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. 1992 Nov 1;287(Pt 3):767–774. doi: 10.1042/bj2870767

Characterization of ruthenium red-binding sites of the Ca(2+)-ATPase from sarcoplasmic reticulum and their interaction with Ca(2+)-binding sites.

S Corbalan-Garcia 1, J A Teruel 1, J C Gomez-Fernandez 1
PMCID: PMC1133074  PMID: 1280106

Abstract

Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5'-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites.

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Selected References

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

  1. Brandl C. J., Green N. M., Korczak B., MacLennan D. H. Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell. 1986 Feb 28;44(4):597–607. doi: 10.1016/0092-8674(86)90269-2. [DOI] [PubMed] [Google Scholar]
  2. Chadwick C. C., Thomas E. W. Inactivation of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide. Biochim Biophys Acta. 1983 May 5;730(2):201–206. doi: 10.1016/0005-2736(83)90334-6. [DOI] [PubMed] [Google Scholar]
  3. Chadwick C. C., Thomas E. W. Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide. Biochim Biophys Acta. 1984 Jan 25;769(2):291–296. doi: 10.1016/0005-2736(84)90309-2. [DOI] [PubMed] [Google Scholar]
  4. Chamberlain B. K., Volpe P., Fleischer S. Inhibition of calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. J Biol Chem. 1984 Jun 25;259(12):7547–7553. [PubMed] [Google Scholar]
  5. Champeil P., Guillain F., Vénien C., Gingold M. P. Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosinetriphosphatase as reflected by organic solvent induced perturbation. Biochemistry. 1985 Jan 1;24(1):69–81. doi: 10.1021/bi00322a012. [DOI] [PubMed] [Google Scholar]
  6. Charuk J. H., Pirraglia C. A., Reithmeier R. A. Interaction of ruthenium red with Ca2(+)-binding proteins. Anal Biochem. 1990 Jul;188(1):123–131. doi: 10.1016/0003-2697(90)90539-l. [DOI] [PubMed] [Google Scholar]
  7. Clarke D. M., Loo T. W., Inesi G., MacLennan D. H. Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase. Nature. 1989 Jun 8;339(6224):476–478. doi: 10.1038/339476a0. [DOI] [PubMed] [Google Scholar]
  8. De Meis L., Suzano V. A., Caldeira T., Mintz E., Guillain F. Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase. Eur J Biochem. 1991 Aug 15;200(1):209–213. doi: 10.1111/j.1432-1033.1991.tb21069.x. [DOI] [PubMed] [Google Scholar]
  9. Dupont Y. Low-temperature studies of the sarcoplasmic reticulum calcium pump. Mechanisms of calcium binding. Biochim Biophys Acta. 1982 May 21;688(1):75–87. doi: 10.1016/0005-2736(82)90580-6. [DOI] [PubMed] [Google Scholar]
  10. Eletr S., Inesi G. Phospholipid orientation in sarcoplasmic membranes: spin-label ESR and proton MNR studies. Biochim Biophys Acta. 1972 Sep 1;282(1):174–179. doi: 10.1016/0005-2736(72)90321-5. [DOI] [PubMed] [Google Scholar]
  11. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  12. Guillain F., Gingold M. P., Champeil P. Direct fluorescence measurements of Mg2+ binding to sarcoplasmic reticulum ATPase. J Biol Chem. 1982 Jul 10;257(13):7366–7371. [PubMed] [Google Scholar]
  13. Herrmann T. R., Shamoo A. E. Ionophorous properties of the 13 000-Da fragment from sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase. Biochim Biophys Acta. 1983 Aug 10;732(3):647–650. doi: 10.1016/0005-2736(83)90242-0. [DOI] [PubMed] [Google Scholar]
  14. Highsmith S. R., Head M. R. Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase. J Biol Chem. 1983 Jun 10;258(11):6858–6862. [PubMed] [Google Scholar]
  15. Highsmith S. Evidence that the ATP binding site of sarcoplasmic reticulum CaATPase has a Mg(2+) ion binding sub-site. Biochem Biophys Res Commun. 1984 Oct 15;124(1):183–189. doi: 10.1016/0006-291x(84)90934-3. [DOI] [PubMed] [Google Scholar]
