Characteristics of MgATP(2-)-dependent electrogenic proton transport in tonoplast vesicles of the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

Membrane vesicles were isolated from mesophyll cells of Mesembryanthemum crystallinum in the C3 state and in the crassulacean acid metabolism (CAM) state. The distribution of ATP-hydrolysis and H(+)-transport activities, and the activities of hydroxypyruvate reductase and Antimycin-insensitive cytochrome-c-reductase on continuous sucrose gradients was studied. For isolations carried out routinely a discontinuous sucrose gradient (24%/37%/50%) was used. Nitrate-sensitive ATP-hydrolysis and H(+)-transport activities increased several-fold during the transition from C3 photosynthesis to CAM. Nitrate-sensitive ATPase showed a substrate preference for ATP with an apparent Km (MgATP(2-)) of 0.19-0.37 mM. In both C3 and CAM states the ATPase showed a concentration-dependent stimulation by the anions chloride and malate. However, the pH optima of the two states were different: the ATPase of C3- M. crystallinum had an optimum of pH 7.4 and that of CAM-M. crystallinum an optimum of pH 8.4. The optical probe oxonol-VI was used to demonstrate the formation of MgATP(2-)-dependent electric-potential gradients in tonoplast vesicles.