Research on Active Calcium Transport Systems of Plant Vacuoles

在植物细胞中，液泡是钙离子的主要储存库。胞质溶胶中Ca^<2+>的浓度保持在较低水平，约为几百纳摩尔。植物液泡膜上存在两种不同的活性Ca^<2+>转运系统。在这项工作中，我们建立了Ca^<2+>进入液泡膜泡的活性测定方法，并分别测定了Ca^<2+>-ATP酶和Ca^<2+>/H^+反转运蛋白的活性。Ca^<2+>/H^+反转运体是由液泡质子泵产生的膜泡的质子梯度驱动的。我们用膜过滤法测定了放射性^<45>Ca的活度。这两种活性均被钙离子载体A23187强烈抑制。这意味着^<45>Ca被积极地运输到膜泡中。Ca^<2+>-ATP酶和Ca^<2+>/H^+反转运蛋白在Ca^<2+>的Km中表现出不同的性质。Ca^<2+>-ATP酶和Ca^<2+>/H^+ Ca^<2+>反转运蛋白的Km值分别约为2muM和20muM。其最大活性是Ca^<2+>-ATP酶的3倍。结果表明，Ca^<2+>-ATP酶是一种高亲和力、低容量的Ca^<2+>转运蛋白，而Ca^<2+>/H^+反转运蛋白是一种低亲和力、高容量的Ca^<2+>转运蛋白。据估计，大量细胞质Ca^<2+>被Ca^<2+>/H^+反转运蛋白占用。Ca^<2+>-ATP酶可使细胞质Ca^<2+>浓度降低小于1微摩尔。现在我们正在对Ca^<2+>/H^+反转运蛋白的cdna进行测序。

In plant cells, vacuole is the main pool of calcium ions. The concentration of Ca^<2+> in the cytosol is kept at low levels, about a few hundred nanomolar. There are two different active Ca^<2+> transport systems on the plant vacuolar membranes. In this work, we established the assay method for the activity of Ca^<2+> uptake into the vacuolar membrane vesicles and determined the activities of Ca^<2+>-ATP ase and Ca^<2+>/H^+ antiporter individually. The Ca^<2+>/H^+ antiporter is driven by the proton gradient of the membrane vesicles generated by the vacuolar proton pumps. We determined the activity by a membrane filtration method with radioactive ^<45>Ca. Both activities were strongly inhibited by a calcium ionophore A23187. It means that ^<45>Ca was actively transported into the membrane vesicles. The Ca^<2+>-ATP ase and Ca^<2+>/H^+ antiporter exhibited different properties in the Km for Ca^<2+>. The Km values of Ca^<2+>-ATP ase and Ca^<2+>/H^+ antiporter for Ca^<2+> were about 2muM,and 20muM,respectively. The maximal activity of the antiporter was 3-times higher than the Ca^<2+>-ATP ase. From our observation, it was concluded that the Ca^<2+>-ATP ase is a high affinity, low capacity Ca^<2+> transporter, and that the Ca^<2+>/H^+ antiporter is a low affinity, high capacity Ca^<2+> transporter. It is estimated that a large quantity of cytoplasmic Ca^<2+> is taken up by Ca^<2+>/H^+ antiporter. The Ca^<2+>-ATP ase may reduce the cytoplasmic Ca^<2+> concentration less than a micromolar. Now we are sequencing the cDNAs for the Ca^<2+>/H^+ antiporter.