Variations on the "dilution" method for reconstituting cytochrome c oxidase into membrane vesicles.
A method for the rapid incorporation of cytochrome c oxidase into membranes has been developed. This method essentially consists of obtaining a preparation of the enzyme in which it is isolated and then dissolving it in a medium containing 0.5% of the detergent Tween 20, which gives a final concentration of 0.0125% after reconstitution. These studies revealed an optimal ratio of 1 microgram of enzyme to 5 mg of phospholipids. A similar optimal ratio was found when the amount of protein was varied. The optimum temperature was found to be 30 degrees C. Without a peak value being reached, it was found that the best reconstitution was obtained at pH 7.0-8.0. When measurements were performed either with a fluorescent cyanine (DiSC3) or by the uptake of tetraphenylphosphonium, it was found that the enzyme, with cytochrome c added to the outside, was capable of generating a membrane potential that was negative inside. Using the same procedure, the enzyme could also be reconstituted into vesicles of yeast plasma membrane. The procedure, then, seems adequate for incorporating cytochrome c oxidase into different kinds of membrane vesicles.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D019826:Cholic Acid; D002793:Cholic Acids; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials; D008929:Mitochondria, Heart / Q000201:enzymology; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010101:Oxygen Consumption; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013057:Spectrum Analysis
D002793:Cholic Acids; D008081:Liposomes; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D003576:Electron Transport Complex IV; D019826:Cholic Acid; C013289:tetraphenylphosphonium