ .n Aw rn‘ ‘- b ... -“ PU i; ’\ v . a; E . . .... a... :u Ad 2. i . .. 5 h~ o . r-u V~" on" ‘ 15 and the supernatant solutions extracted three times with ether. These solutions from the reactions and the pre- acidified controls were adsorbed on Pasteur pipette col— umns of Dowex 50w-XA H+—form cation exchange resin, washed with water, eluted with l M ammonium hydroxide, and con— centrated to dryness under vacuum.' The dry amino acids were dissolved in 0.1 ml water, and 20 ul samples were electro— phoresed (35 volts/cm).on Whatman No. 1 paper using a buf— fer system of 0.25 M sodium acetate, pH 1.3, with which glutamate could be readily separated from proline and orni- - thine. Following electrophoresis, the paper was dried, and the amino acid spots were developed with 0.01% ninhydrin_in acetone in a drying oven. Spots corresponding to the stan- dards (L-glutamate, L—proline, Jsaminovaleric acid, and L- ornithine) were cut out, folded, and covered with toluene— based scintillation fluid (8) in glass vials. All counting was done on a Packard Tri—Carb Liquid Scintillation Spectro- meter. Since proline could not be separated from other neu— tral amino acids by electrophoresis, the amino acids from the above reaction mixtures containing L—[U-JhC]—g1utamate as substrate were also chromatographed (descending) on Whatman No. 3 paper with a butanol-acetic acid—water (60:15:25) sol— vent system. Amino acid spots were developed and cut out for counting as above. To attempt to show the £2 vitro conversion of PSCA ‘. .. . r .. . r. ... . . .... o. .r.. a. u. .. T. .. . .... .. n. v.. ... .. cl» In 0 . w— n. .r. F r. F. .x» n. A “I «\u .. . . . ... I . .. . s u'. we. as: an. .. :- r.. e um . c n.» so. he .. v~ ’- . ¢ ~ ~ I . w— o w .n . a a v -. n- X a .- Q v.- 8» & t sq» 16 1A to glutamate, [U—th]-P5CA prepared from L-[U— C]-ornithine using rat liver ornithine stransaminase was dissolved in 250 mM potassium phosphate, pH 7.5, to give a 15 mM solution of the labeled P5CA. Reaction mixtures of 100 p1 total vol— ume contained 25 mM potassium phosphate, pH 7.5, 10 mM ATP, 10 mM NAD+, 1 mM magnesium chloride, A00 mM [U—luC]-P5CA (specific activity = 0.066 pCi/pmole), varying concentrations of hydrazine sulfate, pH 7.7, and 1.2 mg crude extract of E. gporogenes grown in standard trypticase medium. All reactions were run at A00 for 30 min and stopped with 60 p1 0.A M formic acid. After centrifuging at 18,000 x g for 15 min, 15 pl samples of the reaction mixture and of a pre-acidified control were spotted on Whatman No. 3 paper and chromatographed with a descending butanol-acetic acid- water solvent system. The glutamate spots were cut out and counted for 10 min in the toluene-based scintillation fluid. Fractionation of Cells Grown in Synthetic Media with Labeled Amino Acids Three E. sporogenes cultures of 100 ml each were grown in synthetic medium + 0.05% sodium thioglycollate with 0.23% arginine added as a supplementary amino acid. After auto- claving, 0.5% sterile glucose was added to the media. Each flask was inoculated with A m1 of a growing culture of 2, £223: ogenes (in standard trypticase media), and the flasks were in~ cubated in anaerobic jars. After 5 h, single cultures were supplemented with 10 o I'v; ‘in' .. e 'III 1."... .....|-.- . o - v' u .I._-- ul I. d 1!. I. r. I .2 .. e a» .e C C . I Cu .‘ . as $ v t... Vs ”V ... ..L .. .5“ 4. f. ...a . a p; A. be . n .«4 Q. A a lo v. p“ . . .2 is a. c. . a w. rd 0 u e . n ‘ ...... v.. .s . .~ g :- .. a hi I .. . G» F_ C. is .q u a.» ....I no. ... A r: n- In. .u c on». t t on. aka .5 ~ his f.