Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1985 Nov 1;231(3):737–742. doi: 10.1042/bj2310737

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

J Verbist, F Wuytack, L Raeymaekers, R Casteels
PMCID: PMC1152810  PMID: 2934057

Abstract

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

Full text

PDF
735

Images in this article

740Image
on p.740

Selected References

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

  1. 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]
  2. Caroni P., Carafoli E. The Ca2+-pumping ATPase of heart sarcolemma. Characterization, calmodulin dependence, and partial purification. J Biol Chem. 1981 Apr 10;256(7):3263–3270. [PubMed] [Google Scholar]
  3. Carsten M. E., Miller J. D. Properties of a phosphorylated intermediate of the Ca,Mg-activated ATPase of microsomal vesicles from uterine smooth muscle. Arch Biochem Biophys. 1984 Aug 1;232(2):616–623. doi: 10.1016/0003-9861(84)90581-2. [DOI] [PubMed] [Google Scholar]
  4. Chiesi M., Gasser J., Carafoli E. Properties of the Ca-pumping ATPase of sarcoplasmic reticulum from vascular smooth muscle. Biochem Biophys Res Commun. 1984 Nov 14;124(3):797–806. doi: 10.1016/0006-291x(84)91028-3. [DOI] [PubMed] [Google Scholar]
  5. Clark A., Carafoli E. The stoichiometry of the Ca2+-pumping ATPase of erythrocytes. Cell Calcium. 1983 Apr;4(2):83–88. doi: 10.1016/0143-4160(83)90037-4. [DOI] [PubMed] [Google Scholar]
  6. De Schutter G., Wuytack F., Verbist J., Casteels R. Tissue levels and purification by affinity chromatography of the calmodulin-stimulated Ca2+ -transport ATPase in pig antrum smooth muscle. Biochim Biophys Acta. 1984 Jun 13;773(1):1–10. doi: 10.1016/0005-2736(84)90544-3. [DOI] [PubMed] [Google Scholar]
  7. Gietzen K., Tejcka M., Wolf H. U. Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca+ + Mg2+)-dependent adenosine triphosphatase. Biochem J. 1980 Jul 1;189(1):81–88. doi: 10.1042/bj1890081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gopalakrishna R., Anderson W. B. Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography. Biochem Biophys Res Commun. 1982 Jan 29;104(2):830–836. doi: 10.1016/0006-291x(82)90712-4. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Martonosi A., Halpin R. A. Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes. Arch Biochem Biophys. 1971 May;144(1):66–77. doi: 10.1016/0003-9861(71)90455-3. [DOI] [PubMed] [Google Scholar]
  11. Niggli V., Penniston J. T., Carafoli E. Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column. J Biol Chem. 1979 Oct 25;254(20):9955–9958. [PubMed] [Google Scholar]
  12. Raeymaekers L., Wuytack F., Casteels R. Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum. Biochim Biophys Acta. 1985 May 28;815(3):441–454. doi: 10.1016/0005-2736(85)90372-4. [DOI] [PubMed] [Google Scholar]
  13. Steck T. L., Kant J. A. Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods Enzymol. 1974;31:172–180. doi: 10.1016/0076-6879(74)31019-1. [DOI] [PubMed] [Google Scholar]
  14. Steinbuch M., Audran R. Isolement de l'immunoglobuline IgG du plasma humain à l'aide de l'acide caprylique. Rev Fr Etud Clin Biol. 1969 Dec;14(10):1054–1058. [PubMed] [Google Scholar]
  15. Suko J., Hasselbach W. Characterization of cardiac sarcoplasmic reticulum ATP-ADP phosphate exchange and phosphorylation of the calcium transport adenosine triphosphatase. Eur J Biochem. 1976 Apr 15;64(1):123–130. doi: 10.1111/j.1432-1033.1976.tb10280.x. [DOI] [PubMed] [Google Scholar]
  16. Verbist J., Wuytack F., De Schutter G., Raeymaekers L., Casteels R. Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle. Cell Calcium. 1984 Jun;5(3):253–263. doi: 10.1016/0143-4160(84)90040-x. [DOI] [PubMed] [Google Scholar]
  17. Verma A. K., Gorski J. P., Penniston J. T. Antibodies directed toward human erythrocyte Ca2+-ATPase: effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources. Arch Biochem Biophys. 1982 May;215(2):345–354. doi: 10.1016/0003-9861(82)90095-9. [DOI] [PubMed] [Google Scholar]
  18. Wuytack F., De Schutter G., Casteels R. Purification of (Ca2+ + Mg2+)-ATPase from smooth muscle by calmodulin affinity chromatography. FEBS Lett. 1981 Jul 6;129(2):297–300. doi: 10.1016/0014-5793(81)80187-1. [DOI] [PubMed] [Google Scholar]
  19. Wuytack F., De Schutter G., Verbist J., Casteels R. Antibodies to the calmodulin-binding Ca2+-transport ATPase from smooth muscle. FEBS Lett. 1983 Apr 5;154(1):191–195. doi: 10.1016/0014-5793(83)80901-6. [DOI] [PubMed] [Google Scholar]
  20. Wuytack F., Raeymaekers L., De Schutter G., Casteels R. Demonstration of the phosphorylated intermediates of the Ca2+-transport ATPase in a microsomal fraction and in a (Ca2+ + Mg2+)-ATPase purified from smooth muscle by means of calmodulin affinity chromatography. Biochim Biophys Acta. 1982 Dec 8;693(1):45–52. doi: 10.1016/0005-2736(82)90469-2. [DOI] [PubMed] [Google Scholar]
  21. Wuytack F., Raeymaekers L., Verbist J., De Smedt H., Casteels R. Evidence for the presence in smooth muscle of two types of Ca2+-transport ATPase. Biochem J. 1984 Dec 1;224(2):445–451. doi: 10.1042/bj2240445. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES