Properties of glutamate dehydrogenase purified from green tobacco callus tissue

Abstract

Glutamate dehydrogenase [L-glutamate : NAD(P) oxidoreductase (deaminating) EC 1.4.1.3.] has been purified from the mitochondrial fraction of green tobacco callus tissue. The enzyme was stable at −20°C for several months. The pH optimum for the amination reaction was 7.8. But the optimum for the deamination reaction was indistinct because it was in an extremely alkaline domain. Relative activities of the enzyme for amination were 50 with NADH and 10 with NADPH, and those for deamination were 5 with NAD and 1 with NADP at pH 7.9. The enzyme was inactivated by EDTA, but its activity partially restored by the addition of divalent cations such as Ca2+, Mn2+, Zn2+, Cu2+ and Mg2+. Ca2+, Mn2+ and Zn2+ activated the reductive amination 141, 122 and 39% respectively, but these divalent cations scarcely affected the oxidative deamination. Citrate and fumarate acted as inhibitors for reductive amination, and oxaloacetate for oxidative deamination of the enzyme reaction. These inhibitions were counteracted by the addition of Ca2+. ATP and ADP exerted an inhibitory effect on both directions of the enzyme reaction. The inhibitory effect was hardly prevented by the addition of AMP. Ca2+ caused considerable recovery from the inhibition of ATP and ADP. Amino acids scarcely affected the enzyme activity. Michaelis constants were 0.28 mM for NAD, 0.065 mM for NADH, 2.19 mM for a-ketoglutarate, 43.6 mM for ammonium chloride and 4.24 mM for L-glutamate.