JOURNAL OF BACTERIOLOGY, June 1975, p. 1257-1264 Copyright © 1975 American Society for Microbiology

Vol. 122, No. 3

Printed in U.S.A.

Purification and Properties of Glutamate Dehydrogenase from a Thermophilic Bacillus ISRAEL EPSTEIN AND NATHAN GROSSOWICZ* Department of Bacteriology, The Hebrew University-Hadassah Medical School, Jerusakle, Israel Received for publication 5 March 1975

A 250- to 300-fold purification of a nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (GDH, E.C. 1.4.1.4) with a yield of 60% from a thermophilic bacillus is described. More than one NADP-specific GDH was detected by polyacrylamide gel electrophoresis. The enzyme is of high molecular weight (approximately 2 x 106), similar to that of the beef and frog liver GDH. The pI of the thermophilic GDH is at pH 5.24. The enzyme is highly thermostable at the pH range of 5.8 to 9.0. The purified GDH, unlike the crude enzyme, was very labile at subzero temperatures. An unidentified factor(s) from the crude cell-free extract prevented the inactivation of the purified GDH at -70 C. Various reactants of the GDH system and D-glutamate also protected, to some extent, the enzyme from inactivation at -70 C. From the Michaelis constants for glutamate (1.1 x 10-2 M), NADP (3 x 10-4 M), ammonia (2.1 x 10-2 M), a-ketoglutarate (1.3 x 10- M), and reduced NADP (5.3 x 10-5 M), it is suggested that the enzyme catalyzes in vivo the formation of glutamate from ammonia and a-ketoglutarate. The amination of a-ketoglutarate and deamination of glutamate by the thermophilic GDH are optimal at the pH values of 7.2 and 8.4, respectively.

Glutamate dehydrogenase [L-glutamateNAD(P) oxidoreductase; GDH; E.C. 1.4.1.2-4] catalyzes the reversible reductive amination of a-ketoglutaric acid to glutamic acid. Three different glutamate dehydrogenases have been described: a nicotinamide adenine dinucleotide (NAD) -specific enzyme (E. C. 1.4.1.2), a NADPspecific enzyme (E.C. 1.4.1.4); and a NAD(P)dependent enzyme (E.C. 1.4.1.3). The physiological importance of the enzyme as a link between amino acid and energy metabolism is well documented. In prototrophic microorganisms, GDH catalyzes the conversion of ammonia into the a-amino nitrogen. In various animal tissues (4, 8, 15) only one type of GDH has been demonstrated (E.C. 1.4.1.3). In fungi (3, 6, 11, 16), as well as in a few bacterial species (10, 12), two distinct NADand NADP-dependent enzymes have been found. In the majority of bacteria studied only one GDH, usually the NADP-specific enzyme (9, 17, 19), was found. On the other hand, the anaerobic Rhodospirillum rubrum (2) and certain clostridia (20) possess a NAD-specific GDH. This communication describes the purification and properties of a NADP-specific GDH from a thermophilic bacillus (7).

MATERIALS AND METHODS Cultivation of the microorganism. The previously described (7) thermophilic bacillus was grown in a minimal salt medium (7) in which the glucose was substituted by succinic acid (0.5%). Starter cultures were grown in 2-liter Fernbach flasks containing 1 liter of the minimal medium and were incubated on a rotary shaker for 18 h at 58 C, the optimal temperature for growth (7). For large scale purification of the enzyme, the cells were grown in a fermentor containing 500 liters of the succinate minimal medium. Succinate (pH 7.2) was sterilized separately and added aseptically to the sterile medium. The temperature was adjusted to 58 C, and the medium was inoculated with a starter of 2 to 3%. Aeration of the culture was achieved by an air flow at a rate of 0.5 volume/volume per min, and agitation was at a speed of 300 rpm. The pH of the culture was maintained at 7.0 by the addition of either succinic acid or H2SO4. Growth was measured with a Klett-Summerson colorimeter at 420 nm. The cells were harvested (upon reaching a turbidity of 1,000 Klett units) in a Sharples centrifuge at 10 C, and the packed cells were kept at -70 C. Yields of 5 g (wet wt) per liter of culture were obtained. Preparation of the cell-free extract. The frozen cells were taken up in 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 8.0) containing 1 x 10-2 M 2-mercaptoethanol to obtain a 20% suspension (wet wt/vol). Lysozyme (100

