A PLASMA MEMBRANE-TYPE CA-2+-ATPASE OF 120 KILODALTONS ON THE ENDOPLASMIC-RETICULUM FROM CARROT (DAUCUS-CAROTA) CELLS - PROPERTIES OF THE PHOSPHORYLATED INTERMEDIATE

Cytosolic Ca2+ levels are regulated in part by Ca2+-pumping ATPases that export Ca2+ from the cytoplasm; however, the types and properties of Ca2+ pumps in plants are not well understood. We have characterized the kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca2+-ATPase from suspension-cultured carrot (Daucus carota) cells. Only one Ca2+-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [gamma-P-32]ATP (W.L. Hsieh, W.S. Pierce,and H.Sze [1991] Plant Physiol 97:1535-1544). Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La3+, and decreased by hydroxylamine, confirming its identification as an intermediate of a phosphorylated-type Ca2+-translocating ATPase. The 120-kD Ca2+-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca2+-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca2+-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a K(m) for Ca2+ of 1 to 2 mum and a K(m) for ATP of 67 nm. Relative affinities of substrates, determined by competition experiments, were 0.075 mum f or ATP, 1 mum f or ADP, 1 00 mum for ITP, and 2 50 mum for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.l mum. Calmodulin (1 mum) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca2+-ATPase is similar but not identical to animal plasma membrane-type Ca2+-ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca2+ pump may reside on the cortical endoplasmic reticulum, which is thought to play a major role in anchoring the cytoskeleton and in facilitating secretion.