But Glutamate Dehydrogenase
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Agric. Biol Chem., 48 (7), 1713-1719, 1984 1713 Purification and Properties of a-Hydroxyglutarate Dehydrogenase of Peptococcus aerogenes^ Shigeki Otawara,* Toshihisa Ohshima,** Nobuyoshi Esaki and Kenji Soda Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan Received October 21, 1983 a-Hydroxyglutarate dehydrogenase (NAD+ specific) of Peptococcus aerogenes was purified by manganese chloride treatment, ammoniumsulfate fractionation and chromatographies with DEAE-cellulose, hydroxyapatite and' Sephadex G-200, and then crystallized in a colorless thin plate form by the addition of ammoniumsulfate. The enzyme has a molecular weight of approximately 53,000 and consists of two subunits identical in molecular weight (about 32,000). The isoelectric point of the enzyme is pH 3.7±0.1. The enzymeacts almost exclusively on a-ketoglutarate and a- hydroxyglutarate. The Michaelis constants for a-ketoglutarate, NADH,a-hydroxyglutarate and NAD+are 0.12, 0.028, 0.67 and 0.077him, respectively. Peptococcus aerogenes cells grown in a used for the assay of aspartate aminotrans- glutamate medium, which metabolize gluta- ferase in clinical analysis and related fields.7) mate to butyrate, acetate, CO2, and NH3 through a-hydroxyglutarate,1} produce abun- MATERIALS AND METHODS dant glutamate dehydrogenase1^ (EC 1.4.1.2) and a-hydroxyglutarate dehydro- Materials. DEAE-cellulose was obtained from Brown, genase (EC class 1.1.1).2~4) Acidaminococcus is hydroxyapatite from Seikagaku Kogyo, Sephadex G-200 also a good producer of both enzymes.6) from Pharmacia Fine Chemicals, D,L-a-hydroxyglutarate, a-chymotrypsinogen A, yeast alcohol dehydrogenase, Various other microorganisms tested so far do catalase, trypsin and equine skeletal muscle myoglobin not have a-hydroxyglutarate dehydrogenase from Sigma, and bovine liver glutamate dehydrogenase, but glutamate dehydrogenase. bovine heart lactate dehydrogenase and pyruvate ki- a-Hydroxyglutarate dehydrogenase was pu- nase from Boehringer Mannheim.Other chemicals were rified from Peptococcus aerogenes by Lerud purchased from Nakarai Chemicals and WakoChemicals. and Whitely.1* The purification procedure, Culture conditions. Peptococcus aerogenes ATCC14963 which involves a sucrose gradient fraction- was used throughout, and grown in a medium (100 liters) ation, is tedious and not applicable to large containing 0.4% monosodium glutamate, 1% yeast ex- scale purification. tract, 2% peptone, 0.1% glucose, 0.5% NaCl, 0.25% Wehere report an improved purification K2HPO4 and 0.1% sodium thioglycolate (pH 7.2) at 35~37°C for about two days without aeration in a 130 method for a-hydroxyglutarate dehydrogenase liter fermenter (New Brunswick Scientific Co., Inc.). The and enzymological characterization of the en- cells harvested were suspended in about 1 liter of 20mM zyme. The method is useful for preparing potassium phosphate buffer (pH 7.0), and disrupted with a plenty of the purified enzyme, which can be Dynomill (flow speed, 1 liter/hr). The supernatant fluid A part of this work was presented at the Annual Meeting of the Agricultural Chemical Society of Japan (Nagoya), April, 1978 (The Proceedings, p. 323). * Present address: Research Center, Toyobo Co., Ltd., Katata 2-1, Otsu 520-02, Japan. ** Present address: Department of Chemistry, Kyoto University of Education, Fushimi-ku, Kyoto 612, Japan. 1714 S. Otawara et al: obtained by centrifugation at 12,000 x g for 60min was recorded at intervals of 1 hr to compare and makesure the used as the crude enzyme after dialysis against the equilibrium was established. same buffer. Isoelectric focusing. Isoelectric focusing on 7.5% acryl- Enzymeassays. a-Hydroxyglutarate dehydrogenase ac- amide gels containing 2% ampholyte and 10% glycerol tivity was determined spectrophotometrically by following was done according to the procedure of Wrigley,12>13) changes in the absorbance at 340nmat 25°C with a Union using carrier ampholytes in the pHrange 3.5 to 7.0. The SM401 Spectrophotometer. The reaction mixture for the gels were focused at 4°C, initially at 1 mA/tube until 100 V a-ketoglutarate reduction contained 0. 1 m Tris-HCl buffer and continued for 1 hr at aconstant 100V, followed by 3 hr (pH 8.5), 3mM monosodium a-ketoglutarate, 0.2mM at constant 200V. Equine skeletal muscle myoglobinwas NADH and enzyme in a total volume of 3.05ml. The used as a marker protein. Each gel was sliced into 3mm reaction mixture for the dehydrogenation contained 0. 