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mg/ml, Sigma) was added to the suspension and incubated for 60 min at 37 C with mild agitation. MgCl2 .6H2O (1 x 10-2 M) and deoxyribonuclease (1 Mg/ml, Sigma) were then added, and incubation was continued for another 30 min. Cell debris was removed by centrifugation at 4 C at 15,000 x g for 30 min. The crude cell-free extract was used for enzyme purification. Addition of 2-mercaptoethanol preserved the GDH activity during storage; its removal by dialysis decreased the activity by 40% within 48 h. Protein determination. Protein was determined by the method of Lowry et al. (14) after prior removal of the 2-mercaptoethanol by dialysis; crystalline bovine serum albumin (salt- and lipid-free, Sigma) was used as standard. Isoelectric focusing. Isoelectric focusing was carried out with a LKB 8101 column according to the manufacturer's recommended technique. Ampholine carrier ampholytes (5% final concentration) at the pH range of 3.5 to 10.0 (preliminary run) and 4.0 to 6.0 (final run) were used. At the end of the run (72 h at a constant voltage of 500 V at 8 C), 2-ml fractions were collected and the following determinations were done on each: pH, GDH activity, and sucrose concentration (by measuring the refractive index). GDH activity. Activity of GDH was assayed by recording the changes in absorbance at 340 nm due to the oxidation of NADPH or reduction of NADP (and using a Perkin-Elmer model 137 ultraviolet doublebeam spectrophotometer). Unless otherwise specified, 0.9 ml of the assay solution for reductive amination contained (in Mmol/0.9 ml): Tris-hydrochloride buffer (pH 7.2), 50; (NH4)2SO4, 150; a-ketoglutarate, 50; and NADPH, 0.15. For oxidative deamination, the reaction mixture contained (,umol/0.9 ml): Trishydrochloride buffer (pH 8.4), 50; glutamate, 50; and NADP, 0.33. After equilibration at 55 C, 0.1 ml of the enzyme was added to start the reaction. In the reference beam, the substrate (glutamate or a-ketoglutarate) was omitted from the reaction mixture. One GDH unit catalyzes the oxidation of 1 ,mol of NADPH per min at 55 C under the assay conditions. Specific activities are given in units per milligram of protein. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was carried out at pH 8.4 by the method of Davis (5). All runs were made at 10 C in an apparatus equipped with an insulated circulating pump. The electrode solution was run into a container immersed in a bath of cold water (4 C). A constant current of 5 mA per tube (400 V) was added and run for 3 h until the reference dye migrated approximately 5 mm from the end of the tube (anode). Protein samples (200,ug in 20 gl) were loaded on top (cathode) of the gel (5% acrylamide and 0.15% methylene bisacrylamide in Tris buffer, pH 8.4). Gels were stained for protein at 55 C for 30 min with a 0.1% solution of Coomassie brilliant blue containing ethanol (25%) and glacial acetic acid (10%). Destaining (at 55 C) of the gels was achieved with a solution containing ethanol (25%) and glacial acetic acid (10%). Location of the GDH in the gels was revealed by staining for 30 min at 55 C. The staining solution contained (per ml): Tris-hydrochloride buffer (pH 8.0), 50 ,umol; glutamate, 30 ,mol; NADP, 400 gg;

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phenazine methosulfate, 4 ug; nitro blue tetrazolium, 400 jg. The stained gels were preserved in 10% glacial acetic acid. Preparation of crude GDH from Escherichia coli B. Cells of Escherichia coli B were grown at 37 C on a rotary shaker in the same minimal medium used for the thermophilic bacillus; the cells were harvested by centrifugation upon reaching a turbidity of 400 Klett units (at 420 -nm) and washed twice with 20 mM potassium phosphate buffer (pH 7.0) containing 10 mM 2-mercaptoethanol. Finally, the cells were resuspended in the same buffer and disintegrated in a 60W M.S.E. ultrasonic disintegrator (three 1-min pulses at 4 C); debris was removed by centrifugation, and the supernatant fluid was used as enzyme source. Determination of heat stability of the GDH enzymes from thermophilic and mesophilic bacteria. The various enzyme preparations were incubated at temperatures and times as indicated. At intervals, samples were withdrawn and cooled rapidly to 4 C, the pH was adjusted to 7.2, and the activity was determined. The same procedure was used to assay both enzymes from the thermophilic organism and E. coli except for the temperature which was 55 and 37 C, respectively. The effect of pH on the heat stability of the purified thermophilic GDH was tested using HEPES (N-2hydroxyethylpiperazine-N'-2'-ethanesulfonic acid) and PIPES [piperazine-N-N'-bis(2-ethanesulfonic acid) ]buffers (20 ,umol each) and titrated with KOH to the desired pH, between 4.7 and 8.3. All pH values were determined at 25 C. They were, however, corrected for the effect of temperature on the pH. For example, a buffer solution of a pH value of 6.2 at 25 C gave a value of 5.5 when measured at 75 C. The proper pH of the enzyme was obtained after equilibrium dialysis with the buffer to be tested.

RESULTS Purification of GDH from thermophilic bacillus. The following procedure was employed to purify GDH from thermophilic bacillus. Step 1: (NHJ2SO4 precipitation. The crude extract (10 mg of protein/ml, see above) was brought to pH 6.7 with KH2PO4, 0.01 M final concentration. Solid (NH),2SO4 was added with mild stirring at 35 C until 35% saturation was reached (calculated at 20 C). Stirring was continued for 30 min, and the precipitate formed was discarded (by centrifugation at 5,000 x g for 30 min). The supernatant was cooled to 4 C, the pH was readjusted to pH 6.7, and additional (NH;)2SO4 was added to achieve 45% saturation; the precipitate was discarded. The (NH4)2S04 concentration of the supernatant was increased to 65% saturation and the precipitate (45 to 65% saturation) was collected and dissolved in 0.02 M potassium phosphate buffer (pH 7.0) to a protein concentration of approximately 10 mg/ml. A 100% recovery was obtained (Table 1).

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PURIFICATION OF GLUTAMATE DEHYDROGENASE TABLE 1. Purification of GDH from a thermophilic bacillus b

Total activity Fractionation procedure

Crude extract Ammonium sulfate precipitate between 45-60% saturation Acetone precipitate between 35-45% DEAE-cellulose chromatography eluted at 0.12 M NaCl 2nd Ammonium sulfate precipitation between 45-55% 2nd Acetone precipitation between 55-60%

(units)a

Purification

Recovery (%)

Sp act

(fold)

2,845 2,935

0.13 0.33

2.54

2,915 1,982

5.28 27.6

40.6 212.0

104 70

1,820

38.3

294.6

64

1,564

46.2

353.0

55

aA unit is the amount of enzyme which catalyzes the formation of 1 conditions of the assay. b Specific activity is defined as GDH units per milligram of protein.

Step 2: acetone precipitation. The enzyme solution from step 1 was cooled to 2 C, and cold acetone (-70 C) containing 2-mercaptoethanol (10 mM) was added slowly with constant stirring to a concentration of 35%. The precipitate was allowed to settle for 30 min and centrifuged (15,000 x g at -2 C for 30 min). To the supernatant, more acetone was added slowly to a concentration of 45%. The precipitate, which contained all the GDH activity (Table 1), was resuspended in 0.02 M potassium phosphate buffer (pH 7.0) and dialyzed overnight at 4 C against the same buffer. A 16-fold purification was achieved by this step; despite the fact that not all the material went into solution, the recovery of GDH activity in the supernatant was again above 100% (Table 1). Step 3: DEAE-cellulose column fractionation. The dialyzed preparation from step 2 was loaded upon a column (2.6 by 100 cm) of DEAE-cellulose (DE-52 Whatman) equilibrated with 20 mM potassium phosphate buffer (pH 7.0). The material was eluted using 1-liter batches of the same buffer containing the following concentrations of NaCl (mM): 50, 80, 120, 180, 250, and 350. the absorbance of the effluent solution was monitored with a LKB Uvicord at 256 nm. The bulk of the GDH activity (about 70%) was eluted with 120 mM NaCl; some additional GDH activity was found in the fractions eluted at 180 and 250 mM NaCl (Fig. 1). The GDH activity recovered amounted to 90%. Only the major GDH activity peak (120 mM NaCl effluent) was taken for further purification. Step 4: second (NH4)2SO4 precipitation. Solid (NH4)2S04 was added to the effluent containing the material from step 3 without dialysis; no precipitate was formed until 45% saturation was reached. Most of the GDH activity was precipitated between 45 to 55%

1

100 105

rmol of glutamate per min under the

saturation of (NH4)2SO4. The precipitate was dissolved in 20 mM potassium phosphate buffer (pH 7.0) to a protein concentration of approximately 10 mg/ml and dialyzed for 24 h at 4 C against the same buffer. Step 5: second acetone precipitation. The material of the previous step was precipitated with acetone at 55 to 60% final concentration. Fractions up to 55% acetone showed no activity. Polyacrylamide gel electrophoresis. Homogeneity of the GDH preparation (after step 5) was tested using polyacrylamide gel electrophoresis. As much as 200 ,g of protein was applied to each gel. Different acrylamide concentrations (from 5 to 10%) with the same degree of cross-linkage were used. All protein bands (stained with Coomassie blue) coincided with those showing GDH activity, and no bands appeared when stained in the absence of glutamate or NADP (Fig. 2). The migration characteristics of the enzyme indicated very high molecular weight. After 4 h in 10% acrylamide, the enzyme migrated only 2 mm toward the anode (data not shown), whereas it moved as much as 40 mm in 5% acrylamide. Isoelectric focusing of the purified enzyme. Electrofocusing of GDH was carried out using ampholine solution in the range of pH between 3.5 to 10. A total of 516 U with a specific activity of 45.5 ,umol of protein per mg per min was loaded onto the column. After 72 h, 2-ml fractions were collected, and both the pH and activity were determined. GDH activity was obtained at the pH range between 4.76 and 5.91; maximal activity was found at pH 5.24. The active fractions were pooled and rerun in the presence of 5% ampholine at pH values between 4.0 and 6.0. The results are shown in Fig. 3, and as evident the pI of the GDH was at pH 5.24. Gel filtration of the GDH enzyme. The crude enzyme was loaded onto a Sephadex

