Identification and properties of the nicotinamide adenine dinucleotide (phosphate)+-dependent malic enzyme in mouse ascites tumor mitochondria.

ELD, ELT/B1, Krebs II, and Sarcoma 180 ascites tumor cells were examined for the mitochondria! malic enzymes. The nicotinamide adenine dinucleotide (phosphate) [NAD(P) ]-dependent malic enzyme was present in all mitochondria! fractions at activities that ranged from 21 to 27 nmol reduced nicotinamide adenine dinucleotide formed per min per mg mitochondria! protein. As judged by kinetic properties, the enzyme was essentially identi cal in the different ascites tumors. Activity with both NAD* and NADP was stimulated by fumarate and inhibited by adenosine 5'-triphosphate. In the presence of 5 mu fuma rate, the apparent K : values for malate were about 2 mM with either NAD or NADP* as coenzyme. The apparent Km values for NAD and NADP were approximately 60 (IM, and NAD* and NADP* were shown to compete for reduction. The strictly NADP -linked mitochondria! isoenzyme was not detected in these ascites tumors. The strictly NADP -dependent isoenzyme of the cytosol was present, but its kinetic properties were considerably dif ferent from those during NADP reduction via the mito chondria! NAD(P) -dependent malic enzyme. Pyruvate was formed by ascites tumor mitochondria during malate-supported respiration. Phosphate, which stimulates malate entry via the inorganic orthophosphatemalate transporter, increased the rate of pyruvate accu mulation. Pyruvate accumulation was inhibited by the addition of adenosine 5'-diphosphate, suggesting that pyruvate generated by the NAD(P) -dependent malic en zyme was completely utilized by pyruvate dehydrogenase during State 3.