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Membranes and Bioenergetics

Reconstitution and Characterization of a Calmodulin-Stimulated Ca²⁺-Pumping ATPase Purified from Brassica oleracea L. ¹

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, United Kingdom

Purification and functional reconstitution of a calmodulin-stimulated Ca²⁺-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca²⁺, together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropyl]-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca²⁺ uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 µmol min^–1 (mg protein)^–1, and the Ca²⁺/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca²⁺-ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca²⁺) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca²⁺-pumping ATPase in Brassica oleracea inflorescences.

³ Royal Society 1983 University Research Fellow.

¹ Supported by a grant from the United Kingdom Agricultural and Food Research Council (AFRC) under its Plant Molecular Biology initiative.

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