Malate dehydrogenases of leaf tissue from Spinacia oleracea: Properties of three isoenzymes

Abstract

Starch-gel electrophoresis of leaf homogenates from Spinacia oleracea indicates four forms of malate dehydrogenase ( l-malate: NAD oxidoreductase, EC 1.1.1.37). Three of the isoenzymes (viz., microbody malate dehydrogenase, mitochondrial malate dehydrogenase, and soluble 1 malate dehydrogenase) were completely separated by DEAE-cellulose anion-exchange column chromatography while a fourth isoenzyme (viz., soluble 2 malate dehydrogenase) still contained soluble 1 malate dehydrogenase as a contaminant. The electrophoretic and chromatographic properties of the isoenzymes are significantly different. Of the three resolvable isoenzymes, soluble 1 malate dehydrogenase and microbody malate dehydrogenase are similar and differ from mitochondrial malate dehydrogenase in terms of pH-dependent substrate inhibition by oxaloacetate and in terms of their pH-dependent Michaelis constants (oxaloacetate). Soluble 1 malate dehydrogenase and microbody malate dehydrogenase are inhibited less by oxaloacetate at pH 9.0 when compared to pH 6.0 or 7.5, while mitochondrial malate dehydrogenase is rather constant in its susceptibility to oxaloacetate inhibition at the same pH values. The oxaloacetate Michaelis constants of all the isoenzymes increase with increasing pH, however, soluble 1 malate dehydrogenase and microbody malate dehydrogenase show a sharp increase at pH 9.0 which is not nearly so pronounced with mitochondrial malate dehydrogenase. The thermal stabilities of the isoenzymes are significantly different: soluble 1 > mitochondrial > microbody. Based on gel filtration, the molecular weights of all four isoenzymes were estimated to be approximately identical, at 66,000. NAD analog experiments also suggested significant differences among the isoenzymes.