Na+/H+ antiporter activity of NhaP1 constructs in everted vesicles. (A, B) Two WT constructs with one or two affinity tags had normal activity at pH 6, reduced activity at pH 6.5, and no detectable activity at pH ⩾7.5. Constructs Δ22 and Δ27 lacking helix 1 (C, D) or untransformed KNabc cells (E) showed no detectable activity in the pH range tested. (F) pH-dependent activity expressed as percentage of dequenching. Fluorescence measurements were performed with 40 μl vesicle suspension at 5 mg/ml final protein concentration in a thermostated (22°C), stirred cuvette containing 2 ml of 2 μM acridine orange in MTCM buffer (10 mM MES/Tris titrated to the pH indicated, 140 mM choline chloride, 5 mM MgCl2, 5 mM KCl). (♦) Vesicles preloaded with protons by addition of 2.5 mM Tris-DL-lactate titrated to the pH of the MTCM buffer; (▴) transport initiated with 25 mM NaCl; (▵) pH gradient dissipated with 25 mM NH4Cl. All concentrations are final. The excitation wavelength was 495 nm and emission was recorded at 530 nm.