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FIG. 6. Kinetics of glutamate (A) deamination and NADP reduction (B) by purified GDH at 55 C. Lineweaver-Burk plot. The reaction mixture contained (umoles): Tris buffer (pH 8.2). 50; NADP, 0.3; and different concentrations of L-glutamic acid (A), and for determination of the NADP it contained Tris buffer (pH 8.2); L-glutamic acid, 50; and different concentrations of NADP.

for glutamate (1.1 x 10-2 M) versus a-ketoglutarate (1.3 x 10-3M) and NH4+ (2.1 x 10-2M) and NADP (3 x 10- M) versus NADPH (5.3 x 10--5 M). ACKNOWLEDGMENT This research was supported by research grant 015-0149 from the Joint Research Fund of the Hebrew University and Hadassah. LITERATURE CITED 1. Arkin, H., and N. Grossowicz. 1970. Inhibition by Dglutamate of' growth and glutamate dehydrogenase

activity of Neurospora crassa. J. Gen. Microbiol. 61:255-261. 2. Bachofen, R., and H. Neeracher. 1968. Glutamat-dehy-

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drogenase in photosynthetischen bacterium Rhodospirillum rubrum. Arch. Microbiol. 60:235-245. 3. Barratt, R. W., and W. N. Strickland. 1963. Purif'ication and characterization of a TPN-specific glutamic acid dehydrogenase from Neurospora crassa. Arch. Biochem. Biophys. 102:66-76. 4. Corman, L., L. M. Prescott, and N. 0. Kaplan. 1967. Purification and kinetic characteristics of' dogfish liver glutamate dehydrogenase. J. Biol. Chem. 242:1383-1390. 5. Davis, B. J. 1964. Disc electrophoresis. II. Methods and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:407-427. 6. Dennen, D. W., and D. J. Niederpruem. 1965. Control of glutamate dehvdrogenases in the basidiomycete Schizophyllum commune. Life Sci. 4:93-98. 7. Epstein. I., and N. Grossowicz. 1969. Prototrophic thermophilic bacillus: isolation, properties, and kinetics of' growth. J. Bacteriol. 99:414-417. 8. Frieden, C. 1962. The molecular weight of chicken liver glutamate dehydrogenase. Biochim. Biophys. Acta 62:421-423. 9. Hooper, A. B., J. Hansen, and R. Bell. 1967. Characterization of glutamate dehvdrogenase from the ammoniaoxidizing chemoautotroph Nitrosomonas europaea. J. Biol. Chem. 242:288-296. 10. Joseph, A. A., and R. L. Wixom. 1970. Ammonia incorporation in Hvdrogenomonas eutropha. Biochim. Biophys. Acta 201:295-299. 11. Kato, K., S. Koike, K. Yamada, H. Yamada, and S. Tanaka. 1962. Di- and tri-phosphopyridine nucleotide linked glutamate dehydrogenases of Piricularia orvzae and their behaviors in glutamate media. Arch. Biochem. Biophys. 98:346-347. 12. Le John, H. B., and B. E. McCrea. 1968. Evidence for two species of glutamate dehydrogenase in Thiobacillus novellus. J. Bacteriol. 95:87-94. 13. Lineweaver, H., and D. Burk. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56:658-666. 14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-27-5. 15. Olson, J. A., and C. B. Anfinsen. 1952. The crvstallization and characterization of L-glutamic acid dehvdrogenase. J. Biol. Chem. 197:67-79. 16. Sanwal. B. D., and M. Lata. 1961. The occurrence of two different glutamate dehydrogenases in Neurospora. Can. J. Microbiol. 7:319-328. 17. Shiio, I., and H. Ozaki. 1970. Regulation of' nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Brevibacterium flavum, a glutamate-producing bacterium. J. Biochem. (Tokyo) 68:633-647. 18. Sund, H., and W. Burchard. 1968. Sedimentation coefficient and molecular weight of beef liver glutamate dehydrogenase at the microgram and milligram level. Eur. J. Biochem. 6:202-206. 19. Varrichio, F. 1969. Control of' glutamate dehydrogenase synthesis in Escherichia coli. Biochim. Biophvs. Acta 177:560-564. 20. Winnacker, E. L., and H. A. Barker. 1970. Purification and properties of NAD-dependent glutamate dehydrogenase from Clostridium SB4. Biochim. Biophys. Acta 212:225-242.