Characterization of the vacuolar ATPase activity of the crassulacean‐acid‐metabolism plantKalanchoë daigremontianaReceptor modulating

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Abstract
Plants performing crassulacean acid metabolism show a large nocturnal accumulation of malic acid in the vacuole of the photosynthetic cells. It has been postulated that an H+‐translocating ATPase energizes the transport of malic acid across the tonoplast into the vacuole. In the present work we have characterized the ATPase activity associated with vacuoles of the crassulacean‐acid‐metabolism plantKalanchoë daigremontianaand compare it with other phosphohydrolases.Vacuoles were isolated by polybase‐induced lysis of mesophyll‐cell protoplasts. The vacuoles had a high activity of unspecific acid phosphatase (pH optimum 5.3). The acid phosphatase was strongly inhibited by ammonium molybdate (with 50% inhibition at about 0.5 mmol m3‐), but was not completely inhibited even at much higher ammonium‐molybdate concentrations. In contrast, the vacuolar ATPase activity, assayed in the presence of 100 mmol m−3ammonium molybdate, had a pH optimum of 8.0. ATP was the preferred substrate, but GTP, ITP and ADP were hydrolyzed at appreciable rates. The mean ATPase activity at pH 8.0 was 14.5 mmol h−1(103vacuoles)−1an average 13% of which was attributable to residual acid‐phosphatase activity. Inorganic‐pyrophosphatase activity could not be demonstrated unambiguously.The vacuolar ATPase activity was M2+‐dependent, had an apparentKmfor MgATP2‐of 0.31 mol m−3, and was 32% stimulated by 50 mol m3‐KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO−3were both markedly inhibitory. Dicyclohexylcarbodiimide and tributyltin were also strongly inhibitory. Tributyltin caused a 50% inhibition at about 0.3 mmol m−3. This is taken as evidence that the vacuolar ATPase might function as an H+‐translocating ATPase.It is shown that the measured activity of the vacuolar ATPase would be of the right order to account for the observed rates of nocturnal malic‐acid accumulation inK. daigremontiana.