Abstract
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 μg 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°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 (DiSC 3 (3)) 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.
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