Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles |
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Authors: | John M Wrigglesworth Peter Nicholls |
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Institution: | 1. Department of Biochemistry, Chelsea College, University of London, Manresa Road, London SW3 6LX U.K.;2. Department of Biological Sciences, Brock University, St. Catharines, Ontario L2S 3A1 Canada |
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Abstract: | 1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle.2. The activity of cytochrome c oxidase in the reconstituted vesicles is not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c.3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH? inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externally added ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions. |
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Keywords: | Cytochrome c oxidation Reconstitution Liposome Membrane orientation TMPD DAD FCCP To whom correspondence should be addressed |
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