Purification and properties of malate dehydrogenases from Physarum flavicomum



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Two distinctly different malate dehydrogenases were isolated from Physarum flavicomum by polyacrylamide gel electrophoresis and carboxymethyl cellulose cation exchange column chromatography. Mitochondria isolated by differential centrifugation techniques revealed a mitochondria malate dehydrogenase. The other form was apparently not associated with subcellular particles and was designated supernatant malate dehydrogenase. The malate dehydrogenase isoenzymes were purified by ammonium sulfate fractionation; batch treatment with AGI—X8 anion exchange resin; re fractionation with ammonium sulfate; diethylaminoethyl cellulose column chromatography; carboxymethyl cellulose column chromatography; and Sephadex gel filtration. The mitochondrial malate dehydrogenase was judged highly purified by its specific activity and appeared to be homogeneous by polyacrylamide gel electrophoretic analysis. The supernatant malate dehydrogenase was partially purified. The mitochondrial and supernatant forms of malate dehydrogenase could readily be differentiated with respect to inhibition by oxalacetate and L-malate; oxalacetate and L-malate optima concentrations; pH optima profiles; thermolability; and reactivity with coenzyme analogues. Further dissimilarities were demonstrated by Michealis constants for oxalacetate and L-malate.