Abstract
A procedure to reconstitute CF0CF1 proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced ΔpH of 2.5 to 3.5 units. ΔpH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF0CF1 proteoliposomes prepared by conventional techniques catalyzed an ATP-induced Δψ formation, but were unable to catalyze an ATP-induced ΔpH even in the presence of valinomycin. The ATP-induced ΔpH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced ΔpH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF0CF1 preparation. The ammonium acted as an internal buffer, and thus prevented an observable ΔpH formation.
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