Purification and properties of cytoplasmic and mitochondrial malate dehydrogenases of Physarum polycephalum.
Two isoenzymes of malate dehydrogenase (MDH) were demonstrated in plasmodia of Physarum polycephalum by polyacrylamide-gel electrophoresis. The more "cathodal" form was uniquely associated with mitochondria (M-MDH) and the other form was found in the soluble cytoplasm (S-MDH). The isoenzymes were separated by acetone fractionation of soluble plasmodial homogenates acidified to pH 5.0. The M-MDH was purified 201-fold by cetylpyridinium chloride treatment, fractionation with ammonium sulfate, gradient elution from sulfoethyl cellulose at pH 6.0, and Sephadex G-100 chromatography. The S-MDH was purified 155-fold by ammonium sulfate fractionation, diethylaminoethyl cellulose chromatography, gradient elution from sulfoethyl cellulose at pH 5.5, and Sephadex G-100 chromatography. The optimal cis-oxalacetate concentrations were 0.35 mM for M-MDH and 0.25 mM for S-MDH, and the optimal pH for both isoenzymes was 7.6 for oxalacetate reduction. The optimal l-malate concentrations were 5 mM for S-MDH and 6 mM for M-MDH, and both isoenzymes exhibited an optimal pH of 10.0 for L-malate oxidation. The Michaelis constants of S-MDH and M-MDH served to discriminate between the isoenzymes. The S-MDH was more heat-stable than the M-MDH. High concentrations of oxalacetate and malate inhibited S-MDH more than M-MDH. The isoenzymes were further distinguished by their utilization of analogues of nicotinamide adenine dinucleotide. Many properties of the Physarum isoenzymes were similar to those of more complex organisms, especially vertebrates.
D000096:Acetone; D000645:Ammonium Sulfate; D002594:Cetylpyridinium; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003584:Cytological Techniques; D003593:Cytoplasm / Q000201:enzymology*; D003956:Dialysis; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology*; D009235:Myxomycetes / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism
D007527:Isoenzymes; D011506:Proteins; D009243:NAD; D000096:Acetone; D002594:Cetylpyridinium; D008291:Malate Dehydrogenase; D000645:Ammonium Sulfate