Malate dehydrogenase from physarum polycephalum and physarum flavicomum : a physical, chemical and functional study of the mitochondrial and supernatant isozymes in growing and differentiated stages

Two isozymes of malate dehydrogenase have been demonstrated for Physarum polycephalum and Physarum flavicomum plasmodia as well as P. flavicomum haploid cells by the techniques of polyacryla- mide-gel disc electrophoresis and isoelectric focusing. Analyses of mitochondria from these sources by polyacrylamide-gel electrophoresis revealed that only the more cathodalprotein was associated with these organelles (m-MDH). The other, more anodal form is found in the soluble cytoplasm (s-MDH). The isozymes from P. polycephalum and P. flavicomum plasmodia have been purified to homogeneity as confirmed by gel filtration chromatography, polyacrylamide-gel disc electrophoresis and analytical ultracentrifugation. Final specific activities, as measured by oxaloacetate reduction for P. polycephalum were 1, 020 for m-MDH and 810 for s-MDH. The specific activities for P. flavicomum were 1,080 for m-MDH and 839 for s-MDH. The isozymes from both species were composed of dimeric, size equivalent subunits as demonstrated by sodium dodecyl sulfate gel electrophoresis. The sedimentation coefficients and molecular weights were determined. The molecular weights as established by sedimentation equilibrium were 69,500 for both P. polycephalum isozymes, 70,000 for P. flavicomum m-MDH, and 65,000 for P. flavicomum s-MDH. Total amino acid analyses revealed the isozymes from each species were different. In P. polycephalum the s-MDH contained more isoleucine, serine, threonine and valine but less arginine, glutamate, glycine, half cystine, lysine and methionine than m-MDH. In P. flavicomum the m-MDH had higher values for arginine, aspartate, glutamate, glycine, isoleucine, lysine, methionine, serine and threonine but much lower values for phenylalanine. The determination of optimum pH and substrate concentrations as well as Michaelis constants revealed the Physarum isozymes to be similar to each other as well as to a variety of other malate dehydrogenases from other sources. Analyses of the catalytic mechanism using substrate analogues revealed the mechanism was similar in all Physarum isozymes. Dicarboxylic acids with alpha carbon unsaturation were the best inhibitors of the reactions. The m-MDH isozymes from Physarum were inhibited by adenosine phosphates with ATP giving the highest level of inhibition. The s-MDH isozymes were not inhibited. Other nucleoside phosphates had no effect.