1 m sections, eluted into 0.5ml of deionized, distilled and Tris-HCl buffer (pH 8.8), 3mM D-a-hydroxyglutarate, degassed water, and the pHof the eluate was measuredat 0.7mMNAD+and enzyme in a total volume of 3.05ml. 4°C. The enzymeactivity in gel slices was determined after The enzymewas replaced by water for a blank. Oneunit of standing for 12 hr in 0.1 m potassium phosphate buffer (pH enzyme is defined as the amount of enzyme which cat- 7.0). alyses the degradation of 1 /zmol ofNADHper min in the a-ketoglutarate reduction. Specific activity is expressed as RESULTS units per mgof protein. Protein concentration was de- termined by measurement of the absorbance at 280nm, Purification of the enzyme using an E\0/°m value of 10 obtained by measurements of The purification of the enzyme was per- dry weight and absorbance. formed at 0~ 5°C, unless otherwise specified. Polyacrylamide gel electrophoresis. Analytical gel elec- Manganese chloride was added gradually to trophoresis was carried out by the method ofDavis.8) The the crude enzyme solution (final concentration enzymeactivity in the gel was localized by coupling the of MnCl2; 0.7%). The precipitate was removed activity to the reduction ofnitro-tetrazolium blue. The gels by centrifugation. Ammonium sulfate was were incubated at 37°C for 15min in a reaction mixture containing 0.1m Tris-HCl buffer (pH 8.8), 2mM D-a- added to the supernatant solution to give 35% hydroxyglutarate, 0.2mM NAD+, 0.002% phenazine saturation. After centrifugation, (NH4)2SO4 methosulfate and 0.04% nitro-tetrazolium blue. was added to the supernatant solution (70% The sodium dodecyl sulfate gel electrophoresis was saturation). The precipitate was dissolved in carried out according to the method of Weber and 10mMpotassium phosphate buffer (pH 7.0). Osborn.9) The molecular weights of marker proteins are: catalase, 60,000; pyruvate kinase, 57,000; bovine liver The enzyme solution was dialyzed against 100 glutamate dehydrogenase, 53,000; ovalbumin, 43,000; volumes of the same buffer, and applied to a yeast alcohol dehydrogenase, 37,000; bovine heart lactate column (6x53cm) of DEAE-cellulose. dehydrogenase, 36,000; a-chymotrypsinogen, 25,700; and Elution was achieved with 20mMpotassium trypsin, 23,300. phosphate buffer (pH 7.0) supplemented with KC1. The concentrations of KC1 were in- Determination of sedimentation constant. The sedimen- creased stepwise. a-Hydroxyglutarate dehy- tation constant was determined with a Spinco Model E analytical ultracentrifuge equipped with a phase plate as a drogenase was eluted with the buffer contain- Schlieren diaphragm. The top speed of ultracentrifu- ing 0.23m KC1, and almost completely sepa- gation was 59,780 rpm.10) rated from glutamate dehydrogenase, which was eluted with the buffer containing 0.27m Molecular weight measurements. The molecular weight of the enzyme was determined by the sedimentation KC1.The active fractions were collected and equilibrium method according to the procedure of Van the enzymewas precipitated by 70%saturated Hold and Baldwin.11} The measurements were made in a (NH4)2SO4. After the precipitate was dissolved Spinco Model E ultracentrifuge equipped with Rayleigh in 10 mMpotassium phosphate buffer (pH 6.8) interference optics. Multicell operation was employed in order to perform the experiment on three samples of and dialyzed against the same buffer, the en- different initial concentrations ranging from 0.95 to zymesolution wasapplied to a hydroxyapatite 2.71 mg/ml with the use of An-G rotor and double sector column (4.4 x 33 cm), previously equilibrated cells of different side-wedge angles. The rotor was centri- with the buffer. The enzymewas eluted with fuged at 12,590rpm at 20°C. Interference patterns were the 50mMbuffer (pH 6.8) and separated from Bacterial a-Hydroxyglutarate Dehydrogenase 1715 Table I. Purification of a-HYDRoxYGLUTARATEDehydrogenase Volume T°tal Totalfl SPecific Yield Steps protein activity activity 1 } (g) (103 units) (units/mg) (/o) Crude extract 2130 133 367 2.8 100 Mn+ + treatment 1980 46.9 275 5.9 74.8 (NH4)2SO4 fractionation 150 15.2 266 17.5 72.3 DEAE-cellulose 240 3.38 1 39 41.2 37.8 Hydroxyapatite 57 0.34 10 1 298 27.4 Sephadex G-200 205 0.080 88.5 1 100 24. 1 The activity was measured by a-ketoglutarate reduction. Fig. 2. Photomicrograph of Crystalline a-Hydroxy- (A) (B) (C) glutarate Dehydrogenase. Fig. 1. Polyacrylamide Gel Electrophoresis and Sedi- mentation Analyses of Purified a-Hydroxyglutarate De- nydrogenase. with an over-all yield of about 24%. It can be stored as a suspension in 70%-saturated A: The purified enzyme was subjected to electrophoresis at a current of 2mA. B: The enzyme incubated with 1% (NH4)2SO4 for a few months with little loss of sodium dodecyl sulfate and 1% 2-mercaptoethanol at activity. 37°C for 12hr was subjected to electrophoresis in the presence of 0.1% sodium dodecyl sulfate at a current of Purity and absorption spectrum 8mAper tube. The gels were stained with Coomassie Brilliant Blue R-250. C: Sedimentation analyses were The purity of the enzymewas examined by polyacrylamide gel electrophoresis (Fig. 1A) performed as described in Materials and Methods. The enzyme concentrations were 2.36mg/ml (upper pattern) and ultracentrifugation (Fig. 1C). The enzyme and 7.85 mg/ml (lower pattern). The pictures were taken at was found to be homogeneous. 94min after achieving top speed (59,780 rpm). The enzyme was crystallized in a colorless thin plate form by addition of (NH4)2SO4 (Fig.