Characteristics of glutamate dehydrogenase in mitochondria prepared from corn shoots

The amination of a-ketoglutarate (α-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca²⁺. The NADH-GDH purified 161-fold with ammonium sulfate, DEAE-Toyopearl, and Sephadex G-200 was also activated by Ca²⁺ in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca²⁺ had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca²⁺. About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADHGDH, Ca²⁺ had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca²⁺. In a simulated in vitro system using concentrations of 6A millimolar NAD, 0.21 millimolar NADH, 5 millimolar α-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca²⁺, 10 and 50 millimolar NH₄⁺, and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.