  16. Inesi G. Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase. J Biol Chem. 1987 Dec 5;262(34):16338–16342. [PubMed] [Google Scholar]
  17. Inesi G., Sumbilla C., Kirtley M. E. Relationships of molecular structure and function in Ca2(+)-transport ATPase. Physiol Rev. 1990 Jul;70(3):749–760. doi: 10.1152/physrev.1990.70.3.749. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. 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]
  21. Lin T. I., Morales M. F. Application of a one-step procedure for measuring inorganic phosphate in the presence of proteins: the actomyosin ATPase system. Anal Biochem. 1977 Jan;77(1):10–17. doi: 10.1016/0003-2697(77)90284-6. [DOI] [PubMed] [Google Scholar]
  22. Luft J. H. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. Anat Rec. 1971 Nov;171(3):347–368. doi: 10.1002/ar.1091710302. [DOI] [PubMed] [Google Scholar]
  23. Lüdi H., Hasselbach W. Separation of the tryptic fragments of sarcoplasmic reticulum ATPase with high performance liquid chromatography. Identification of the calcium binding site. FEBS Lett. 1984 Feb 13;167(1):33–36. doi: 10.1016/0014-5793(84)80827-3. [DOI] [PubMed] [Google Scholar]
  24. MacLennan D. H., Brandl C. J., Korczak B., Green N. M. Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature. 1985 Aug 22;316(6030):696–700. doi: 10.1038/316696a0. [DOI] [PubMed] [Google Scholar]
  25. Madeira V. M., Antunes-Madeira M. C. Interaction of ruthenium red with isolated sarcolemma. J Membr Biol. 1974;17(1):41–50. doi: 10.1007/BF01870171. [DOI] [PubMed] [Google Scholar]
  26. Moore C. L. Specific inhibition of mitochondrial Ca++ transport by ruthenium red. Biochem Biophys Res Commun. 1971 Jan 22;42(2):298–305. doi: 10.1016/0006-291x(71)90102-1. [DOI] [PubMed] [Google Scholar]
  27. Moutin M. J., Dupont Y. Interaction of potassium and magnesium with the high affinity calcium-binding sites of the sarcoplasmic reticulum calcium-ATPase. J Biol Chem. 1991 Mar 25;266(9):5580–5586. [PubMed] [Google Scholar]
  28. Munkonge F., East J. M., Lee A. G. Positions of the sites labeled by N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodiimide on the (Ca2+ + Mg2+)-ATPase. Biochim Biophys Acta. 1989 Feb 13;979(1):113–120. doi: 10.1016/0005-2736(89)90530-0. [DOI] [PubMed] [Google Scholar]
  29. Murphy A. J. Kinetics of the inactivation of the ATPase of sarcoplasmic reticulum by dicyclohexylcarbodiimide. J Biol Chem. 1981 Dec 10;256(23):12046–12050. [PubMed] [Google Scholar]
  30. Nakamura J. Calcium-dependent non-equivalent characteristics of calcium binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Biochim Biophys Acta. 1986 Apr 22;870(3):495–501. doi: 10.1016/0167-4838(86)90258-x. [DOI] [PubMed] [Google Scholar]
  31. Orlowski S., Champeil P. Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase. Biochemistry. 1991 Jan 15;30(2):352–361. doi: 10.1021/bi00216a007. [DOI] [PubMed] [Google Scholar]
  32. Petithory J. R., Jencks W. P. Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum. Biochemistry. 1988 Nov 15;27(23):8626–8635. doi: 10.1021/bi00423a018. [DOI] [PubMed] [Google Scholar]
  33. Pick U., Karlish S. J. Indications for an oligomeric structure and for conformational changes in sarcoplasmic reticulum Ca2+-ATPase labelled selectively with fluorescein. Biochim Biophys Acta. 1980 Nov 20;626(1):255–261. doi: 10.1016/0005-2795(80)90216-0. [DOI] [PubMed] [Google Scholar]
  34. Pick U., Racker E. Inhibition of the (Ca2+)ATPase from sarcoplasmic reticulum by dicyclohexylcarbodiimide: evidence for location of the Ca2+ binding site in a hydrophobic region. Biochemistry. 1979 Jan 9;18(1):108–113. doi: 10.1021/bi00568a017. [DOI] [PubMed] [Google Scholar]
