Proton-pumping adenosine triphosphatase in membrane vesicles of tobacco callus: Sensitivity to vanadate and K+

H⁺-pumping adenosinetriphosphatases (ATPases, EC 3.6.1.3) were demonstrated in sealed microsomal vesicles of tobacco callus. Quinacrine fluorescence quenching was induced specifically by MgATP and stimulated by EGTA and Cl^?. Fluorescence quenching reflected a relative measure of pH gradient formation (inside acid), as it could be reversed by gramicidin (an H⁺/cation conductor) or 10 mM NH₄Cl (an uncoupler). H⁺ pumping was inhibited by tributyltin (an ATPase inhibitor) and sodium vanadate, but it was insensitive to oligomycin or fusicoccin. The vanadate concentration required to inhibit pH gradient formation was similar to that needed to inhibit KCl-stimulated Mg²⁺-ATPase activity and generation of a membrane potential (measured by ATP-dependent ³⁵SCN^? uptake). About 45% of all three activities (ATPase, pH gradient, membrane potential generation) were vanadate-insensitive, supporting the idea that non-mitochondrial membranes of plants have at least two types of electrogenic H⁺ pump.A vanadate-insensitive, H⁺-pumping ATPase previously shown by methylamine accumulation was characterized to be anion-sensitive and possibly enriched in vacuolar membranes (Churchill, K.A. and Sze, H. (1983) Plant Physiol. 71, 610–617). Yet, pH gradient formation determined by quinacrine fluorescence quenching was decreased by monovalent cations with a sequence K⁺, Rb⁺, Na⁺ > Cs⁺,Li⁺> choline, bisTris-propane. Since K⁺ stimulated ATPase activity more than Bistris-propane, K⁺ appeared to collapse formation of the pH gradient by an H⁺/K⁺ countertransport. The sensitivity to vanadate and K⁺ provides evidence that the plasma-membrane ATPase is an electrogenic H⁺ pump.