Leucine Dehydrogenase of a Thermophilic Anaerobe

Agric. BioL Chern., 51 (12), 3375-3381, 1987 3375

Leucine Dehydrogenase of a Thermophilic Anaerobe, Clostridium thermoaceticum: Gene Cloning, Purification and Characterization Hiroko Shimoi, Shinji Nagata,1" Nobuyoshi Esaki, Hidehiko Tanaka and Kenji Soda* Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan Received July 30, 1987

The leucine dehydrogenase (L-leucine: NAD+ oxidoreductase, deaminating, EC 1.4. 1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzymeoverproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of46%. The enzyme ofE. coli- pICD242was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is.not inactivated by heat treatment: at pH 7.2 and 75°C for 15min; at 55°C and various pH's between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain L-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD+and NADH,respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADHis exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407 (1985)].

Leucine dehydrogenase (L-leucine: NAD+ thermophilic bacteria, we have found that oxidoreductase, deaminating, EC1.4. 1.9) cat- the enzyme occurs also in several strains of alyzes the reversible deamination of L-leucine, a moderately thermophilic anaerobe, Clostri- and several other branched-chain and straight- dium thermoacticum. Wehave chosen C. chain L-amino acids.1} The enzyme occurs thermoaceticum AN28-4, which produces mainly in Bacillus species,2) and was purified leucine dehydrogenase most abundantly, as from B. cereus,3) B. subtilis^ B. sphaericus,2) an enzyme source. We here describe the and B. stearothermophilus5) to homogeneity. cloning and expression for the gene of C. The enzyme is applicable to the produc- thermoaceticum AN28-4 enzyme, in Escheri- tion3'^ and determination^ of branched chia coii, the rapid and simple purification, chain L-amino acids, and the assay of leucine and characterization of enzyme. This is the amino peptidase (EC 3.4.ll.1).8'9) The en- first report of leucine dehydrogenase of an- zyme has an antineoplastic activity against Ehrlich's ascites carcinoma.10) Ohshima et al. aerobic bacteria. have purified leucine dehydrogenase from a MATERIALS AND METHODS thermophile, B. stearothermophilus, and shown that the enzyme is very stable.5) In Materials. NAD+ and NADH were obtained from the course of screening for the enzyme in Kojin, Tokyo, Japan; 2-oxo acids (sodium salts) from

* To whomall correspondence should be addressed. f Present address: Department of Agricultural Chemistry, Kochi University, Nangoku, Kochi 783, Japan. 3376 H. Shimoi et al.

Sigma Chemical; amino acids from Nakarai Chemicals,phenazine methosulfate, and 0.24him nitroblue tetra- Kyoto, Japan. Other chemicals were analytical grade zolium. reagents. Purification of the enzyme. The leucine dehydrogenase of Strains and media. All the C. thermoaceticum strainsB. stearothermophilus was purified as described pre- used were isolated and identified by Dr. Akihiko viously.16) The enzyme was purified from the E. coli clone Nakayama,n) Tokai Regional Fisheries Research cells as follows. The cells were desrupted by ultrasonic Laboratory, Tokyo. C. thermoaceticum AN 28-4, whichoscillation at 0°C for lO min. The extracts were dialyzed at produces leucine dehydrogenase most abundantly, was4°C for 12hr against lOmMpotassium phosphate buffer used throughout this study. The cells were grown statically(pH 7.2) containing 0.02% 2-mercaptoethanol. This buffer at 60°C for 20h under anaerobic conditions in a mediumwas used as the standard buffer. All operations were (pH 7.2) containing 1.7% BBL-Trypticase, 0.3% BBL-performed at 0° to 5°C. Phytone, 0.6% glucose, 0.25% NaCl, 0.05% sodium thio-Step I. Preparation ofa crude extract. The washedE. glycolate, 0.025% L-cystine, 0.01% Na2SO3, and 0.07%coli clone cells (about 10 g wet weight) were suspended in agar. The cells, harvested by centrifugation, were washed20 ml of the buffer and disrupted by sonication. The intact with 0.85% NaCl and then with 10mM potassium phos-cells and debris were removedby centrifugation. phate buffer (pH 7.2) containing 0.02% 2-mercapto- Step 2. Heat treatment. The pH of the supernatant ethanol. The recombinants of E. coli C600 (F~, thi-1, solutionthr- was adjusted to 5.4 with 1 Macetate buffer (pH 1, leuB6) were grown in Luria-Bertani (L broth) medium5.0). The enzymesolution was incubated at 75°Cfor at 37°C. When necessary, ampicillin (25 jug/ml) and tetra-30 min, and then cooled in ice. The precipitate formed was cycline (15 /zg/ml) were added to the medium. removed by centrifugation. Step 3. Preparative gel electrophoresis. Preparative Enzyme andprotein assays. Leucine dehydrogenase wasslab gel electrophoresis was performed as described pre- assayed at 55°C. The standard assay mixture for oxidativeviously.^ The gel was crushed with a Teflon homogenizer, deamination contained 20 iumol of l-leucine, 2.5 /mioland ofthe enzyme was extracted with the buffer, followed by NAD+, 120 /miol of glycine-KCl-KOH buffer (pH 10.5),centrifugation. and enzyme in a final volume of 1.0ml. The assay systemStep 4. Gel filtration. The enzyme was applied to two for reductive amination consisted of 10 /imol of 2-oxoiso-tandem columns of Superose 12á" (1.0x30cm), in an caproate, 0.1/onol of NADH, 750/miol of NH4C1- Pharmacia FPLC system, with 0.15m NaCl and 0.01% 2- NH4OH buffer (pH 9.5), and enzyme in a final volumemercaptoethanol of as the mobile phase at a flow rate of 1.0ml. One unit of the enzyme is defined as the amount0.4ml/min. of The active fractions were pooled and con- enzyme that catalyzes the formation of 1 ,umol of NADHcentrated by ultrafiltration with an Amicon PM10 per min in the oxidative deamination with a molar absorption coefficient for NADH (6220M^cm"1). Spe- membrane. cific activity is expressed as units per mg of protein. Pro-Ultracentrifugal analysis and molecular weight determi- tein was assayed by the method ofLowry et al.,12) withnation. eggThe purity of the enzyme and its sedimentation albumin as a standard. Protein elution patterns were es-coefficient were determined with a Spinco model E ultra- timated by the 280-nm absorption. The concentration centrifugeof in the same manner as described previous- the purified enzyme was determined with an absorptionly.1^ The molecular weight of the enzyme was also esti- £=9.\\),coefficient which {A\° was determined by the mated with Superose 12á"under the same conditions method of Perlman and Longworth.13) described above. Pig muscle lactate dehydrogenase (Mr, 142,000), bovine liver catalase (Mr, 232,000), horse Cloning of the gene for C. thermoaceticum leucine dehy-spleen ferritih (Mr, 440,000), and bovine thyroid thyro- drogenase. C. thermoaceticum AN 28-4 cells were harvest-globulin (Mr, 669,000) were used as marker proteins. ed in the late log phase (at about 20hr). They were The subunit molecular weight was determined by the incubated with lOmg/ml of lysozyme at 37°C for 30min,method of Laemmli.18) The marker proteins used were: and then the suspension was frozen and thawed repeatedlysoybean trypsin inhibitor (Mr, 20,100), carbonic anhy- twice. The chromosomal DNA (10fig), which was isolateddrase (Mr, 30,000), ovalbumin (Mr, 43,000), and bovine essentially according to the procedure of Saito and serum albumin (Mr, 67,000). Miura,14) was digested with Hindlll, and the resulting fragments were ligated into the Hindlll site of pBR322 Immunochemical analysis. Antiserum against the C. (3 fig). The recombinant E. coli library was screened thermoaceticumfor leucine dehydrogenase was obtained from the expression of the C. thermoaceticum leucine dehy-an adult male rabbit by subcutaneous injection of the drogenase gene essentially by the method of Inagaki ethomogeneous enzyme (0.5mg in lml of 0.9% NaCl) al.15) in a reaction mixture containing 50mM L-leucine,emulsified in an equal volume of Freund's complete 50mM Tris-HCl buffer (pH 9.0), 0.625mM NAD+, 64^madjuvant (Difco). Booster injections were given twice Leucine Dehydrogenase 3377

(1 mg each), after 2 and 3 weeks. Oneweek after the final it by nick translation, and then hybridized it injection, blood was collected from the heart and serum with C. thermoaceticum and E. coli C600chro- was obtained. Immunodiffusionanalyses were performed mosomal DNAsby the method of Southern.20* according to the procedure of Ouchterlony.19) The probe could hybridize only to the 5.4kb Stereochemical analysis of hydrogen transfer from C-4 of Hindlll fragment from C. thermoaceticum. the dihydronicotinamide ring of NADH. [4S-3H] NADH, This shows that the isolated DNAfragment is with a specific activity of 2.9 x 105cpm/mmol,was pre- derived from C. thermoaceticum. The extract pared by the previous method.2) Hydrogen transfer of [4S- prepared from E. coli-plCD242 cells showed 3H] NADHto 2-oxoisocaproate was examined in a reac- about 900-fold higher leucine dehydrogenase tion mixture (2 ml) containing 2 /miol of [4S-3H] NADH, 20/miol of sodium 2-oxoisocaproate, 0.3mmol of activity than that from C. thermoaceticum cells NH4C1/NH4OH buffer (pH 9.5) and 0.01 mg of leucine (Table I). As the cell growth of E. coli C600- dehydrogenase. After incubation at 30°C for 2hr, the pICD242 was much higher than that of C. reaction was stopped by adjusting the pH to 5 with 4n acetic acid. Leucine and NADHwere isolated as described thermoaceticum, the productivity of leucine previously.2) The radioactivity was measured with a dehydrogenase by E. coli-plCD242 in terms of Packard Tri-Carb 300C liquid scintillation system. unit medium volume was about 3000-fold higher than that by C. thermoaceticum. RESULTS Purification of leucine dehydrogenase from E. Cloning of the gene for C. thermoaceticum coli-pICD242 and its stability leucine dehydrogenase Leucine dehydrogenase was purified by heat The recombinant E. coli library of Hindlll treatment, which is very effective for removing fragments of C. thermoaceticum DNAwas impurities, and another two steps (Table II). screened for the expression of the gene for C. The final enzymepreparation was found to be thermoaceticum leucine dehydrogenase as de- homogeneous by the criteria of polyacryl- scribed above. The procedure is based on the amide gel electrophoresis and analytical ultra- fact that E. coli has no leucine dehydrogenase centrifugation. The enzyme can be stored in activity.2) Among750 ampicillin resistant and the standard buffer containing 0.02% sodium tetracycline sensitive colonies, 4 distinctly azide at 4°C for more than 1 year without loss showed leucine dehydrogenase activity. The of activity. The enzyme retained its full activity recombinant plasmids of these transformants of heating at 75°C for 30min, but the activity were designated as pICD241, pICD242, was substantially lost on heating at 85°C for pICD243, and pICD244. pICD242 contained 5 min. The activity was not lost on incubation a single 5.4kb Hindlll fragment, and was used between pH 6.0 to 10.0 at 55°C for 10min. for further study. To confirm that the cloned DNAfragment was derived from the C. ther- Immunochemical analysis moaceticum chromosomal DNA,we isolated Whenthe antiserum raised against leucine the HindWl fragment of pICD242, 32P-labeled dehydrogenase of E. co//-pICD242 reacted Table I. Leucine Dehydrogenase Activity in Crude Extracts of C. thermoaceticum and E. coli C600-pICD242fl

T otal Total c .,, Culture . ^. v Specific Strain ,.. protein activity . . conditions * /iZn , v ,L activity (mg/lft) (units/ F) C. thermoaceticum 60°C, 30 hr 88 0.9 0.01 E. coli C600-pICD242 37°C, 16 hr 320 3,020 9.4

The cells were disrupted by ultrasonic oscillation at 0°C for lOmin. The extracts were dialyzed at 4°C for 12 hr against 10 mMpotassium phosphate buffer (pH 7.2) containing 0.02% 2-mercaptoethanol. Values expressed per liter of the culture medium. 3378 H. Shimoi et al.

