Purification and characterization of cold-active L-glutamate dehydrogenase independent of NAD(P) and oxygen.
L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp. L101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5.7. Maximum activity was obtained at 55 degrees C and pH 8.5. The activity of GLDH at 4 and 20 degrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[alpha]phenoxazin-7-ium chloride (meldola's blue), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H tetrazolium (INT). The presence of NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.
Yamamura
A
A
School of Materials Science, Japan Advanced Institute of Science & Technology, Tatsunokuchi, Ishikawa, 923-1292, Japan. yamamura@jaist.ac.jp.
Sakaguchi
T
T
Murakami
Y
Y
Yokoyama
K
K
Tamiya
E
E
D000333:Aeromonas / Q000201:enzymology / Q000302:isolation & purification; D000818:Animals; D003080:Cold Temperature; D004795:Enzyme Stability; D005399:Fishes / Q000382:microbiology; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD; D009249:NADP; D017685:Oncorhynchus keta / Q000382:microbiology; D010100:Oxygen; D013379:Substrate Specificity; D013696:Temperature
D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase; D010100:Oxygen