Further characterization of gradient-fractionated submitochondrial membrane fragments from beef heart mitochondria.
We have recently reported that with a linear sucrose density gradient centrifugation two distinct types of membrane fragments, designated as X- and Y-fragments are obtained (Huang, C. H., Keyhani, E. and Lee, C. P. (1973) Biochim. Biophys. Acta 305, 455-473). Further characterization of these two membranes fragments is reported. (1) Potassium chloride at the concentration of 0.15 m extracts 7% and 30% of cytochrome c from the X- and Y-fragments, respectively. (2) When cytochrome c was added to the mitochondrial suspension prior to sonication, the cytochrome c content was increased by 6-8-fold in both X- and Y-fragments. Subsequently KC1 extraction resulted in loss of cytochrome c by 1/4 in the X- and by 2/3 in the Y-fragments. (3) With partially inhibitory concentrations of KCN, cytochrome c in either the X- or the KC1 extracted X-fragments showed uncoupler-sensitive, biphasic reduction kinetics upon the addition of NADH to the oligomycin-supplemented system. Under identical conditions rapid first order reduction kinetics were seen for cytochrome c in Y-fragments supplemented with either oligomycin or oligomycin + carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (4) When cytochrome c was added to the mitochondrial suspension after sonication, a significant amount of cytochrome c was bound to both X- and Y-fragments, but was readily removed with a high ionic strength medium. (5) Lubrol had little effect on the ATPase activity of the X- and the Y-fragments, suggesting a lack of membrane-buried ATPase. (6) Partial depletion of ATPase in X-fragments did not induce an increase in reactivity towards externally added cytochrome c. (7) Both the X- and the Y-fragments showed an energy-linked fluorescence enhancement of 8-anilinonaphthalene-1-sulfonate and an energy-linked fluorescence decrease of quinacrine. (8) In the presence of K-+ nigericin alone or in combination with valinomycin exhibited a stimulating effect on the rate of NADH oxidase of the oligomycinsupplemented X- and Y-fragments.
D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003580:Cytochromes / Q000032:analysis; D008566:Membranes / Q000032:analysis / Q000648:ultrastructure*; D008931:Mitochondria, Muscle / Q000032:analysis / Q000648:ultrastructure*; D009206:Myocardium; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D013010:Sonication; D013053:Spectrophotometry; D013696:Temperature; D014451:Ubiquinone / Q000032:analysis
D000963:Antimetabolites; D003580:Cytochromes; D010743:Phospholipids; D014451:Ubiquinone; D009247:NADH, NADPH Oxidoreductases; D000251:Adenosine Triphosphatases