PLASMODIUM-KNOWLESI - PARTIAL-PURIFICATION AND CHARACTERIZATION OF NADP-GLUTAMATE DEHYDROGENASE

An NADP-linked glutamate dehydrogenase (L-Glutamate dehydrogenase NADP -oxidoreductase, EC 1.4.1.4.) was purified 181 folds from Plasmodium k nowlesi (simian malaria parasite) by ammonium sulfate fractionation, g el filtration and hydroxylapatite column chromatography. The molecular weight of the enzyme was found to be 295,000 as determined by gel fil tration. The enzyme appeared to be heat stable (4 h at 56-degrees-C) a nd activated about 39% and 14% by KCl and NaCl respectively. It cataly sed the amination of is-proportional-to-ketoglutarate and the deaminat ion of glutamate with optimum activity at pH 7.4 and 8.6 respectively. Hyperbolic kinetics were observed for the substrates and cofactors yi elding Km values of 0.25 +/- 0.02mM for is-proportional-to-ketogultrat e, 1.3 +/- 0.2mM for ammonium acetate, 0.011 +/- 0.001mM for NADPH, 1. 8+/-0.1mM for glutamate and 0.050+/-0.002 mM for NADP. The amination r eaction was about 10 times more active as compared to the deamination reaction. Purine nucleotides did not show any effect on enzyme activit y. These results suggest that the amination reaction may predominate i n P.knowlesi parasites.