Figure 5. V-ATPase activity is blocked by Cu²⁺.

(A) Acridine orange (AO) fluorescence quenching assays were used to monitor H⁺ pumping into the vacuole lumen. Vacuoles were incubated with dose curve of CuCl₂ or buffer (± ATP) and 15 μM AO. AO fluorescence quenching was measured using a plate reader. Fluorescence was measured every 40 seconds and plotted against time. (B) Quantitation of average maximum AO fluorescence. (C) AO fluorescence quenching in the presence of PS buffer (Buf.), 100 μM MgCl₂, ZnCl₂ or CoCl₂. Error bars are S.E.M. (n=3). (D) Log phase BJ3505 cells were incubated with 50 μM CuCl₂ for 1h. Vacuoles were stained with 2 μM FM4–64 and 200 μM quinacrine. DIC (Differential Interference Contrast). Scale bar = 5 μm.