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

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Abstract
Glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase (deaminating); EC 1.4.1.2) was purified from P. 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 MW of 49,000 .+-. 500 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 6 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 Km for the substrates NH3, 2-oxoglutarate, NADH, NAD+ and glutamate were 18.4, 0.82, 0.066, 0.031 and 6 mM, respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength.