Characterization of mitochondrial glutamate dehydrogenase from dark-grown soybean seedlings

Purified preparations of NAD(H)-glutamate dehydrogenase (GDH, EC 1.4.1.2.) were assayed to determine the effects of mono- and divalent cations, nucleotides and select carbon compounds on NAD(H)-dependent GDH activity. The amination reaction was stimulated 2- to 17-fold by divalent cations (Ca2+ > Cd2+ > Co2+ > Mg2+ > Mn2+ > Zn2+ between 1 and 1000 mu M), but the reaction was unaffected by monovalent cations (Na+ and K+). The amination reaction was most responsive to changes in Ca2+ in a NADH-dependent manner. The addition of EDTA or EGTA nullified the stimulatory effects of Ca2+. Calmodulin alone or in combination with calmodulin antagonists did not affect the amination reaction. Divalent cations (at 1 mM) inhibited the rate of the deamination reaction by 15 to 25%, while monovalent cations had no effect. ATP inhibited the amination reaction by 10 to 60%, while ADP had little or no effect. ATP or ADP decreased the rate of the deamination reaction 23 to 60 or 20 to 38%, respectively. Many tricarboxylic acid cycle intermediates inhibited the amination reaction, 20 to 50% of the inhibition could be attributed to the chelating capacity of intermediates. Conversely, most of the carbon sources tested did not affect the deamination reaction. the only appreciable differences were increases in activity with sucrose (21%) and glucose (41%) and a decrease in activity with pyruvate (34%). Inhibitors of sulfhydryl groups were used to examine the importance of reduced thiol groups in the amination or deamination reactions. The amination was not dependent on reduced thiol groups, whereas the deamination reaction was dependent on reduced thiol groups.