Evidence for two distinct mitochondrial malic enzymes in human skeletal muscle: purification and properties of the NAD(P)+-dependent enzyme.
Human muscle mitochondria reduced either NADP+ or NAD+ in the presence of L-malate and Mn2+ or Mg2+. After polyacrylamide slab gel electrophoresis and agarose gel isoelectrofocusing, two bands were seen in mitochondrial extract, one strictly NADP+-dependent and the other reacting with either NAD+ or NADP+. The two mitochondrial malic enzymes were separated by DEAE-Sepharose chromatography. The NAD+/NADP+-dependent enzyme was purified 1600-fold with a final yield of 34% and a final specific activity of 32.9 units/mg of protein by employing affinity chromatography on Agarose-ATP. SDS electrophoresis revealed a single band having an apparent Mr = 64,000. Estimates of the native apparent molecular weight upon gel filtration yielded a value of 140,300. Kinetic characterization showed that succinate and ATP were activator and inhibitor, respectively. In the absence of succinate the Km values for malate, NAD+ and NADP+ were 3.7, 0.13 and 0.78 mM, respectively; in the presence of succinate the Km value for malate was 1.9 mM. ATP was found to be an inhibitor competitive with malate, with a Ki (ATP) of 0.2 mM. This is the first report to show that mammalian skeletal muscle mitochondria contains two distinct malic enzymes, one active with either NAD+ or NADP+ and the other active only with NADP+.
Taroni
F
F
Laboratory of Biochemistry, Istituto Neurologico C. Besta, Milan, Italy.
Gellera
C
C
Di Donato
S
S
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D008931:Mitochondria, Muscle / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*
D007527:Isoenzymes; D009243:NAD; D009249:NADP; D008291:Malate Dehydrogenase