Purification and properties of cytosolic and mitochondrial malic enzyme isolated from human brain.
Three isoforms of malic enzyme have been described in mammalian tissues: a cytosolic NADP(+)-dependent enzyme, a NADP(+)-dependent mitochondrial isoform and a mitochondrial isozyme which can use both NAD+ and NADP+ but is more effective with NAD+. We purified mitochondrial and cytosolic malic enzyme from human brain extract to apparent homogeneity in order to compare properties of these isozymes and to verify whether mitochondria contain one or two malic enzyme. Specific activities of both isoforms are approx. 90 mumol/min/mg of protein, which corresponds to about 1900-fold purification. The two isozymes have identical native molecular mass (257 kDa) and are presumably tetramers composed of four identical subunits (M(r) = 64 kDa). The isoelectric point of cytosolic isozyme is 5.65, and that of mitochondrial one is 7.0. The isozymes show a substantial difference in their capability to catalyse the reductive carboxylation of pyruvate to malate: the maximal carboxylation rate approaches 80% that of decarboxylation velocity for the cytosolic enzyme, and only 17% for the mitochondrial isozyme. The coenzyme specificity of both isozymes is not stringent; NADP+ is the preferred and NAD+ can substitute it, although with much lower efficiency. The homogenous cytosolic malic enzyme catalysed decarboxylation of oxaloacetate and NADPH-dependent reduction of pyruvate at about 24 and 0.5% of the maximum rate of NADP-dependent oxidative decarboxylation of malate respectively. Decarboxylation of oxaloacetate catalysed by mitochondrial malic enzyme has not been detectable, while NADP-linked reduction of pyruvate approaches only 0.15% of the maximum rate of NADP-linked oxidative decarboxylation of malate.(ABSTRACT TRUNCATED AT 250 WORDS)
Bukato
G
G
Department of Biochemistry, Academic Medical School, Gdańsk, Poland.
Kochan
Z
Z
Swierczyński
J
J
D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight
D007527:Isoenzymes; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating)