REVERSIBLE INACTIVATION OF GLUTAMATE DEHYDROGENASE IN BACTEROIDES FRAGILIS: PURIFICATION AND CHARACTERIZATION OF HIGH ACTIVITY- AND LOW ACTIVITY-ENZYMES

Abstract

The NAD(P)⁺-specific glutamate dehydrogenase (GDH) in the strictly anaerobic bacterium Bacteroides fragilis is reversibly inactivated depending on ammonia concentrations in the culture (YAMAMOTO et al., J. Gen. Microbiol., 133, 2773 (1987)). A high-activity form of GDH was purified to homogeneity from B. fragilis grown on 1mM ammonia, and a low-activity form from the cells exposed to 50mM ammonia immediately
before harvesting the ammonia-limited culture. The specific activities of the high- and low-activity forms were respectively 32.3 and 5.9 μmol/min/mg protein in NADP⁺-dependent deamination. The molecular weight of each form of GDH was 290, 000 daltons. The weight of the subunit was 49, 000 daltons. The isoelectric point was pH 4.5 in each form of GDH. There was no difference in the amino acid composition of the two forms. The optimum pH and the K_m values for ammonia, 2- oxoglutarate, and L-glutamate were almost the same in the two forms of GDH. The two GDHs had different affinities for coenzymes; the K_m values for coenzymes were slightly different in the two forms, and they were eluted by NaCl at different concentrations in affinity chromatography on Reactive red-agarose.