Purification and properties of cytosolic malic enzyme from human skeletal muscle

Franco Taroni, Stefano Di Donato

Research output: Contribution to journalArticlepeer-review


1. 1.An NADP*-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for l-malate and NADP+ were 0.246 mM and 5.8 μM, and 0.304 mM and 5.8 μM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 μM and 22.2 mM, respectively. 4. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. 5. The following physical parameters were established: s20,w 0=10.48, Stokes' radius = 5.61 nm, pI = 5.72, Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f{hook}/f{hook}0 = 1.33 by combining the Chromatographie data with the sedimentation measurements. 6. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme.

Original languageEnglish
Pages (from-to)857-866
Number of pages10
JournalInternational Journal of Biochemistry
Issue number8
Publication statusPublished - 1988

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'Purification and properties of cytosolic malic enzyme from human skeletal muscle'. Together they form a unique fingerprint.

Cite this