Fig. 2.

Activities of the hybrid F₁F_o (Left) compared with TF₁F_o (Right). (A) H⁺-pumping activity of inverted E. coli membrane vesicles containing the hybrid F₁F_o [hF₁F_o⁽⁺ⁱ⁾] and TF₁F_o. NaCl or KCl was added into the assay mixture at the concentrations indicated. The reaction was initiated by adding 1 mM K⁺-ATP and terminated with FCCP. %ACMA shows the fluorescent intensity of ACMA, in which the initial intensity before the addition of ATP is calibrated as 100%. (B) H⁺-pumping activity of PLs containing hF₁F_o⁽⁺ⁱ⁾ and TF₁F_o. The assay conditions were the same as A. (C) Na⁺-pumping activity of PLs containing hF₁F_o⁽⁺ⁱ⁾ and TF₁F_o. As indicated, PLs were reacted with 50 μM DCCD for 1 h and used for the measurement. The reaction was initiated by the addition of 1.3 mM Na⁺₂-ATP, and the increase in Na⁺ concentration inside the PLs was monitored with an increase in sodium green fluorescence at 540 nm. (D) ATP synthesis activity of membrane vesicles containing hF₁F_o⁽⁺ⁱ⁾ and TF₁F_o. Electrochemical potential of H⁺ was generated by respiratory H⁺ pumps in the membrane vesicles driven by NADH oxidation. ATP synthesis was monitored in real time at 35°C by luciferase reaction at 560 nm. As indicated, a Na⁺/H⁺ antiporter, monensin (5 μM) was added to convert some amount of H⁺ gradient into Na⁺ gradient. Traces indicated by “-Pi” represent negative controls without addition of P_i. Light (100%) in D corresponds to the initial luminescence intensity in the cuvette before the addition of NADH.