A. Yamamura et al., Purification and characterization of cold-active L-glutamate dehydrogenaseindependent of NAD(P) and oxygen, J BIOCHEM, 125(4), 1999, pp. 760-769
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 b
y a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sep
harose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determ
ined 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 d
egrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was co
upled to cytochrome c and several redox dyes including 1-methoxy-5-methylph
enazinium methylsulfate (l-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; NET), and 2-(4-iod
ophenyl) -3-(4-nitrophenyl) -5-phenyl-2H tetrazolium (INT). The presence of
NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profil
e and ICP data indicated ab-type cytochrome containing iron.