Characteristics of MgATP2--dependent electrogenic proton transport in tonoplast vesicles of the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

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Abstract
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 (MgATP2-) 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 MgATP2--dependent electric-potential gradients in tonoplast vesicles.