Two Proton Pumps Operate in Parallel Across the Tonoplast of Vacuoles Isolated from Suspension Cells ofChenopodium rubrumL.*

The kinetics of vacuolar acidification upon addition of ATP and/or pyrophosphate (PPi) has been assayed on single immobilized vacuoles by computer‐aided microfluorimetry of 9‐aminoacridine, and by acridine orange absorption photometry on vacuole suspensions isolated from green suspension cells of Chenopodium rubrum L. Two proton pumps at the tonoplast, an ATPase and a pyrophosphatase (PPase), operate in parallel to acidify the vacuole with different contributions adding up to a transtonoplast Δ pH of 2.6 pH units at external pH 7.2. The saturable components of proton pumping reach half maximal velocity with 0.32 ± 0.06 mM ATP and 23 ± 2.5 μM PPi, respectively. At saturating substrate concentrations, ATPase and PPase hydrolyse ATP and PPi, respectively, at a ratio of 2.3. The same ratio holds for the corresponding proton fluxes maintaining a given steady‐state vacuolar pH. We conclude that both pumps operate at the same stoichiometry.