Table II. Purification of Leucine Dehydrogenase Table III. Substrate Specificity for the from E. co//-pICD242 OXIDATIVE DEAMINATION

Total Total .n ..... c u. . a Relative Km . . Specific Yield Substrate activity (mM) Steps protein/ x activity/ à".\ activity (%) (mg) (units) v/0/ L-Leucine 100 8. 5* Crude extract 520 5,000 9.6 100 L-Valine 93 6.8" Heat treatment 1 10 4,800 44 96 L-Isoleucine 88 3. 9" Preparative L-a-Aminobutyrate 1 9 1. 5C electrophoresis 25 2,800 112 56 L-Norleucine 1 5 2. 3C FPLC-Superose 20 2,300 115 46 L-Norvaline 1 1 5.6C L-Alanine 2. 8 L-Methionine 2. 6 5- Methyl-L-cysteine 2. 4 with the extract of C. thermoaceticum, a single L-Cysteine 2. 1 line of precipitation formed with complete 20mM. fusion. Nocross reaction was observed with Determined from the secondary plots of intercepts the extract of E. coll. Thus, the enzyme of E. versus reciprocal concentrations of the substrate. coli-plCD242 is immunochemically identical Determined by Lineweaver-Burk plots with the reaction system containing 2.5 mMNAD+. with that of C. thermoaceticum. The antiserum Inert: cycloleucine, L-cysteic acid, L-glutamate, l- also produced a single line of precipitation aspartate, L-threonine, L-serine, glycine, L-phenyl- with complete fusion against the B. stearother- alanine, L-tryptophan, L-proline, L-lysine, L-argi- mophilus enzyme. The same result was ob- nine, L-isoglutamate, D-leucine, D-norleucine, d- valine, L-norvaline, D-allo-isoleucine, D-a-amino- tained when the antiserum against the B. butyrate, /?-alanine, and e-amino-«-caproate. stearothermophilus enzyme reacted with C thermoaceticum enzyme. Therefore, the enzymes of C. thermoaceticum and B. stearother- threonine, L-phenylalanine, D-amino acids, mophilus are identical immunochemically. and co-amino acids were not utilized as substrates. The pH optimum for the reductive Molecular weight and subunit structure amination of 2-oxoisocaproate was 9.5 in the The sedimentation coefficient of the enzyme presence of 750him NH4C1/NH4OHbuffer. (^2o,w) was calculated to be 1 1.5 S. The molec- The 2-oxo analogues of branched-chain amino ular weight was determined as 360,000 by the acids served as good substrates for the re- sedimentation equilibrium method with the ductive amination (Table IV). The enzyme assumption that its partial specific volume is requires NAD+and NADHas coenzymes; 0.74 ml/mg, and as 350,000 by the gel filtration NADP+and NADPHare inert. Double re- method. Sodium lauryl sulfate gel electro- ciprocal plots of initial velocity against NAD+ phoresis of the enzyme gave only one band, concentration in the presence of various fixed and the molecular weight of the peptide was concentrations of L-leucine' gave intersecting estimated to be 56,000. These results show that straight lines. From the secondary plots of the the enzyme consists of six subunits identical in intercepts against the reciprocals of the fixed molecular weight. NAD+ concentrations, the Km value for NAD+was calculated to be 0.61 niM. At a high Substrate specificity and kinetics concentration of ammonia (750 mM),the dou- The enzyme showed maximumreactivity at ble reciprocal plots of the velocities against 2- pH 10.5 as to the oxidative deamination of l- oxoisocaproate concentrations at several fixed leucine, when examined in the presence of concentrations of NADHalso gave intersect- 0. 