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Reconstitution and Characterization of a Calmodulin-Stimulated Ca-Pumping ATPase Purified from Brassica oleracea L
Jönköping University, School of Education and Communication, HLK, Disciplinary Research. Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.ORCID iD: 0000-0003-0117-2974
Department of Plant Sciences, University of Oxford, Oxford, United Kingdom.
1992 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 100, no 4, p. 1670-1681Article in journal (Refereed) Published
Abstract [en]

Purification and functional reconstitution of a calmodulin-stimulated Ca(2+)-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 Ca(2+), 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 Ca(2+) uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 mumol min(-1) (mg protein)(-1), and the Ca(2+)/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca(2+)-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 Ca(2+)) 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 Ca(2+)-pumping ATPase in Brassica oleracea inflorescences.

Place, publisher, year, edition, pages
1992. Vol. 100, no 4, p. 1670-1681
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Biochemistry and Molecular Biology
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URN: urn:nbn:se:hj:diva-2178PubMedID: 16653183OAI: oai:DiVA.org:hj-2178DiVA, id: diva2:32998
Available from: 2007-10-17 Created: 2007-10-17 Last updated: 2017-12-12Bibliographically approved

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