Purification and properties of glutamate dehydrogenase from Vigna unguiculata (L.) walp.

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Abstract
Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating), EC 1.4.1.2) from 2-day-old seedlings of Vigna unguiculata was purified more than 500-fold. The enzyme gave one diffuse protein band on polyacrylamide gel electrophoresis and its subunit molecular weight was 51,000±5% as determined by sodium-dodecyl-sulfate-gel electrophoresis. The enzyme was active with NADH, NAD+ and NADPH in the ratio of 126:1:2 with pH optima of 8.0, 10.0 and 6.0 respectively. Inhibition of enzyme by EDTA was reversed by Ca2+ and Mn2+ while reduced glutathione reversed inhibition by p-hydroxymercuribenzoate. The enzyme was highly specific for L-glutamate and α-ketoglutarate showing very little or no activity with a range of other amino acids and keto acids. Values for Michaelis constants were: NAD+, 0.285 mM; NADH, 0.059 mM; α-keto-glutarate, 1.79 mM; ammonium sulphate, 28.6 mM; L-glutamate, 16.7 mM.