Improved purification for thermophilic F 1F 0 ATP synthase using n-dodecyl β- d-maltoside

Abstract

Here we report a fast, simple purification for thermophilic F 1F 0 ATP synthase (TF 1F 0) that utilizes a cocktail of stabilizing reagents and the detergent n-dodecyl β- d-maltoside to yield enzyme with an ATPase activity of 41 μmol/min/mg, 2.5-fold higher than that previously reported. ATPase activity was 80% inhibited by the F 0-reactive reagent dicyclohexylcarbodiimide, indicating that F 1–F 0 interactions were largely intact. To measure ATP-driven proton pumping activity, purified TF 1F 0 was incorporated into liposomes, and the ATP-induced change in internal pH was measured using the fluorescent probe pyranine. In the presence of valinomycin, a maximum ATP-driven ΔpH of 0.8 units was obtained. To measure ATP synthesis activity, TF 1F 0 was incorporated into liposomes with the light-dependent proton pump bacteriorhodopsin. Proteoliposomes were illuminated to generate an electrochemical gradient, after which ADP and inorganic phosphate were added to initiate ATP synthesis. A steady state ATP synthesis activity of 490 nmol/min/mg was achieved after an initial ∼30-min lag phase.