Partial Purification and Some Kinetic Properties of NAD-linked and NADP-linked Isocitrate Dehydrogenases from Candida tropicalis

Abstract

NAD- and NADP-linked isocitrate dehydrogenases were purified 280-fold and 54-fold, respectively, from the cell-free extract of n-alkane-grown Candida tropicalis by means of ammonium sulfate fractionation, and column chromatography on Sephadex G-200 and DEAE-cellulose. NAD-linked isocitrate dehydrogenase was activated by AMP, ADP or citrate, whereas the NADP-linked enzyme was not affected by these compounds. The NAD-linked enzyme showed the kinetics of Michaelis-Menten type at a low concentration of MgCl₂ (3.3mM), while allosteric nature of the enzyme was observed at a high MgCl₂ concentration (33.3mM). Concomitant presence of glyoxylate and oxalacetate inhibited the reactions of both dehydrogenases. Especially, the NADP-linked enzyme was completely inhibited by these acids at a concentration of as low as 1mM each, showing a mixed pattern of inhibition.