Beef-heart malic dehydrogenases: II. Preparation and properties of crystalline supernatant malic dehydrogenase

Abstract

Differential extraction of beef-heart muscle revealed the presence of two malic dehydrogenases. One, apparently derived from the supernatant fraction, is easily extractable and exhibits no inhibition of oxidation of DPNH by elevated concentrations of oxaloacetate. The other, presumably mitochondrial in origin, requires more drastic means of extraction and under similar defined conditions of assay is inhibited by elevated concentrations of oxaloacetate. A method of purifying the supernatant malic dehydrogenase is described. By a combination of ammonium sulfate fractionation, heat inactivation, chromatography on DEAE-cellulose, starch-block electrophoresis, and finally dialysis against ammonium sulfate solutions, a crystalline malic dehydrogenase has been prepared. Electrophoretic studies over a wide range of pH values revealed that the preparation was essentially homogeneous. The enzyme was also homogeneous in the ultracentrifuge, and had an s 20, w 0 of 5.1·10 −13 sec and a D 20, w 0 of 9.1·10 −7 cm 2/sec. From these data, assuming a partial specific volume of 0.74 ml/g, a molecular weight of 52 000 was calculated. Some kinetic constants of the enzyme have been determined and its specificity examined.