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Identification and Properties of the Nicotinamide Adenine Dinucleotide (Phosphate)⁺-dependent Malic Enzyme in Mouse Ascites Tumor Mitochondria¹

The Mary Imogene Bassett Hospital, Cooperstown, New York 13326

ELD, ELT/B1, Krebs II, and Sarcoma 180 ascites tumor cells were examined for the mitochondrial malic enzymes. The nicotinamide adenine dinucleotide (phosphate) [NAD(P)⁺]-dependent malic enzyme was present in all mitochondrial fractions at activities that ranged from 21 to 27 nmol reduced nicotinamide adenine dinucleotide formed per min per mg mitochondrial protein. As judged by kinetic properties, the enzyme was essentially identical 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 mM fumarate, the apparent K_m values for malate were about 2 mM with either NAD⁺ or NADP⁺ as coenzyme. The apparent K_m values for NAD⁺ and NADP⁺ were approximately 60 µM, and NAD⁺ and NADP⁺ were shown to compete for reduction. The strictly NADP⁺-linked mitochondrial isoenzyme was not detected in these ascites tumors. The strictly NADP⁺-dependent isoenzyme of the cytosol was present, but its kinetic properties were considerably different from those during NADP⁺ reduction via the mitochondrial 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 accumulation. Pyruvate accumulation was inhibited by the addition of adenosine 5'-diphosphate, suggesting that pyruvate generated by the NAD(P)⁺-dependent malic enzyme was completely utilized by pyruvate dehydrogenase during State 3.

¹ This research was supported by NIH Grant CA 18262 and by the Stephen Carlton Clark Research Fund of The Mary Imogene Bassett Hospital, Cooperstown, N. Y. This is Paper 2 in a series on the mitochondrial malic enzymes. Paper 1 is Ref. 15.

Received 12/15/77. Accepted 3/14/78.