Abstract
An NADP-linked glutamate dehydrogenase (L-Glutamate dehydrogenase NADP-oxidoreductase, EC 1.4.1.4.) was purified 181 folds from Plasmodium knowlesi (simian malaria parasite) by ammonium sulfate fractionation, gel filtration and hydroxylapatite column chromato-graphy. The molecular weight of the enzyme was found to be 295,000 as determined by gel filtration. The enzyme appeared to be heat stable (4h at 56℃) and activated about 39% and 14% by KCl and NaCl respectively. It catalysed the amination of α-ketoglutarate and the deamination of glutamate with optimum activity at pH 7.4 and 8.6 respectively. Hyperbolic kinetics were observed for the substrates and cofactors yielding Km values of 0.25±0.02ｍM for α-ketoglutarate, 1.3±0.2mM for ammonium acetate, 0.011±0.001mM for NADPH, 1.8±0.1mM for glutamate and 0.050±0.002mM for NADP. The amination reaction was about 10 times more active as compared to the deamination reaction. Purine nucleotides did not show any effect on enzyme activity. These results suggest that the amination reaction may predominate in P. knowlesi parasites.
