title: Calcium-pumping ATPases in vesicles from carrot cells : stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme.
abstract: Ca(2+)-ATPases keep cytoplasmic [Ca(2+)] low by pumping Ca(2+) into intracellular compartments or out of the cell. The transport properties of Ca(2+)-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca(2+) transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase) partially inhibited oxalate-stimulated Ca(2+) transport activity; however, it had no effect on calmodulin-stimulated Ca(2+) uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca(2+)-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma(32)P]ATP resulted in the formation of a single acyl [(32)P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca(2+), but independent of Mg(2+). Its enhancement by La(3+) is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca(2+) transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca(2+) transport, similar to their effect on the erythrocyte plasma membrane Ca(2+)-ATPase. These results would indicate that the calmodulin-stimulated Ca(2+) transport originated in large part from a plasma membrane-type Ca(2+) pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca(2+)-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested.
authors: Department of Botany, University of Maryland, College Park, Maryland 20742.
authors: W L
authors: WL
authors: Hsieh
authors: W S
authors: WS
authors: Pierce
authors: H
authors: H
authors: Sze