Malic enzymes of rabbit heart mitochondria. Separation and comparison of some characteristics of a nicotinamide adenine dinucleotide-preferring and a nicotinamide adenine dinucleotide phosphate-specific enzyme.

Abstract Two distinct malic enzymes have been separated from rabbit heart mitochondria. Both enzymes require Mn2+ or Mg2+ for activity. One of these enzymes (Enzyme 1) catalyzes the oxidative decarboxylation of malate in the presence of either NAD+ or NADP+, the former being much the more effective. After purification 100- to 200-fold by Sephadex and DEAE-cellulose chromatography and removal of contaminating malic dehydrogenase, some kinetic and physical characteristics of this enzyme were evaluated. The pH optimum of activity is near 7.0, and its isoelectric pH is 5.4. The enzyme appears not to be reversible and does not catalyze the decarboxylation of oxalacetate at pH 4.5 or 7.4. This enzyme is inhibited by ATP competitively with respect to malate, and noncompetitively by oxalate. A similar or identical enzyme is also present in mitochondria from guinea pig and pigeon heart and from pigeon breast muscle. The second enzyme (Enzyme 2), which is readily separated from Enzyme 1 by ammonium sulfate precipitation, is specific for NADP+. This enzyme, which is similar to the enzyme isolated from bovine heart mitochondria (Frenkel, R. (1971) J. Biol. Chem. 246, 3069–3074) also requires low concentrations of Mn2+ or Mg2+ for activity. Enzyme 2 carries out the reverse reaction (malate synthesis) in the presence of high concentrations of CO2 and pyruvate at about 6% of the rate of the forward reaction and catalyzes the decarboxylation of oxalacetate at pH 4.5 at about one-half the rate of forward reaction. The pH optimum (malate oxidation) for Enzyme 2 is between 7.0 and 8.0, and its isoelectric pH is 7.26. In contrast to the bovine heart mitochondrial enzyme, Enzyme 2 showed classical saturation kinetics and was not stimulated by succinate or fumarate at low concentration of malate. Estimates of the molecular weights using Sephadex G-200 indicated values of approximately 180,000 (range in two experiments, 165,000 to 195,000) and 270,000 (range, 260,000 to 280,000) for Enzyme 2 and Enzyme 1, respectively.