Smith J A, Uribe E G, Ball E, Heuer S, Lüttge U
Eur J Biochem. 1984 Jun 1;141(2):415-20. doi: 10.1111/j.1432-1033.1984.tb08207.x.
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 plant Kalanchoë daigremontiana and 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 m-3), 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-3 ammonium 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 nmol h-1 (10(3) vacuoles)-1, an average 13% of which was attributable to residual acid-phosphatase activity. Inorganic-pyrophosphatase activity could not be demonstrated unambiguously. The vacuolar ATPase activity was Mg2+-dependent, had an apparent Km for MgATP2- of 0.31 mol m-3, and was 32% stimulated by 50 mol m-3 KCl. Of the inhibitors tested, oligomycin slightly inhibited the vacuolar ATPase activity and diethylstilbestrol and NO-3 were 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 in K. daigremontiana.
进行景天酸代谢的植物在光合细胞的液泡中夜间会大量积累苹果酸。据推测，一种H⁺转运ATP酶为苹果酸跨液泡膜进入液泡的运输提供能量。在本研究中，我们对景天酸代谢植物落地生根液泡相关的ATP酶活性进行了表征，并将其与其他磷酸水解酶进行了比较。通过多碱诱导叶肉细胞原生质体裂解来分离液泡。这些液泡具有高活性的非特异性酸性磷酸酶（最适pH为5.3）。酸性磷酸酶受到钼酸铵的强烈抑制（在约0.5 mmol m⁻³时抑制50%），但即使在更高的钼酸铵浓度下也不会被完全抑制。相比之下，在100 mmol m⁻³钼酸铵存在下测定的液泡ATP酶活性，其最适pH为8.0。ATP是首选底物，但GTP、ITP和ADP也能以可观的速率被水解。在pH 8.0时的平均ATP酶活性为14.5 nmol h⁻¹（10³个液泡）⁻¹，其中平均13%可归因于残留的酸性磷酸酶活性。无机焦磷酸酶活性无法明确证实。液泡ATP酶活性依赖于Mg²⁺，对MgATP²⁻的表观Km为0.31 mol m⁻³，并且受到50 mol m⁻³ KCl的32%的刺激。在所测试的抑制剂中，寡霉素对液泡ATP酶活性有轻微抑制作用，己烯雌酚和NO₃均有明显抑制作用。二环己基碳二亚胺和三丁基锡也有强烈抑制作用。三丁基锡在约0.3 mmol m⁻³时导致50%的抑制。这被视为液泡ATP酶可能作为H⁺转运ATP酶发挥作用的证据。结果表明，所测得的液泡ATP酶活性大小足以解释在落地生根中观察到的夜间苹果酸积累速率。