12 m glycine/KCl/KOH buffer. In addition to ing straight lines. From the secondary plots of L-leucine, L-valine and L-isoleucine were pre- the intercepts against the reciprocals of the ferred substrates (Table III). L-Glutamate, l- fixed NADHconcentrations, the Kmvalue Leucine Dehydrogenase 3379 for NADHwas calculated to be 25jum. similar to that of the B. stearothermophilus enzyme(Table V). Statistical analysis of the Stereospecificity of hydrogen transfer between amino acid compositions on a mole percent coenzymeand substrate basis by the method of Harris et al.21) showed When [4S-3H] NADH (8,100dpm, 100%) the low deviation functions (D= was oxidized enzymatically in the presence of E(^i,i-*2,i)2l1/2) between the two enzymes: 2-oxoisocaproate and ammonia, the tritium was mostly incorporated into leucine Table V. Amino Acid Compositions of (7,800dpm, 96%) but not retained substan- LEUCINE DEHYDROGENASESa tially in NAD+(460dpm, 6%). This shows C. thermoaceticum that the pro-S hydrogen at C-4 of the dihydro- B. stearo- nicotinamide ring of NADH is exclusively Amino acid no. of residues thermophilus transferred to the substrate without exchange (mol/mol of (mol%) (mol%) with protons of the medium; the enzyme is B- subunit) stereospeciflc. The same stereospecificity was Aspartic acid 55 1 1.0 1 1.8 observed for the B. sphaericus enzyme.2) Threonine* 22 4. 3 4.2 Serine" 1 9 3.7 2.7 Glutamic acid 67 13.3 1 1.5 Aminoacid composition and N-terminal amino Proline 14 2.8 2.4 acid sequence Glycine 53 10.4 10.0 The amino acid composition of the C. ther- Alanine 59 ll.7 1 1.7 moaceticumenzymewas found to be quite Half-cystinec 2 0. 38 0.3 1 Valine 30 5.9 6.6 Methionine 1 3 2.5 3.3 Table IV. Substrate Specificity for Isoleucine 36 7.2 7.8 the Reductive Amination Leucine 29 5.7 5.6 Re lative Km Tyrosined 19 3.7 4.6 Phenylalanine 1 6 3. 1 2.9 Sub strate sa activity (him) Lysine 28 5.6 5.8 2-Oxoisocaproate 1 00 0. 8b Histidine 12 2.3 2. 1 2-Oxoisovalerate 1 30 4.0* Arginine 29 5.8 5.9 2-Oxovalerate 67 1.0* Tryptophand 3 0. 58 0.43 2-Oxocaproate 44 6. 2C 2-Oxobutyrate 35 7. 3C The enzymes (15pmol), dialyzed against distilled Pyruvate 1.9 water, were hydrolyzed with 6n HC1in a Waters PICO-TAGautomatic acid hydrolysis system at 10him. 115°C for 24, 48, and 72hr. The hydrolysates were Determined from the secondary plots of intercepts dried, and the residues were dissolved in 0.2 m citrate versus reciprocal concentrations of the substrate. buffer (pH 2.2) and then analyzed with a Beckman Determined by Lineweaver-Burk plots with the 7300 high performance amino acid analyzer. reaction system containing 750mM ammonium Obtained by extrapolation to zero hydrolysis time. chloride and 0.1 mMNADH. Determined as S-carboxymethylcysteine by the Inert: 2-oxoglutarate, phenylpyruvate, oxalacetate, method of Hirs.29) Determined spectrophotometrically.30) glyoxylate, and benzylpyruvate. Table VI. N-Terminal Amino Acid Sequences of the Leucine Dehydrogenases0

1 10 C. thermoaceticum Met-Glu-Leu-Phe-Lys-Tyr-Met-Glu-Thr-Tyr- B. stearothermophilus Met-Glu-Leu-Phe-Lys-Tyr-Met-Glu-Thr-Tyr- 20 Asp-Tyr-Glu-Gln-Val-Leu-Phe-? -Gln-Asp-Lys-Glu-Ser-Asn- Asp-Tyr-Glu-Glu-Val-Leu-Phe-Cys-Glu-Asp-Lys-Glu-Ser

About 4nmol of the purified enzymes was subjected to automated Edmandegradation with an Applied Biosystems gas-phase protein sequencer 470 A equipped with an on-line PTHanalyzer 120A. 3380 H. Shimoi et al.

D=0.028. We have also observed high ho- unit structures of the three enzymes are the mology in the TV-terminal amino acid se- same (hexamers of identical peptides).2'5) We quences of the two enzymes (Table VI). have found high homology between the two thermostable leucine dehydrogenases; the DISCUSSION amino acid composition and the N-terminal amino acid sequence of the C. thermoaceticum Clostridium thermoaceticum is a moderately enzymeare closely similar to those of the B. thermophilic anaerobe, and the cells are only stearothermophilus enzyme. Moreover, both poorly produced than those of aerobic bac- the enzymes are identical immunochemically. teria. The amount of leucine dehydrogenase As shown by the molecular weights of the produced also is very low. Wehave cloned the subunits (C. thermoaceticum enzyme, 56,000; gene for leucine dehydrogenase of C. ther- B. stearothermophilus enzyme, 49,000),5) the C. moaceticumto E. coli cells to increase the thermoaceticumenzymehas extra aminoacid enzyme productivity. Based on the specific residues over the B. stearothermophilus en- activity in the extract of the recombinant cells, zyme. We are currently studying the DNA the amount of enzyme in the cells was esti- sequences of these leucine dehydrogenase mated to be about 8.3% of the soluble cellular genes to clarify the encoded primary protein protein. Thus, we have increased the pro- sequences. ductivity of the enzyme by 900-fold over that of the wild-type cells by means of gene cloning. Acknowledgments. We wish to thank Dr. Akihiko The enzyme purified to homogeneity from the Nakayama, Tokai Regional Fisheries Research clone cells was immunologically identical with Laboratory, Tokyo, for generously giving us the strains of that of C. thermoaceticum. C. thermoaceticum and the helpful suggestion. Weare also grateful to Dr. Seiki Kuramitsu and Professor Hiroshi The E. coli clone cells expressing the gene Kagamiyama, Osaka Medical College, for the peptide for a thermostable enzymeare very useful for sequence analyses. the rapid effective purification of the enzyme by heat treatment. A few thermostable en- REFERENCES zymes have been purified from clone cells by heat treatment: 3-isopropylmalate dehydro- 1) B. D. Sanwal and M. W. Zink, Arch. Biochem. genase of Thermus thermophilus,22'23) alanine Biophys., 94, 430 (1961). 2) T. Ohshima, H. Misono and K. Soda, /. Biol. Chem., racemase15) and alanine dehydrogenase240 of 253, 5719 (1978). B. stearothermophilus. Wehere have shown 3) H. Schiitte, W. Hummel, H. Tsai and M.-R. Kula, Appl. Microbiol. Biotechnol, 22, 306 (1985). that leucine dehydrogenase was purified sev- 4) J. Hermier, J. M. Lebeault and C. Zevaco, Bull. Soc. eral times from the recombinant cells by heat Chim. Biol, 52, 1089 (1970). treatment in a high yield. The enzyme was 5) T. Ohshima, S. Nagata and K. Soda, Arch. purified to homogeneity by the subsequent Microbiol, 141, 407 (1985). preparative gel electrophoresis. The C. ther- 6) R. Wichmann, C. Wandrey, A. F. Biickmann and moaceticum enzyme is stable enough to be M.-R. Kula, Biotechnol. Bioeng., 23, 2789 (1981). 7) T. Ohshima, T. Yamamoto, H. Misono and K. Soda, electrophoresed for a long time with little loss Agric. Biol. Chem., 42, 1739 (1978). of activity. 8) S. Takamiya, T. Ohshima, K. Tanizawa and K. Alanine dehydrogenases from thermophiles Soda, Agric. Biol. Chem., 47, 893 (1983). have higher molecular weights than those from 9) S. Takamiya, T. Ohshima, K. Tanizawa and K. mesophiles.25~28) This is also the case for Soda, Anal Biochem., 130, 266 (1983). leucine dehydrogenases; the molecular weights 10) T. Oki, M. Shirai, M. Ohshima, T. Yamamoto and of the C. thermoaceticum enzyme (350,000) K. Soda, FEBS Lett., 33, 286 (1973). ll) A. Nakayama, H. Kadota and J. Sonobe, /. Food and the B. stearothermophilus enzyme Hyg. Soc. Japan, 24, 297 (1984). (300,000) are higher than that of the B. 12) O. H. Lowry,N.J. Rosebrough,A. L. FarrandR.J. sphaericus enzyme. (245,000), although the sub- Randall, /. Biol Chem., 193, 265 (1951). Leucine Dehydrogenase 3381

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