  35. Pick U., Weiss M. Spectral and catalytical properties of the sarcoplasmic reticulum Ca-ATPase labeled with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)-carbodiimide. Eur J Biochem. 1985 Oct 1;152(1):83–89. doi: 10.1111/j.1432-1033.1985.tb09166.x. [DOI] [PubMed] [Google Scholar]
  36. Reed K. C., Bygrave F. L. The inhibition of mitochondrial calcium transport by lanthanides and ruthenium red. Biochem J. 1974 May;140(2):143–155. doi: 10.1042/bj1400143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scofano H. M., Barrabin H., Lewis D., Inesi G. Specific dicyclohexylcarbodiimide inhibition of the E-P + H2O equilibrium E + Pi reaction and ATP equilibrium Pi exchange in sarcoplasmic reticulum adenosinetriphosphatase. Biochemistry. 1985 Feb 12;24(4):1025–1029. doi: 10.1021/bi00325a033. [DOI] [PubMed] [Google Scholar]
  38. Shamoo A. E., Ryan T. E., Stewart P. S., MacLennan D. H. Localization of ionophore activity in a 20,000-dalton fragment of the adenosine triphosphatase of Sarcoplasmic reticulum. J Biol Chem. 1976 Jul 10;251(13):4147–4154. [PubMed] [Google Scholar]
  39. Squier T. C., Bigelow D. J., Fernandez-Belda F. J., deMeis L., Inesi G. Calcium and lanthanide binding in the sarcoplasmic reticulum ATPase. J Biol Chem. 1990 Aug 15;265(23):13713–13720. [PubMed] [Google Scholar]
  40. Stephens E. M., Grisham C. M. Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance, and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase. Biochemistry. 1979 Oct 30;18(22):4876–4885. doi: 10.1021/bi00589a016. [DOI] [PubMed] [Google Scholar]
  41. Stewart P. S., MacLennan D. H. Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum. J Biol Chem. 1976 Feb 10;251(3):712–719. [PubMed] [Google Scholar]
  42. Sumbilla C., Cantilina T., Collins J. H., Malak H., Lakowicz J. R., Inesi G. Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase. J Biol Chem. 1991 Jul 5;266(19):12682–12689. [PubMed] [Google Scholar]
  43. Teruel J. A., Inesi G. Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase. Biochemistry. 1988 Aug 9;27(16):5885–5890. doi: 10.1021/bi00416a010. [DOI] [PubMed] [Google Scholar]
  44. Teruel J. A., Kurzmack M., Inesi G. Kinetic and thermodynamic control of ATP synthesis by sarcoplasmic reticulum adenosinetriphosphatase. J Biol Chem. 1987 Sep 25;262(27):13055–13060. [PubMed] [Google Scholar]
  45. Thorley-Lawson D. A., Green N. M. Studies on the location and orientation of proteins in the sarcoplasmic reticulum. Eur J Biochem. 1973 Dec 17;40(2):403–413. doi: 10.1111/j.1432-1033.1973.tb03209.x. [DOI] [PubMed] [Google Scholar]
  46. Vale M. G., Carvalho A. P. Effects of ruthenium red on Ca2+ uptake and ATPase of sarcoplasmic reticulum of rabbit skeletal muscle. Biochim Biophys Acta. 1973 Oct 19;325(1):29–37. doi: 10.1016/0005-2728(73)90147-3. [DOI] [PubMed] [Google Scholar]
  47. Vasington F. D., Gazzotti P., Tiozzo R., Carafoli E. The effect of ruthenium red on Ca 2+ transport and respiration in rat liver mitochondria. Biochim Biophys Acta. 1972 Jan 21;256(1):43–54. doi: 10.1016/0005-2728(72)90161-2. [DOI] [PubMed] [Google Scholar]
  48. Volpe P., Salviati G., Chu A. Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers. J Gen Physiol. 1986 Feb;87(2):289–303. doi: 10.1085/jgp.87.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Watson E. L., Vincenzi F. F., Davis P. W. Ca 2+ -activated membrane ATPase: selective inhibition by ruthenium red. Biochim Biophys Acta. 1971 Dec 3;249(2):606–610. doi: 10.1016/0005-2736(71)90140-4. [DOI] [PubMed] [Google Scholar]
  50. de Ancos J. G., Inesi G. Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase. Biochemistry. 1988 Mar 8;27(5):1793–1803. doi: 10.1021/bi00405a061. [DOI] [PubMed] [Google Scholar]
  51. de Meis L. Fast efflux of Ca2+ mediated by the sarcoplasmic reticulum Ca2(+)-ATPase. J Biol Chem. 1991 Mar 25;266(9):5736–5742. [PubMed] [Google Scholar]

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