Purification and characterization of electron-transfer flavoprotein:rhodoquinone oxidoreductase from anaerobic mitochondria of the adult parasitic nematode, Ascaris suum
1993
Ma, Y.C. | Funk, M. | Dunham, W.R. | Komuniecki, R.
Electron-transfer flavoprotein:rhodoquinone oxidoreductase (ETF-RO) was purified to homogeneity from anaerobic mitochondria of the parasitic nematode, Ascaris suum. The enzyme has a subunit molecular mass of 64.5 kDa and is similar in many respects to the electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-UO) characterized in mammalian tissues. EPR spectroscopy of the purified enzyme revealed signals at g = 2.076, 1.936, and 1.883, arising from an iron-sulfur center, as well as signals attributable to a flavin semiquinone. Potentiometric titration on the enzyme with dithionite yielded an oxidation-reduction midpoint potential (Em.) for the iron-sulfur center of +25 mV at pH 7.4. The reduction of flavin occurred in two distinct steps, with a flavin semiquinone radical detected as an intermediate. The E. values for the two steps in the complete reduction of flavin were +15 mV and -9 mV, respectively. Physiologically, the ascarid ETF-RO accepts electrons from a low potential quinone, rhodoquinone, and functions in a direction opposite to that of the ETF-UO. Incubations of A. suum submitochondrial particles with NADH, 2-methylcrotonyl-CoA, purified A. suum electron-transfer flavoprotein and 2-methyl branched-chain enoyl-CoA reductase resulted in significant 2-methylbutyryl-CoA formation, which was inhibited by both rotenone and antisera to the purified ETF-RO. Quinone extraction of the submitochondrial particles with dry pentane resulted in almost the complete loss of 2-MBCoA formation by the system. However, the reincorporation of rhodoquinone, but not ubiquinone, restored over 50% of the NADH-dependent 2-MBCoA formation.
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