Glutamate Dehydrogenase of Lupin Nodules: Purification and Properties

Glutamate dehydrogenase, GDH (L-glutamate:NAD+ oxidoreductase (deaminating) EC 1.4.1.2) was purified from the plant fraction of lupin nodules and the purity of the preparation established by gel electrophoresis and electrofocusing. The purified enzyme existed as 4 charge isozymes with a MW of 270000. The subunit MW, as determined by dodecyl sulphate electrophoresis, was 45000. On the basis of the results of the MW determinations a hexameric structure is proposed for lupin-nodule GDH. The pH optima for the enzyme were pH 8.2 for the amination reaction and pH 8.8 for the deamination reaction. GDH from lupin nodules showed a marked preference for NADH over NADPH in the amination reaction and used only NAD+ for the deamination reaction. Pyridoxal-5’-P and EDTA inhibited activity. The enzyme displayed Michaelis-Menten kinetics with respect to all substrates except NAD+. When NAD+ was the varied substrate, there was a deviation from Michaelis-Menten behaviour towards higher activity at high concentrations of NAD+