Taroni 1987 Biochim Biophys Acta: Difference between revisions
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Latest revision as of 15:10, 27 May 2020
Taroni F, Gellera C, Di Donato S (1987) Evidence for two distinct mitochondrial malic enzymes in human skeletal muscle: Purification and properties of the NAD(P)+-dependent enzyme. Biochim Biophys Acta 916: 446-454. |
Taroni F, Gellera C, Di Donato S (1987) Biochim Biophys Acta
Abstract: 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+.
Labels:
Organism: Human
Tissue;cell: Skeletal muscle
Preparation: Enzyme
Regulation: Substrate
Malic enzyme, Malate, Succinate, ATP, NAD, NADP, Anaplerosis