|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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.