- 17 1A PCi of sterile L—[U-th]-proline, L—[U— C]—ornithine, or L—[U-th]-glutamate. Growth was allowed to continue for 15 more hours. All cultures were harvested at 18,000 x g for 15 min, the pellets washed twice with 10 ml volumes of cold 0.05 M potassium phosphate, pH 7.5, and then treated with the following procedures: (a) cold TCA extraction; (b) hot TCA extraction; and (c) complete protein hydrolysis. (a) Cold TCA extraction: Five ml of cold 5% TCA were added to each pellet and held at 0C for 5 min before centrifuging. The pellets were washed a second time-and the two 5—m1 supernatant solutions combined to give the cold TCA extracts. (b) Hot TCA extraction: Each pellet was extracted with 2.5 ml 5% TCA at 90C for 30 min and centrifuged. The pellets were then washed with 2.5 m1 warm TCA and the wash- ings discarded. (c) Protein hydrolysis: The pellets were then sus- pended in 1 ml 6 N HCl and transferred to vials. The cen— trifuge tubes were rinsed with another 1 m1 of the HCl and added to the vials. The vials were flushed with argon, sealed, and incubated at 110C for 2A h to give the protein hydrolysate. The cold TCA extracts were extracted three times with equal volumes of ether, adsorbed on a Pasteur pipette column of Dowex 50W-XA H+—form resin, washed with water, and eluted . eat. -v v -.o>" .9. ;~ I—v-l'.‘ . H... , .... _, o. ,.- "up .-.-~~‘ A . .4 ‘5‘»,- .. I . ‘ ‘ ‘ "C." u.‘l _ ‘ 0,; A’\‘ -_L g' --.. . V—...,‘ _ .- Co‘; I A Q : "mh ' - ’n u a v» n5 “.1 5.4.,‘ a”. . ,, u- 18 with 1 M sodium hydroxide. The eluates were dried under a vacuum, washed once with 5 ml water, and dried again. The dried pellets were dissolved in 0.5 ml water and electro- phoresed as previously described. The protein hydrolysates were washed from their vials with 3 ml water and dried under vacuum. The pellets were picked up in 1 ml water and adsorbed on Pasteur pipette Dowex columns as described above and eluted with 1 M sodium hydroxide. These eluates were dried under vacuum, washed with water, and dried again. The dry amino acids were dis- solved in 0.5 ml water and electrophoresed as described above. The cold TCA extract and the protein hydrolysate of the cells from each culture were also chromatographed as pre— viously described. The paper was then dried, sprayed with 0.1% ninhydrin in acetone, and the spots cut out which cor— responded toamino acid standards. These spots were counted for 10 min in toluene—based scintillation fluid. RESULTS Studies on the Role of Proline Dehydrogenase in Cells of Clostridium sporogenes Effect of growth medium on proline dehydrogenase ac— tivity: If proline dehydrogenase is involved in the dissimi- lation of proline in E. sporogenes, one might expect the en— zume to be repressed in a glucose medium anC induced in a me— dium supplemented with exceSs proline. Conversely, if the enzyme is involved in the biosynthesis of proline from glu- tamate, one might expect it to be repressed in a high pro— line medium. To test these possibilities, E, sporogenes cells were grown in 1 liter batches of standard trypticase and syn— thetic media supplemented with various amino acids and with glucose. Crude extracts of cells were prepared from each medium and these extracts assayed for proline dehydrogenase activity. The presence of glucose in the medium resulted in a decrease of about 50% in enzyme activity (Table 1). There is also some indication that the presence of proline or of amino acids readily converted to proline (arginine and orni- thine) in the medium enhanced proline dehydrogenase activity. The extract with the highest enzyme activity was of the cells grown in standard trypticase medium supplemented with 0.1% L-proline. -While not definitive, these data indicate that proline dehydrogenase may be involved in the dissimilation of 19 ,- 20 Table l.--Effect of growth medium on proline dehydrogenase activity.* Growth Medium Specific Activity Standard trypticase 0.0119 Standard trypticase + 0.1% L-ornithine 0.01h7 Standard trypticase + 0.1% L-arginine 0.0159 Standard trypticase + 0.5% D-glucose 0.0076 Standard trypticase + 0.5% D-glucose + 0.1% L-ornithine 0.0095 Standard trypticase + 0.1% L—proline 0.018h Synthetic 0.0093 Synthetic + 0.5% DL— proline 0.0163 Synthetic + 0.1% L— glutamate 0.0150 Synthetic + 0.5% D-glucose 0.0058 Synthetic + 0.5% DL— proline + 0.1% L—glutamate 0.0lh8 Synthetic + 0.5% DL- proline + 0.5% D-glucose 0.007h *Assays were performed by measuring the rate of in— crease in absorbancy at hh3 nm. Enzyme was crude extract, between 0.55 and 1.3 mg protein per reaction. 21 proline in these cells. Interconversion of proline and glutamate: Since the most likely function of the proline dehydrogenase in cells of g. sporogenes would be in the interconversion of proline and glutamic acid, a number of experiments were conducted to attempt to demonstrate such conversions. In an effort to show . . . 1h - . in the £2 v1tro convers1on of L—[U— CJ—proline to L—[U— C]— glutamate, reactions were conducted as described in MATERIALS AND METHODS with whole cells of C. sporogenes, grown in a synthetic medium supplemented with L—proline and/or D-glu- 11+C]-—proline. Counting the cose, in the presence of L—[U- glutamate spots from paper electrophoresis in toluene-based scintillation fluid revealed a significant conversion of the labeled proline to L—[U-th]—glutamate by cells grown in the synthetic medium + 0.5% L—proline and some conversion to L-[U—th]-glutamate by cells grown in synthetic medium + 0.5% L-proline and 0.5% D-glucose (Table 2). These data agree with the specific activities noted for the proline dehydrogen- ase from cells grown in different media (Table l). The re~ sults suggest that proline dehydrogenase is indeed involved in the conversion of proline to glutamate. To attempt to demonstrate the conversion of L-[U-th]~ glutamate to L—[U-lqu-proline, reactions were conducted as described above but with L-[U-th]-glutamate as substrate, using whole cells or crude extracts of cells of g. sporogenes grown in synthetic medium supplemented with a variety of amino acids, including 0.1% L—glutamate. Conversion of the labeled 22 TatflLe 2.-—Conversion of L-[U—th]— proline to L-[U—th]- glutamate by cells of g. sporogenes? Growth Medium L—[U-th]-glutamate Spots Reaction Control (cpm) (cpm) Synthetic h67 h08 Synthetic + 0.5% L—proline lShh - 286 Synthetic + 0.5% D—glucose 378 h22 Synthetic + 0.5% L-proline + 897 60h 0.5% D—glucose *Reaction mixtures were as follows: 25 mM potassium phosphate, pH 7.5; 15 mM ATP; 15 mM NAD+; 130 mM L-[U—th]— proline (specific activity = 0.0032 uCi/umole ); and 25 mg (dry weight) whole cells of g. Eporogeggg. Controls were acidified prior to adding cells. .n D. 2 . w ._ .r L.— c c. . is. .I o . a... u . Pp— .. . TI. '0 -... '0 r .h r\ . . L ~3- r” 9.» . e A... VL- .14 .- l. ‘,U u 1‘ . , t— n a E .v u. ”H en ru ..e t e Hi we at ... um .. u . e As. H. .- d 23 glintamate to labeled proline could not be shown in any in- st;ance with theSe reaction mixtures. Incubation was conducted berth aerobically and under an argon atmosphere. The next step was to attempt the conversion of the pro— lixae dehydrogenase reaction product, P5CA, to glutamate. Re- acrtions were conducted as described in MATERIALS AND METHODS, 'wixth crude extracts of cells of g. sporogenes grown in stan- d11rd.trypticase medium, and [U—th]-P5CA in the presence of vwirious concentrations of hydrazine sulfate. The hydrazine Sitlfate was used because previous experiments had demonstra- ined that it strongly inhibited the interconversion of L— proline and P5CA. In this case, the hydrazine was used to Irrevent the reduction of the [Uéth]—P5CA to L-[U—th]—pro— liune. After paper chromatography of the final reaction mix— tirres, counting the glutamate spots in toluene-based scintil- Ilation fluid revealed significant conversion of [U—th1—P5CA tc> L—[U—th]-glutamate, with the extent of conversion drop- Piuug off as the concentration of hydrazine increased (Table 3). These data indicate that g. sporogenes does possess a functional P5CA dehydrogenase which can operate ig_vitro. To attempt to determine the interconversion of L-pro— line, L-glutamate, and L-ornithinc in growing cells of g. .Eggrogenes, cells were grown in synthetic medium supplemented with the ltic-{Labeled amino acids and fractionated as described in MATERIALS AND METHODS. Table A summarizes the results of this study. The data are not altogether consistent, however there is some evidence that labeled proline may have been 2h . it I Tatale 3.—-Conver31on of [U- C]—PSCA to L-[U—th]-glutamate in the presence of various concentrations of hydrazine sulfate.* Hydrazine sulfate L—[U—th]—glutamate Spots (cpm) Control 17h 10 mM 68l 15 mM 57A 20 mM 350 25 mM 320 *Reactions contained 25 mM potassium phosphate, pH '7.5, 10 mM ATP; 1 mM magnesium chloride; hOO mM [U-1 C]- IHSCA.(specific activity = 0.066 pCi/umole); 10 mM NAD+; hywirazine sulfate as above; and 1.2 mg crude extract from celds.of g, sporogenes grown in standard trypticase. The (nontrol contained 10 mM hydrazine sulfate and was acidified Prixn‘to adding cell extract. ... - ~ ‘tu'l. ( 25 .eHch soHpsHHHpsHOh censpucscsHop sH an OH hoe ecu Issoo use meQSem Hi om use 0H mo hsmestpwaoano semen mqwesoomoe so mHmoMOQQOHpooHo non lam mcHSOHHom pso pdo macs mponm @How osfied .890 aw woMSmems we? >9H>Hpoe0Hceme _m O O OH m O O msHspHnno A.hmopes0hscv OOO Om: om OH Omm Ohm csHHohe menusHohcss mOmH OOm mmem mmms omH ms msssnnuHm chponm OOH mm m n O m eHusn A.hosm0hpechV NONH mom OOH mm new ONm Hsnescs cunnsHoncss mmHH mmm owmw mac: 0 o mpeEepSHm sfioponm OH O m m O O anansho A.nwopss0hscv o o o o o o ccHHoam pocnpxo ONH mm OONH osm O O cesseusHm «Os eHOO : o OH NH 0 o canes A.aonaonpooHov Om mm OO Om m m Henpucs possess emH OO OHmH mam OO ms censneus eoe cHoO mmm ONH OH e oeH mm .chep A.hosnonpecHuV ONH: mmOH mme ONm meow OOm Hsnpsms pesuschcesn omm mHH mmeOH mmmm mes oem censnpus copshpnmoeoo Ha ON H1 OH H1 ON H1 OH H1 ON H1 OH seem much csHseHsno-HO -Ol-a cessepsHm- O .2 -g Oe ogg. . u s i s i s e: e .1 as see assesses o % £930hm msfihse unexpwnho use $.85 .mpesepsWme owpmspnAm a ma mustachomn OGHHOHQ cwo GOHmhw>fioohmpGHll.# QHDQB 26 partially converted to glutamate, which either was incor- In porated into protein or leaked back into the medium. contrast, labeled ornithine appeared to have been converted tc> proline and glutamate to a rather large extent and then Relatively little conversion of ichorporated into protein. and there glnrtamate to proline was indicated by the data, 1:31; no indication of any conversion of glutamate to basic amino acids . Partial Purification of Proline Dehydrogenase Production of cells and preparation of crude Extract: Cealils of g. sporogenes were grown in two 20-liter batches After 17 h in— ass (described by Costilow and Laycock (10). Clltyaxion, the cells were harvested with a Sharples continu- flow centrifuge, model AS—l2; 86 g of cell paste were ob— .0118 This pellet was suspended in an equal volume of 10 i35li.rled. Inna Ipotassium phosphate, pH 7.5, with 10% glycerol and ex- The cell tI‘Etczted by ultrasonic oscillation of 8 ml batches. dSBID-lris was removed by centrifugation at 20,000 x g for 20 min to’ ZVPield 6h ml of crude extract. The crude extract was dialyzed Dialyzed extract: age-:‘Lnst 2 liters of 10 mM potassium phosphate, pH 7.5, for 12 h’* 'czhanging the buffer every h h, to yield 78 ml of dialyzed 93th :r-act. It was interesting that the dialysis of crude extracts o . . . . . if (zells conSlstently resulted in a dramatic increase 1n ac— ‘hiivfiity of proline dehydrogenase. A possible explanation of 27 this phenomenon is that glutamate present in the crude ex- tracts may have been inhibiting the enzyme, with dialysis renmving the glutamate and thus relieving the inhibition. IPrcfline dehydrogenase in crude extracts of cells prepared ‘flith the aid of a French press had the same specific activ- iisy'as proline dehydrogenase from sonicated cells. Streptomycin sulfate treatment: The dialyzed ex- tlract was diluted with 10 mM potassium phosphate, pH 7.5, tuith 10% glycerol,to a final protein concentration of ap— plroximately 25 mg/ml. An equal volume of 5% streptomycin Slllfate in the same buffer was added slowly with stirring, 811d the mixture was allowed to stand overnight at hC. Af- tear centrifuging at 36,000 x g for 30 min, 172 ml of ex- tract were collected. Ammonium sulfate precipitation: The streptomycin Sinlfete-treated extract was fractionated by precipitation 'wiiih solid ammonium sulfate between h5 and 80% saturation. The: ammonium sulfate was added very slowly with stirring in an ice bath and allowed to continue stirring for 30 min. ThiLs h5-80% fraction was centrifuged at 36,000 x g for 30 mint and the supernatant solution (which gave a strong reac- tiCHn with geaminobenzaldehyde due to the high concentration 0f tammonium sulfate) was discarded. The pellet was resus— Perrded.in 20 m1 of 25 mM Tris~HCl, pH 8.0, and dialyzed agadJufl;l.liter of the same buffer for 16 h, changing the buffer after 8 h. This dialysis yielded 38 ml of extract Which was divided into 5 ml aliquots and frozen at —18C. 28 DEAE-cellulose chromatography: A column of precy- cled DEAE—cellulose (Whatman DE52) was poured to 1.5 x 15 cm and equilibrated with 50 mM Tris-H01, pH 8.0. One 8-ml aliquot of the dialyzed h5-80% ammonium sulfate cut (168 mg protein) was layered onto the column and eluted with a gra- client of Tris-HCI, pH 8.0, from 0.2 M to 0.3 M (100 ml of eacfln). Five—m1 fractions were collected with a flow rate of‘ 30 ml/h. The column was further eluted with hO m1 of 0.3 M Tris and then with ho ml of 0.35 M Tris-HCl, pH 8.0. -Mosrt of the protein was eluted with the first 50 ml of the grwadient (fractions 1 to 10), as determined by absorbancy at 280 nm. The proline dehydrogenase eluted with the 0.35 1M {Pris (fractions 37 to MO), and these fractions were pooled, coalcentrated to 0.8 ml in an Amicon ultrafiltration cell, model 12, and frozen at —18C. Two more DEAE—cellulose columns were run exactly as abcrve using 12 ml of the dialyzed h5—80% ammonium sulfate- IPreacipitated fraction in each. Ultrafiltration of the frac— tixarls which had proline dehydrogenase activity yielded 1.3 ml :from one of these columns and 2.3 ml from the other. The thrwee DEAE-cellulose columns yielded proline dehydrogenase PreIParations with specific activities of 0.111, 0.126, and 0.11.5 units/mg protein. The purity of the concentrated fractions was visual- izeél by analytical polyacrylamide disc gel electrophoresis (anixanic) by the method of Davis (11). With each gel, a pro- tein stain of 1% amido black in 7% acetic acid revealed a 1" n:" uoH‘ Q. Q ' ha .y. r: F- .c“ ... . "I “-4.... \‘fi .Fn wk h n .‘Ic .29 small band with a relative mobility (RF) of 0.18, a large band with an RF of 0.3, and a diffuse, continuous band be- tween RF values of 0.36 and 0.9. Because of this evidence of impurity in the DEAE—cellulose preparations, another pur- ification step was necessary. The procedure chosen was Sephadex G-200 gel filtration. Sephadex G-200 gel filtration: After running several snuall Sephadex test columns with the concentrated DEAE—cellu- 1x3se fractions, the remaining volumes of these fractions wexre pooled to a volume of 3.66 ml (about 22 mg protein). A SEfiphadex G-200 column, 2.5 x 33 cm was poured and equilibrated with hoo ml of 0.25 M Tris—H01, pH 8.0. Two ml (about 12 mg prwotein) of the DEAE-cellulose fraction were layered onto the ccfllumn and eluted with the equilibration buffer, collecting 5 :fractions of 5 ml and then MO fractions of 2 ml with a flcrw rate of 17 ml/h. The absorbancy of the fractions at £28C) nm revealed a protein peak in fraction 20, after about 55 :ml of eluate.(Fig. 2). (The void volume of the column hami been determined to be about 50 ml by the use of Blue Deirtran 2000.) The proline dehydrogenase eluted in the trail— in€§ edge of the initial protein peak, showing a maximum ac- tinvisty in fraction 2h (Fig. 2). Fractions 22 to 29 were P0CfiLed.and concentrated by ultrafiltration to a volume of 3'8 Inl and frozen in 80 pl aliquots at -l8C. Specific activ— ities of concentrated Sephadex 0—200 fractions from different COJJdmns ranged from 0.319 to 0.337. Table 5 summarizes the Partial purification of the proline dehydrogenase. 30 Fig. 2.-- Sephadex G—200 gel filtration of DEAR-cellulose fraction. Protein (A280),(); proline dehydrogen- ase (A24143/15 min), 9. mmmzzz onHu w n GEL l Q E: GEL 2 ('3 GEL 3 :Filg. 3.--Polyacrylamide disc gel electrophoresis of the Sephadex G—200 fraction. Gel l, stained with amido black; A: RF = 0.18; B: RF = 0.3; C: dye front. Gel 2, stained with L—proline + NAD+; A: RF = 0.29; C: dye front. Gel 3, stained with L-glutamate + NAD+; A: RF = 0.0 -0.3; B: RF = 0.31; C: dye front. 35 Separation of proline dehydrogenase and glutamate de— hydrogenase by polyacrylamide gel electrophoresis: Although the proline dehydrogenase and glutamate dehydrogenase mi— grated closely together on polyacrylamide gels, an attempt 'was made to separate the two enzymes by taking very thin slices of these gels and assaying each for proline and glu- ‘tamate dehydrogenase activities. Two dialyzed h5-80% ammon- ilun sulfate preparations and the Sephadex G—200 fraction vnere electrophoresed as before, and the gels were sliced into 3 Inm sections. Each slice was put into a 13 mm tube contain- irlg 0.2 ml of 0.1 M Tris—HCl, pH 8.0. After equilibration fxpr 2 hours at AC, the slices were assayed for enzyme activi— ties. To assay for glutamate dehydrogenase, the rate of re- éhuction of 10 mM NAD+ was monitored as the increase in absor— bantny at 3h0 nm, using 5 mM L-glutamate as Substrate and 50 )fl.c>f the Tris buffer used to cover each gel slice as enzyme in a. 1 m1 total volume reaction mixture. Results showed that glutaJnate dehydrogenase activity peaked in gel slice no. 5 of the: gels used for the ammonium sulfate fractions and in gel SJLice no. 6 of the gel used for the Sephadex G—200 frac— tion (Irig. h). To assay for proline dehydrogenase, the fol— lowing :reactants were placed into each gel slice tube: 0.1 M Tris—IiCl, pH 8.0, saturated with g—aminobenzaldehyde; 0.375 M IL-proline; and 10 mM NAD+. Enzyme was the Tris solu- tion Covwering each gel, bringing each reaction mixture to 1 m1 t0138.1 ‘volume. After equilibration to 38C, L-proline was 36 added to start the reactions which were stopped after 1 h with an equal volume of 10% TCA. Proline dehydrogenase ac- tivity was measured as absorbancy to 14143 nm against a pre- acidified'control. The enzyme activity peaked in gel slice no. ’4 of the gels with the ammonium sulfate fractions and in slice no. 5 of the gel with the Sephadex G—200 fraction (Fig. 14). These data are in agreement with the positions of the enzymes on stained gels (Fig. 3) and indicate that the proline dehydrogenase and glutamate dehydrogenase are dif— ferent enzymes Since there was little or no proline dehy— drogenase activity in the gel slices which contained the glutamate dehydrogenase peaks. Separation offiproline dehydrogenase and glutamate iehydrogenase by DEAE-cellulose and hydroxylapatite chrom— l__t0graphy: A AO-liter culture of g. sporogenes was pro- duced, harvested, and sonicated as described above to yield a. crude extract. This was dialyzed, treated with strepto— mycin sulfate, fractionated with 50—80% ammonium sulfate, and'dialyzed. The specific activity of the proline dehy— drOgenase increased about lO-fold through these procedures, from 0.0028 units/mg protein in the crude extract to 0.021; umits/mg in the dialyzed ammonium sulfate fraction. The glutamate dehydrogenase in these fractions was purified 2.3 f01d from the crude extract. A DEAE-cellulose column (2.5 x A2 cm) was then poured and equilibrated with 0.15 M Tris~HCl, pH 7.5, with 10% Fig. 37 h.—-Separation of glutamate dehydrogenase (GDH) from proline dehydrogenase (PDH) by polyacrylamide disc gel electrophoresis. Reactions for GDH and PDH were as described in text. GDH from 0.A mg ammonium sulfate fraction,(>; GDH from 0.8 mg ammonium sulfate fraction,£§; GDH from Sepha— dex G—2OO fraction,U ; O, A, u, PDH from same respective fractions. 38 —~-----”-------_‘”_---“-‘---- 0‘... 39 glycerol and 2 mM dithiothreitol (DTT)- AbOUt 1-12'8 protein of the dialyzed ammonium sulfate fraction was layered onto the column, and the column was washed with the equilibra~ 'tion buffer until the A280 of the eluate was below 0.2. The jprotein remaining on the column was eluted with a linear gradient of Tris-HC1, pH 7.5, with 10% glycerol and 2 mM IHNP, from 0.15 to 0.3 M. Eighty lO—ml fractions were col— lexrted with a flow rate of 35 ml/h. The glutamate dehy— diwagenase peaked 17 fractions before the proline dehydrogen_ ases (Fig. 5). On the basis of these data, fractions A3 to 53! (Fig. 5) were pooled and concentrated by ultrafiltration tc> yield a proline dehydrogenase preparation-with a specific axrtivity of 0.133 units/mg protein. This preparation, how~ evwer, still contained a significant concentration of glu_ tamate dehydrogenase. A preliminary run demonstrated that the use of Seph_ euiex G—2OO gel filtration was of no benefit in separating tflie two dehydrOgenases. The peak activities of both enzymes eldlted together from the column. A hydroxylapatite column was prepared by suspending anlxydrous BIO'RAD HTP hydroxylapatite powder in 20 mM potas_ si1xm phosphate, pH 7.3, and packing it into a column (1.5 x 30 cm) as specified by the manufacturer. After equilibrating 'thta column with 100 ml of the same buffer, about 12.5 mg ' PIWDtein of the concentrated DEAE—cellulose fraction were lay“ eIWed onto the column and eluted in Suml fractions with a to from DhAE—cellulose. Protein (A280), 0; proline dehydrOgenase (PDH) activity in 100 pl samples, measured by the 15 min tube assay, A; glutamate dehydrogenase (GDH) activity in 5 pl samples,A. Fig. 5.-—Elution of enzymes hi 2an mOH x OOOOO OH OH ma NN HIQOV rOOH x EZO mmmzzz onHof 1.29 units/mg protein. Glutamate dehydrogenase activi- ‘ty in this preparation was 0.09 units/mg protein. Table 6 srummarizes the purification procedures used to separate pro— ILine dehydrogenase and glutamate dehydrogenase. Characterization of Proline Dehydrogenase Enzyme stability: The proline dehydrogenase protein tippears to be quite stable. The enzyme from each purifica— tixon step lost no activity for at least two months when Stnored at —180. It did gradually lose activity upon re— ?Peated thawing and refreezing. Also, as was reported for 'the pumpkin proline dehydrogenase (32), the g. sporogenes enzyme was suprisingly stable to heat up to 50C. A3 Fig. 6.—-E1ution of enzymes from hydroxylapatite. Protein (A28 ), O; proline dehydrogenase (PDH) in 100 pl samp es, measured by the 15 min tube assay,A; glutamate dehydrogenase in 5 pl samples,ll. (Hfld) 501 X SUN“ 80 (HGBYZOT X 31an 55,52 22.5%... a: N: mm em . om mm mm ma. 3.. 3.. H _ _ _ _ _ H . H . ...d/ :4: \dlldlhnlh41414nlandnl 4 {uuvdlldllddilwdso 4 l \ c r ‘u! \ c / c 4 o \ a I.-. o . .I- . 4 I... _ . z. a c c K r . _ _ _ _ _ _ _ b _ 08zV A5 Table 6.-—Partial purification of proline dehydrogenase II. Total Specific Fraction Vol. Protein activity activity Recovery (ml) (mg) (units) (units/mg) (%) Crude A9 3136 9.09 0.0029 100 £3trep. 180 2700 56.7 0.021 623 sulfate 80% amm. ‘sulfate A2 113A 27.2 0.02A 299 DEAE—cell. ll 28 3.7 0.133 A1 Hydroxyl- apatite 10 0.17 0.22 1.29 2.A A6 .Relationship between enzyme concentration and measured activity: To show the relationship between enzyme concen- tration and enzyme activity, proline dehydrogenase reac- tions were conducted with a Sephadex G—200 fraction, moni— toring the rate of reduction of NAD+ at 3A0 nm using var— ious concentrations of protein. The rate of reduction was directly dependent upon the concentration of protein in the, reaction mixture over the range tested. (Fig. 7). Effect of pH on enzyme activity: The effect of pH .on proline dehydrogenase activity was determined by conducting assays for the enzyme using 0.1 M concentrations of four buffer systems: (a) potassium phosphate; (b) Tris—H01; (c) sodium carbonate-bicarbonate; and (d) glycine—sodium hy- droxide. Enzyme activity was measured as the rate of reduc— tion of NAD+ at 3A0 nm. Maximal activity of proline dehy— drogenase occurred at pH 10 (Fig. 8). At the pH routinely used to assay for proline dehydrogenase, pH 8.0, only about 20% maximal activity was observed. At this pH, however, there was strong P5CA reductase activity which may have masked proline dehydrogenase activity. At pH 10, there was no P5CA reductase activity, possibly allowing greater ex— pression of proline dehydrogenase activity. Therefore, a PH of about 10 would be the optimal pH for assaying proline dehydrogenase lg vitro, but this pH may or may not be the Optimal pH i_ vivo for the conversion of prroline to P5CA. The reason that a pH of 8.0 was routinely used for In Fig. 7.--Correlation of enzyme concentration and measured activity. Proline dehydrogenase a tivity is given in units measured by the NAD reduction assay; enzyme was a Sephadex G—200 fraction. UNITS x 103 w M 1:8 I l l l - a .. /¢ "' o ,/ _. ./ h l 1 I 1 5 10 15 20 )JG PROTEI N/ REACTION .hg Fig. 8.——Effect of pH on proline dehydrogenase activity. Proline dehydrogenase was measured by the NAD+' reduction assay, using 9 pg protein of a Seph— adex G-2OO fraction as enzyme and 100 mM con— centrations of h buffer systems. Potassium phosphate,0; Tris—HCl, 0; sodium carbonate- bicarbonate, A; glycine—sodium hydroxide,A. 50 <1 4 <1 ‘i' <1 00 O .. l l l l 1 4h N C) 00 Lo :1- O H H ZOI x NIw/OWEV v 10 PH 51 proline dehydrogenase assays during this investigation was that initial studies of pH with Tris buffer indicated that a pH of 8.0—8.5 was more optimal than 9.0. After the work was essentially complete, references (21, 31, 32) were dis- covered regarding proline dehydrogenase in eucaryotic cells which led to the reevaluation of the pH optimum for the g. sporogenes enzyme. Effect of temperature on enzyme activity: To mea— sure the activity of proline dehydrogenase over a range of temperatures, the rate of NAD+ reduction was monitored at 3h0 nm using a Sephadex G-200 preparation as enzyme. The activity of the enzyme increased to a maximum at SOC then dropped off sharply above this temperature due to rapid in- activation of the enzyme (Fig. 9). At 370, the proline dehydrogenase showed only about 72% of maximal activity, but this temperature was used throughout to avoid any pos— sible inactivation of the enzyme by heat. Response of enzyme activity to oxygen: Experiments to determine whether or not the in_xi££g enzymatic conversion of L-proline to PSCA was sensitive to oxygen were conducted by adding reducing agents to the proline dehydrogenase reac— tion mixtures and measuring the activity of the enzyme color— imetrically as the rate of increase in absorbancy at hh3 nm. Reactions were run with 10 mM 2—mercaptoethanol, with 1 mM DTT, and with both of these, before and after flushing each reaction mixture with argon. In none of these reactions did the enzyme activity vary from the controls (without reducing 52 Fig. 9.-—Effect of temperature on proline dehydrogenase activity. Proline dehydrogenase was measured by the NAD reduction assay, using 9 pg pro- tein of a Sephadex G—200 fraction as enzyme{ 53 _ 0 .3 0 . 2 .NDH x ZHZ\osM~ Glutamic valeric acid X—semialdehyde P2CA P5CA Proline Fig. l3.——Postulated relationships of arginine, ornithine, proline, and glutamate in g. sporogenes.. Dashed arrows represent reactions not known to occur in this organism. 70 conversion of lyC-proline to glutamate. No significant label 1A from C-glutamate was found in either proline or in the ba— sic amino acids. Previous reports indicate that ornithine cannot be directly converted to glutamate (10, 23) nor to glutamic F-semialdehyde by an ornithine J—transaminase re- action (10). Therefore, it appears likely that glutamate was primarily derived from the conversion of proline. This is the most probable physiological role of proline dehy— drogenase in Q. sporogenes. Glutamate dehydrogenase, present in high levels in species of Clostridium (A9), was shown to copurify to a great extent with proline dehydrogenase in 9, sporogenes. However, the most highly purified fraction obtained from hydroxylapatite chromatography, contained only low levels of glutamate dehydrogenase, showing that these two enzyme activities were catalyzed by different proteins in g. EREE' ogenes. Non-specific glutamate dehydrogenases have been identified in other organisms (A3, A9), but Luproline has never been shown to be a suitable substrate for these en- zymes. The proline dehydrogenase activity in cell extracts of g. sporogenes is strongly inhibited by very low levels of L—glutamate (1 mM). This inhibition was shown to occur in fractions from all stages of purification. Doubling and tripling the already high (10 mM) concentrations of NAD+ in the reaction mixtures could not reverse this inhibition. 71 This indicates that the inhibition observed was not merely due to the glutamate dehydrogenase reaction competing with the proline dehydrogenase for the available NAD+. Studies on the precise mechanism of inhibition will require more highly purified proline dehydrogenase preparations. If indeed the primary role of the proline dehydrogenase is in the biosynthesis of glutamate, the glutamate inhibition observed may well be of the feedback type. Another control of proline dehydrogenase in 2. £221- ogenes appears to be catabolite repression by D—glucose. Simmons and Costilow (3A) have shown that the enzymes of the Emden—Meyerhof-Parnass pathway of glucose metabolism are induced by D—glucose. Also, Stern and Bambers (3S) dem- onstrated that in Clostridium kluyyeri,ci—ketoglutarate can be formed through a partial tricarboxylic acid cycle. 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