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. 1992 Dec;100(4):1670–1681. doi: 10.1104/pp.100.4.1670

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

Per Askerlund 1,2, David E Evans 1,3
PMCID: PMC1075850  PMID: 16653183

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Askerlund P., Laurent P., Nakagawa H., Kader J. C. NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase. Plant Physiol. 1991 Jan;95(1):6–13. doi: 10.1104/pp.95.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borke J. L., Caride A. J., Yaksh T. L., Penniston J. T., Kumar R. Cerebrospinal fluid calcium homeostasis: evidence for a plasma membrane Ca2+-pump in mammalian choroid plexus. Brain Res. 1989 Jun 12;489(2):355–360. doi: 10.1016/0006-8993(89)90870-6. [DOI] [PubMed] [Google Scholar]
  3. Briars S. A., Evans D. E. The calmodulin-stimulated ATPase of maize coleoptiles forms a phosphorylated intermediate. Biochem Biophys Res Commun. 1989 Feb 28;159(1):185–191. doi: 10.1016/0006-291x(89)92421-2. [DOI] [PubMed] [Google Scholar]
  4. Briskin D. P. Ca-translocating ATPase of the plant plasma membrane. Plant Physiol. 1990 Oct;94(2):397–400. doi: 10.1104/pp.94.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buckhout T. J. Characterization of Ca Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots. Plant Physiol. 1984 Dec;76(4):962–967. doi: 10.1104/pp.76.4.962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carafoli E. The calcium pumping ATPase of the plasma membrane. Annu Rev Physiol. 1991;53:531–547. doi: 10.1146/annurev.ph.53.030191.002531. [DOI] [PubMed] [Google Scholar]
  7. Carafoli E., Zurini M. The Ca2+-pumping ATPase of plasma membranes. Purification, reconstitution and properties. Biochim Biophys Acta. 1982 Dec 31;683(3-4):279–301. doi: 10.1016/0304-4173(82)90004-0. [DOI] [PubMed] [Google Scholar]
  8. Cornelius F. Functional reconstitution of the sodium pump. Kinetics of exchange reactions performed by reconstituted Na/K-ATPase. Biochim Biophys Acta. 1991 Mar 7;1071(1):19–66. doi: 10.1016/0304-4157(91)90011-k. [DOI] [PubMed] [Google Scholar]
  9. Giannini J. L., Gildensoph L. H., Reynolds-Niesman I., Briskin D. P. Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum. Plant Physiol. 1987 Dec;85(4):1129–1136. doi: 10.1104/pp.85.4.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gräf P., Weiler E. W. Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L. Plant Physiol. 1990 Oct;94(2):634–640. doi: 10.1104/pp.94.2.634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Helmke S. M., Howard B. D. Fractionation and reconstitution of the sarcoplasmic reticulum Ca2+ pump solubilized and stabilized by CHAPS/lipid micelles. Membr Biochem. 1987;7(1):1–22. doi: 10.3109/09687688709029426. [DOI] [PubMed] [Google Scholar]
  12. Hsieh W. L., Pierce W. S., Sze H. Calcium-pumping ATPases in vesicles from carrot cells : stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme. Plant Physiol. 1991 Dec;97(4):1535–1544. doi: 10.1104/pp.97.4.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kaplan R. S., Pedersen P. L. Sensitive protein assay in presence of high levels of lipid. Methods Enzymol. 1989;172:393–399. doi: 10.1016/s0076-6879(89)72025-5. [DOI] [PubMed] [Google Scholar]
  14. Kasai M., Muto S. Solubilization and reconstitution of ca pump from corn leaf plasma membrane. Plant Physiol. 1991 Jun;96(2):565–570. doi: 10.1104/pp.96.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Neugebauer J. M. Detergents: an overview. Methods Enzymol. 1990;182:239–253. doi: 10.1016/0076-6879(90)82020-3. [DOI] [PubMed] [Google Scholar]
  17. Penniston J. T., Filoteo A. G., McDonough C. S., Carafoli E. Purification, reconstitution, and regulation of plasma membrane Ca2+-pumps. Methods Enzymol. 1988;157:340–351. doi: 10.1016/0076-6879(88)57089-1. [DOI] [PubMed] [Google Scholar]
  18. Pohl T. Concentration of proteins and removal of solutes. Methods Enzymol. 1990;182:68–83. doi: 10.1016/0076-6879(90)82009-q. [DOI] [PubMed] [Google Scholar]
  19. Preiss J., Danner S., Summers P. S., Morell M., Barton C. R., Yang L., Nieder M. Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits. Plant Physiol. 1990 Apr;92(4):881–885. doi: 10.1104/pp.92.4.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Racker E. Reconstitution of membrane processes. Methods Enzymol. 1979;55:699–711. doi: 10.1016/0076-6879(79)55078-2. [DOI] [PubMed] [Google Scholar]
  21. Rasi-Caldogno F., Carnelli A., De Michelis M. I. Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin. Plant Physiol. 1992 Mar;98(3):1202–1206. doi: 10.1104/pp.98.3.1202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Seidler N. W., Jona I., Vegh M., Martonosi A. Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. J Biol Chem. 1989 Oct 25;264(30):17816–17823. [PubMed] [Google Scholar]
  23. Stoscheck C. M. Quantitation of protein. Methods Enzymol. 1990;182:50–68. doi: 10.1016/0076-6879(90)82008-p. [DOI] [PubMed] [Google Scholar]
  24. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tsien R. Y., Rink T. J. Ca2+-selective electrodes: a novel PVC-gelled neutral carrier mixture compared with other currently available sensors. J Neurosci Methods. 1981 Jun;4(1):73–86. doi: 10.1016/0165-0270(81)90020-0. [DOI] [PubMed] [Google Scholar]

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