Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Jul;82(14):4573–4577. doi: 10.1073/pnas.82.14.4573

Direct demonstration of structural changes in soluble, monomeric Ca2+-ATPase associated with Ca2+ release during the transport cycle.

J P Andersen, P L Jørgensen, J V Møller
PMCID: PMC390427  PMID: 3161073

Abstract

The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state of membrane-bound and soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum have been examined at pH 8.0, 2 degrees C. The transition from ADP-sensitive to ADP-insensitive phosphoenzyme occurs in the soluble monomer as well as in membranous Ca2+-ATPase and is accompanied by an increase in fluorescence from 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine diphosphate bound to the catalytic site and change in tryptic cleavage pattern. A decrease of Ca2+ affinity occurs simultaneously with the fluorescence rise, suggesting a single-step mechanism for energy transfer between the catalytic site and the Ca2+ transport sites. This is in accordance with the tryptic degradation pattern that suggests proximity between the phosphorylation site and Ca2+ transport sites on the peptide. The structural changes occurring in the soluble monomeric Ca2+-ATPase show that a single polypeptide chain is the functional unit in energy transduction.

Full text

PDF
4573

Images in this article

4576Image
on p.4576
4576Image
on p.4576

Selected References

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

  1. Andersen J. P., Fellmann P., Møller J. V., Devaux P. F. Immobilization of a spin-labeled fatty acid chain covalently attached to Ca2+-ATPase from sarcoplasmic reticulum suggests an oligomeric structure. Biochemistry. 1981 Aug 18;20(17):4928–4936. doi: 10.1021/bi00520a019. [DOI] [PubMed] [Google Scholar]
  2. Andersen J. P., Lassen K., Møller J. V. Changes in Ca2+ affinity related to conformational transitions in the phosphorylated state of soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum. J Biol Chem. 1985 Jan 10;260(1):371–380. [PubMed] [Google Scholar]
  3. Andersen J. P., Møller J. V., Jørgensen P. L. The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements. J Biol Chem. 1982 Jul 25;257(14):8300–8307. [PubMed] [Google Scholar]
  4. Andersen J. P., le Maire M., Møller J. V. Properties of detergent-solubilized and membranous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum as studied by sulfhydryl reactivity and ESR spectroscopy. Effect of protein-protein interactions. Biochim Biophys Acta. 1980 Dec 2;603(1):84–100. doi: 10.1016/0005-2736(80)90393-4. [DOI] [PubMed] [Google Scholar]
  5. Dean W. L., Gray R. D. Effect of solubilization on adenosine 5'-triphosphate induced calcium release from purified sarcoplasmic reticulum calcium adenosinetriphosphatase. Biochemistry. 1983 Jan 18;22(2):515–519. doi: 10.1021/bi00271a039. [DOI] [PubMed] [Google Scholar]
  6. Dupont Y., Pougeois R. Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase. FEBS Lett. 1983 May 30;156(1):93–98. doi: 10.1016/0014-5793(83)80255-5. [DOI] [PubMed] [Google Scholar]
  7. Ikemoto N. Structure and function of the calcium pump protein of sarcoplasmic reticulum. Annu Rev Physiol. 1982;44:297–317. doi: 10.1146/annurev.ph.44.030182.001501. [DOI] [PubMed] [Google Scholar]
  8. Imamura Y., Saito K., Kawakita M. Conformational change of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum upon binding of Ca2+ and adenyl-5'-yl-imidodiphosphate as detected by trypsin sensitivity analysis. J Biochem. 1984 May;95(5):1305–1313. doi: 10.1093/oxfordjournals.jbchem.a134736. [DOI] [PubMed] [Google Scholar]
  9. Jorgensen P. L. Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool. Biochim Biophys Acta. 1975 Sep 2;401(3):399–415. doi: 10.1016/0005-2736(75)90239-4. [DOI] [PubMed] [Google Scholar]
  10. Kosk-Kosicka D., Kurzmack M., Inesi G. Kinetic characterization of detergent-solubilized sarcoplasmic reticulum adenosinetriphosphatase. Biochemistry. 1983 May 10;22(10):2559–2567. doi: 10.1021/bi00279a037. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lumry R. Conformational mechanisms for free energy transduction in protein systems: old ideas and new facts. Ann N Y Acad Sci. 1974 Feb 18;227:46–73. doi: 10.1111/j.1749-6632.1974.tb14373.x. [DOI] [PubMed] [Google Scholar]
  13. Martin D. W. Active unit of solubilized sarcoplasmic reticulum calcium adenosinetriphosphatase: an active enzyme centrifugation analysis. Biochemistry. 1983 Apr 26;22(9):2276–2282. doi: 10.1021/bi00278a034. [DOI] [PubMed] [Google Scholar]
  14. Martin D. W., Tanford C., Reynolds J. A. Monomeric solubilized sarcoplasmic reticulum Ca pump protein: demonstration of Ca binding and dissociation coupled to ATP hydrolysis. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6623–6626. doi: 10.1073/pnas.81.21.6623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martin D. W., Tanford C. Solubilized monomeric sarcoplasmic reticulum Ca pump protein. Phosphorylation by inorganic phosphate. FEBS Lett. 1984 Nov 5;177(1):146–150. doi: 10.1016/0014-5793(84)81000-5. [DOI] [PubMed] [Google Scholar]
  16. Møller J. V., Andersen J. P., le Maire M. The sarcoplasmic reticulum Ca2+-ATPase. Mol Cell Biochem. 1982 Feb 5;42(2):83–107. doi: 10.1007/BF00222696. [DOI] [PubMed] [Google Scholar]
  17. Nakamoto R. K., Inesi G. Studies of the interactions of 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine nucleotides with the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase active site. J Biol Chem. 1984 Mar 10;259(5):2961–2970. [PubMed] [Google Scholar]
  18. Tanford C. Mechanism of free energy coupling in active transport. Annu Rev Biochem. 1983;52:379–409. doi: 10.1146/annurev.bi.52.070183.002115. [DOI] [PubMed] [Google Scholar]
  19. Tanford C. Simple model for the chemical potential change of a transported ion in active transport. Proc Natl Acad Sci U S A. 1982 May;79(9):2882–2884. doi: 10.1073/pnas.79.9.2882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Taylor K., Dux L., Martonosi A. Structure of the vanadate-induced crystals of sarcoplasmic reticulum Ca2+-ATPase. J Mol Biol. 1984 Mar 25;174(1):193–204. doi: 10.1016/0022-2836(84)90372-3. [DOI] [PubMed] [Google Scholar]
  21. Wang C. T., Saito A., Fleischer S. Correlation of ultrastructure of reconstituted sarcoplasmic reticulum membrane vesicles with variation in phospholipid to protein ratio. J Biol Chem. 1979 Sep 25;254(18):9209–9219. [PubMed] [Google Scholar]
  22. Yamamoto T., Tonomura Y. Desensitization to Mg2+ of the phosphoenzyme in sarcoplasmic reticulum Ca2+-ATPase by the addition of a nonionic detergent and the restoration of sensitivity after its removal. J Biochem. 1982 Feb;91(2):477–486. doi: 10.1093/oxfordjournals.jbchem.a133720. [DOI] [PubMed] [Google Scholar]
  23. Yamamoto T., Yantorno R. E., Tonomura Y. Comparative study of the kinetic and structural properties of monomeric and oligomeric forms of sarcoplasmic reticulum ATPase. J Biochem. 1984 Jun;95(6):1783–1791. doi: 10.1093/oxfordjournals.jbchem.a134791. [DOI] [PubMed] [Google Scholar]
  24. Yantorno R. E., Yamamoto T., Tonomura Y. Energy transfer between fluorescent dyes attached to Ca2+,Mg2+-ATPase in the sarcoplasmic reticulum. J Biochem. 1983 Oct;94(4):1137–1145. doi: 10.1093/oxfordjournals.jbchem.a134458. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES