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. 1991 Dec;97(4):1535–1544. doi: 10.1104/pp.97.4.1535

Calcium-Pumping ATPases in Vesicles from Carrot Cells 1

Stimulation by Calmodulin or Phosphatidylserine, and Formation of a 120 Kilodalton Phosphoenzyme

Wen-Ling Hsieh 1, Wayne S Pierce 1,2, Heven Sze 1
PMCID: PMC1081197  PMID: 16668581

Abstract

Ca2+-ATPases keep cytoplasmic [Ca2+] low by pumping Ca2+ into intracellular compartments or out of the cell. The transport properties of Ca2+-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca2+ 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 Ca2+-ATPase) partially inhibited oxalate-stimulated Ca2+ transport activity; however, it had no effect on calmodulin-stimulated Ca2+ uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca2+-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma32P]ATP resulted in the formation of a single acyl [32P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca2+, but independent of Mg2+. Its enhancement by La3+ is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca2+ transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca2+ transport, similar to their effect on the erythrocyte plasma membrane Ca2+-ATPase. These results would indicate that the calmodulin-stimulated Ca2+ transport originated in large part from a plasma membrane-type Ca2+ pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca2+-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested.

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Selected References

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