Mitochondrial and cytosolic variants of malic enzyme (EC 1.1.1.40 L-malate: NADP+ oxidoreductase, decarboxylating) occur in mammalian tissues. This study concerns characterization of these isoenzymes in rat brain. Isolation of subcellular fractions was carried out by Ficoll-sucrose discontinuous gradient centrifugation and enzyme activity was measured spectrophotometrically. Specific activities were 34.9 and 9.1 nmol/min/mg protein in free mitochondria and in cytosol, respectively. Both mitochondrial and cytosolic enzymes were strictly NADP+-dependent and more active with Mn2+ than with Mg2+. The two variants had similar kinetic properties. Km values were 6.82 and 7.38 x 10-4 M for malate, and 1.36 and 1.26 x 10-5 M for NADP+, respectively. Mitochondrial enzyme was strongly inhibited by 5,5'-dithiobis (2-nitrobenzoic acid) at concentrations which did not affect the cytosolic enzyme. The former was also more sensitive to dicumarol inhibition. Cooperativity and NAD+-dependent activity, which occur in other mammalian tissues, were not found in rat brain.
RAT-BRAIN MALIC ENZYME - CHARACTERIZATION AND SUBCELLULAR-DISTRIBUTION / G. DEMICHELE, A. FILLA, M. POPOLI, V. MORRA, V. PALMA, G. DIGERONIMO, G. CAMPANELLA. - In: MEDICAL SCIENCE RESEARCH. - ISSN 0269-8951. - 15:7-8(1987), pp. 365-366.
RAT-BRAIN MALIC ENZYME - CHARACTERIZATION AND SUBCELLULAR-DISTRIBUTION
M. POPOLI;
1987
Abstract
Mitochondrial and cytosolic variants of malic enzyme (EC 1.1.1.40 L-malate: NADP+ oxidoreductase, decarboxylating) occur in mammalian tissues. This study concerns characterization of these isoenzymes in rat brain. Isolation of subcellular fractions was carried out by Ficoll-sucrose discontinuous gradient centrifugation and enzyme activity was measured spectrophotometrically. Specific activities were 34.9 and 9.1 nmol/min/mg protein in free mitochondria and in cytosol, respectively. Both mitochondrial and cytosolic enzymes were strictly NADP+-dependent and more active with Mn2+ than with Mg2+. The two variants had similar kinetic properties. Km values were 6.82 and 7.38 x 10-4 M for malate, and 1.36 and 1.26 x 10-5 M for NADP+, respectively. Mitochondrial enzyme was strongly inhibited by 5,5'-dithiobis (2-nitrobenzoic acid) at concentrations which did not affect the cytosolic enzyme. The former was also more sensitive to dicumarol inhibition. Cooperativity and NAD+-dependent activity, which occur in other mammalian tissues, were not found in rat brain.
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