Characterization of Peptostreptococcus asaccharolyticus Glutamate Dehydrogenase Purified by Dye-ligand Chromatography

Department of Biochemistry, University of Sheffield, Western Bank, Sheffield S10 2TN, UK ABSTRACT Glutamate dehydrogenase ( L -glutamate: NAD + oxidoreductase (deaminating); EC 1.4.1.2 ) has been purified from Peptostreptococcus asaccharolyticus in a single step using dye-ligand chromatography. The enzyme (GDH) was present in high yields and was stabilized in crude extracts. A subunit molecular weight of 49000 ± 500 was determined by SDS polyacrylamide gel electrophoresis and six bands were obtained after cross-linking the subunits with dimethyl suberimidate. This bacterial GDH was predominantly NAD + -linked, but was able to utilize both NADP + and NADPH at 4% of the rates with NAD + and NADH, respectively. An investigation of the amino acid specificity revealed some similarities with GDH from mammalian sources and some clear differences. The values of apparent K m for the substrates ammonia, 2-oxoglutarate, NADH, NAD + and glutamate were 18.4, 0.82, 0.066, 0.031 and 6 m M , respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength. Keywords: GDH, glutamate dehydrogenase