MALIC DEHYDROGENASE ISOENZYEMS IN GREEN STEM TISSUE OF OPUNTIA - ISOLATION AND CHARACTERIZATION

By starch gel electrophoresis and DEAE-cellulose anion exchange column chromatography, three distinctly different malic dehydrogenase (l-malate:NAD oxidoreductase, EC 1.1.1.37) isoenzymes were isolated from green stem tissue of Opuntia ficus indica. Chloroplasts isolated and purified by nonaqueous density gradient centrifugation techniques indicated that one isoenzyme was unique to the chloroplast fraction. Mitochondria isolated and purified by sucrose density gradient centrifugation techniques revealed a mitochondrial-malic dehydrogenase. Another was apparently not associated with subcellular particles and was designated as a soluble-malic dehydrogenase. Hence, these data suggest that green stem tissue of this cactus has at least three malic dehydrogenase isoenzymes; a chloroplast-malic dehydrogenase, a mitochondrial-malic dehydrogenase, and a soluble-malic dehydrogenase. Although the physical properties of the three isoenzymes were distinctly different, kinetic properties such as Km values for oxaloacetate, malate, NAD, and NADH at pH's of 7.4 and 8.5 were quite similar. Similarities were demonstrated with NAD analogs such as thionicotinamide-NAD and 3-acetylpyridine-NAD. The chloroplast-malic dehydrogenase, however, was considerably more active in the presence of 3-acetylpyridinedeamino-NAD and deamino-NAD than were the mitochondrial or soluble forms. Hence, the latter analogs demonstrated kinetic differences. The thermostabilities of the three isoenzymes were distinctly different with the chloroplast-malic dehydrogenase being the most stable and the soluble-malic dehydrogenase being the least stable. Metabolites such as α-ketoglutarate, cis-aconitate, citrate, dl-isocitrate, succinate, fumarate, aspartate, glutamate, and maleate partially inhibited the isoenzymes. Kinetic data suggested that α-ketoglutarate was a partially competitive inhibitor with respect to both the substrates, NADH and oxaloacetate. In the presence of α-ketoglutarate and cis-aconitate, a cumulative" type of inhibition was observed. It was concluded that α-ketoglutarate and cis-aconitate bind at sites distinct from each other and at a site distinct from the substrate binding site. © 1969."