ABSTRACT
Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.
Subject(s)
Electron Transport Complex IV/metabolism , Membrane Lipids/physiology , Mitochondria, Heart/enzymology , Mitochondria, Liver/enzymology , Animals , Buffers , Cattle , Electron Transport Complex IV/antagonists & inhibitors , Kinetics , Liposomes/metabolism , Peptide Hydrolases , Polarography , SpectrophotometrySubject(s)
Electron Transport Complex IV/isolation & purification , Isoenzymes/isolation & purification , Mitochondria/enzymology , Amino Acid Sequence , Animals , Chromatography, Ion Exchange/methods , Detergents , Electron Transport Complex IV/metabolism , Indicators and Reagents , Isoenzymes/metabolism , Kinetics , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Octoxynol , Polyethylene Glycols , Sequence Homology, Nucleic AcidABSTRACT
Isolated beef heart cytochrome c oxidase was reconstituted in liposomes by the cholate dialysis method with 85% of the binding site for cytochrome c oriented to the outside. Trypsin cleaved specifically subunit VIa and half of subunit IV from the reconstituted enzyme. The kinetic properties of the reconstituted enzyme were changed by trypsin treatment if measured by the spectrophotometric assay but not by the polarographic assay. It is concluded that subunit VIa and/or subunit IV participate in the electron transport activity of cytochrome c oxidase.
