Identification and Characterization of the Ca2+-ATPase which Drives Active Transport of Ca2+ at the Plasma Membrane of Radish Seedlings

Abstract

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca(2+) uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca(2+)-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca(2+)-dependent ATPase is stimulated by the Ca(2+) ionophore A(23187) and is localized at the plasma membrane. Ca(2+)-dependent ATPase activity and ATP-dependent Ca(2+) uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca(2+) (half-maximal rate at about 70 nanomolar), and MgATP (K(m) 15-20 micromolar). Ca(2+) uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca(2+) uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca(2+)-ATPase described in this paper is the enzyme which drives active transport of Ca(2+) at the plasma membrane of higher plants.