Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria.
The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D005260:Female; D007700:Kinetics; D007942:Leukemia, Experimental / Q000201:enzymology; D008114:Liver Neoplasms, Experimental; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology; D051379:Mice; D008815:Mice, Inbred Strains; D008928:Mitochondria / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000201:enzymology*; D051381:Rats; D013945:Thymoma / Q000201:enzymology; D013953:Thymus Neoplasms / Q000201:enzymology
D009243:NAD; D009249:NADP; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)