Determination of the membrane potential of vacuoles isolated from red-beet storage tissue : Evidence for an electrogenic ATPase.

The membrane potential of isolated vacuoles of red beet (Beta vulgaris L.) was estimated using several methods. The quenching of the fluorescence of the cyanine dyes 3,3'-diethylthiodicarbocyanine iodide (DiS-C2-(5)) and 3,3'-dipropylthiodicarbocyanine iodide (DiS-C3-(5)) in vacuoles indicated a transmembrane potential difference, negative inside at low external potassium concentrations. The Δψ was found to be-55 mV with two other methods, the distribution of (204)T1(+) in the presence of valinomycin and the distribution of the lipophilic cation triphenylmethylphosphonium. Uncouplers reduced this value to-35 mV. High external potassium concentrations, comparable to cytosolic values, abolished the membrane potential almost completely. The addition of 1 mM Tris-Mg(2+)-ATP markedly hyperpolarized the membrane to-75 mV. This effect was prevented by inhibitors of the ATPase activity located in isolated vacuole membranes.