Abstract
The ubiquinone reductase activity of a soluble extract of beef heart submitochondrial particles can be separated into two active fractions by chromatography on a hydroxylapatite column. Fraction I was further purified by ammonium sulfate fractionation and Fraction II by chromatography on DEAE-cellulose. Both fractions catalyze the oxidation of NADH by ubiquinone-10, ubiquinone-6, ubiquinone-1, menadione, cytochrome c, and ferricyanide at nearly equal rates. Both contain FMN, nonheme iron, and acid-labile sulfide in the approximate ratio of 1:4:5, and have nearly identical spectra. Sedimentation and electrophoretic analyses indicate that the fractions are better than 95% homogeneous. The molecular weights of the two preparations are very similar (near 90,000), although their electrophoretic mobilities differ. NADH causes anaerobic bleaching of the flavin (450 mμ), but the absorption peak at 550 mμ is not altered. The reductases are inhibited by Amytal and rotenone, the latter showing an unusual biphasic curve with maximal inhibition at a rotenone to enzyme flavin ratio of nearly one. Piericidin A, dicumarol, o-phenanthrolene, or Tiron do not inhibit reductase activity.
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