PLANT PHYSIOLOGY , Vol 102, Issue 2 651-661, Copyright © 1993 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
A Plasma Membrane-Type Ca2+-ATPase of 120 Kilodaltons on the Endoplasmic Reticulum from Carrot (Daucus carota) Cells (Properties of the Phosphorylated Intermediate)
F. H. Chen, D. M. Ratterman and H. Sze
Department of Botany and The Maryland Agricultural Experiment Station, University of Maryland, College Park, Maryland 20742
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]-32P]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 Km for Ca2+ of 1 to
2 [mu]M and a Km for ATP of 67 nM. Relative affinities of substrates,
determined by competition experiments, were 0.075 [mu]M for ATP, 1 [mu]M
for ADP, 100 [mu]M for ITP, and 250 [mu]M 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 <0.1 [mu]M. Calmodulin (1 [mu]M)
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.
