Formations of Electrochemical Proton Gradient and Adenosine Triphosphate in Proteoliposomes Containing Purified Adenosine Triphosphatase and Bacteriorhodopsin

Abstract

Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H⁺-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF₀.F₁ were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (Δμ¯H⁺) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (ΔpH) component and 8-anilinona- phthalene sulfonate for the electrical (Δψ) component. In illuminated bacteriorhodopsin-vesicles the Δμ¯H⁺ reached 180–190 mV when reconstituted by the dialysis method and 210–220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF₀.F₁ and bacteriorhodopsin by the dialysis method generated a Δμ¯H⁺ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little Δμ¯H⁺ if any. These vesicles generated similar Δμ¯H⁺ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF₀.F₁ and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to Δμ¯H⁺ formation. It was necessary to generate a Δμ¯H⁺ of above 170 mV for demonstration of appreciable formation of ATP and the greater the Δμ¯H⁺ the faster the rate of ATP synthesis.