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 (Δ_??_H⁺) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (ΔpH) component and 8-anilinonaphthalene sulfonate for the electrical (Δφ) component. In illuminated bacteriorhodopsinvesicles the Δ_??_H⁺ reached 180-190mV when reconstituted by the dialysis method and 210-220mV when reconstituted by the sonication method. Vesicles reconstituted from both TF₀•F₁ and bacteriorhodopsin by the dialysis method generated a Δ•H⁺ of about 200mV on addition of ATP, while vesicles prepared by the sonication method generated very little Δ_??_H⁺, if any. These vesicles generated similar ΔH⁺ 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 Δ_??_H⁺ formation. It was necessary to generate a ΔH⁺ of above 170mV for demonstration of appreciable formation of ATP and the greater the Δ_??_H⁺, the faster the rate of ATP synthesis.