查看文章

Vacuolar-type rotary H⁺-ATPase/synthase (V_oV₁) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V₁ (A₃B₃D₁F₁) and V_o (C₁E₂G₂I₁L₁₂) subcomplexes in vitro. A₃B₃D and A₃B₃ also reconstituted with V_o, resulting in a holoenzyme-like complexes. However, A₃B₃D-V_o and A₃B₃-V_o did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V₁ or A₃B₃D and a donor dye attached to subunit C in V_o. The estimated dissociation constants K_d for V_oV₁ and A₃B₃D-V_o were ∼0.3 and ∼1 nm at 25 °C, respectively. These results suggest that the A₃B₃ domain tightly associated with the two EG peripheral stalks of V_o, even in the absence of the central …