Anion‐sensitive ATPase activity and proton transport in isolated vacuoles of species of the CAM genus Kalanchoë
Jochem P., Rona J., Smith JAC., Lüttge U.
Vacuoles were isolated from leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bathie, and the ionic sensitivity of the vacuolar ATPase was studied in vacuole homogenates desalted on Sephadex G‐25. The ATPase activity was dependent on the presence of divalent cations (Mg2+≥ Mn2+≥ Ca2+, Co2+; Zn2+ had no effect). Mg2+‐dependent ATPase activity was stimulated by anions (Cl− > malate2+, HCO−3), with maximal stimulation at concentrations above 50 mM. Mg2+‐Dependent activity was inhibited by NO−3 above 2 mM, but no saturation was observed up to 100 mM. No stimulation by K+ or Na+ was detected; stimulation by NH+4 was abolished by 0.01% (w/v) Triton X‐100, suggesting that the NH+4 effect was due to the permeability of vacuolar membrane vesicles to NH3. Trans‐tonoplast electrical potentials (Δψ) and intra‐vacuolar pH were measured with glass microelectrodes and antimony covered glass micro‐pH‐electrodes, respectively. Free vacuofes isolated from Kalanchoë tubiflora (Harv.) Hamet were slightly positive with respect to the suspension medium. This Δψ was insensitive to the protonophore FCCP and depolarized by about 4 mV on addition of 50 mM KCl, still remaining about +5 mV. Upon addition of 7 mM Mg‐ATP, vacuoles showed an FCCP‐sensitive increase of Δψ from +9.2 ± 2.8 (13) to +17.8 ± 3.7 (12) mV [given as x̄± sd (n)] and an internal acidification from pH 5.4 ± 0.2 (11) to pH 4.3 ± 0.4 (12). Mg‐ADP and ATP without Mg2+ had no effect on Δψ. It is concluded that the H4 pumping at the tonoplast is due to the functioning of the anion‐sensitive vacuolar ATPase and that this is an essential part of the mechanism of nocturnal acid accumulation in CAM. Copyright © 1984, Wiley Blackwell. All rights reserved