Evidence for two distinct mitochondrial malic enzymes in human skeletal muscle: purification and properties of the NAD(P)+-dependent enzyme

Franco Taroni, Cinzia Gellera, Stefano Di Donato

Research output: Contribution to journalArticlepeer-review

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+.

Original languageEnglish
Pages (from-to)446-454
Number of pages9
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume916
Issue number3
DOIs
Publication statusPublished - Dec 18 1987

Keywords

  • (Human skeletal muscle)
  • (Muscle mitrochondrion)
  • ATP affinity chromatography
  • Malic enzyme
  • NAD
  • NADP

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

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