Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Oct;77(4):2217–2225. doi: 10.1016/S0006-3495(99)77062-1

Characterization of calcium, nucleotide, phosphate, and vanadate bound states by derivatization of sarcoplasmic reticulum ATPase with ThioGlo1.

S Hua 1, D Fabris 1, G Inesi 1
PMCID: PMC1300502  PMID: 10512841

Abstract

Sarcoplasmic reticulum vesicles were incubated with the maleimide-directed probe ThioGlo1, resulting in ATPase inactivation. Reacted ThioGlo1, revealed by its enhanced fluorescence, was found to be associated with the cytosolic but not with the membrane-bound region of the ATPase. The dependence of inactivation on ThioGlo1 concentration suggests derivatization of approximately four residues per ATPase, of which Cys(364), Cys(498), and Cys(636) were identified in prominently fluorescent peptide fragments. These cysteines reside within the phosphorylation and nucleotide-binding region of the ATPase. Accordingly, protection is observed in the presence of ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-AMP), or an fluoroisothiocyanate label of Lys(515). Furthermore, protection is observed in the presence of vanadate (or decavanadate), but not in the presence of phosphate. Labeling occurs equally well in the presence or in the absence of Ca(2+) and thapsigargin, excluding a role of the E1-to-E2 transition in the protective effect of vanadate. It is concluded that protection by vanadate is due to formation of a pentacoordinated orthovanadate complex at the phosphorylation site, corresponding to a stable transition state analog of the phosphorylation reaction, with intermediate characteristics of the EP1 and EP2 states. The lack of protection by phosphate is attributed to instability of its complex with the enzyme (EP2). These findings are discussed with respect to different structural images obtained from diffraction studies of ATPase in the presence or in the absence of Ca(2+) and/or decavanadate (Ogawa et al., 1998, Biophys. J. 75:41-52).

Full Text

The Full Text of this article is available as a PDF (393.0 KB).

Selected References

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

  1. Bigelow D. J., Inesi G. Contributions of chemical derivatization and spectroscopic studies to the characterization of the Ca2+ transport ATPase of sarcoplasmic reticulum. Biochim Biophys Acta. 1992 Dec 11;1113(3-4):323–338. doi: 10.1016/0304-4157(92)90005-u. [DOI] [PubMed] [Google Scholar]
  2. Bigelow D. J., Inesi G. Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum. Biochemistry. 1991 Feb 26;30(8):2113–2125. doi: 10.1021/bi00222a016. [DOI] [PubMed] [Google Scholar]
  3. Chaloub R. M., Guimaraes-Motta H., Verjovski-Almeida S., de Meis L., Inesi G. Sequential reactions in Pi utilization for ATP synthesis by sarcoplasmic reticulum. J Biol Chem. 1979 Oct 10;254(19):9464–9468. [PubMed] [Google Scholar]
  4. 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]
  5. Csermely P., Varga S., Martonosi A. Competition between decavanadate and fluorescein isothiocyanate on the Ca2+-ATPase of sarcoplasmic reticulum. Eur J Biochem. 1985 Aug 1;150(3):455–460. doi: 10.1111/j.1432-1033.1985.tb09043.x. [DOI] [PubMed] [Google Scholar]
  6. Dupont Y. Fluorescence studies of the sarcoplasmic reticulum calcium pump. Biochem Biophys Res Commun. 1976 Jul 26;71(2):544–550. doi: 10.1016/0006-291x(76)90821-4. [DOI] [PubMed] [Google Scholar]
  7. Dux L., Martonosi A. Two-dimensional arrays of proteins in sarcoplasmic reticulum and purified Ca2+-ATPase vesicles treated with vanadate. J Biol Chem. 1983 Feb 25;258(4):2599–2603. [PubMed] [Google Scholar]
  8. 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]
  9. Fernandez-Belda F., Kurzmack M., Inesi G. A comparative study of calcium transients by isotopic tracer, metallochromic indicator, and intrinsic fluorescence in sarcoplasmic reticulum ATPase. J Biol Chem. 1984 Aug 10;259(15):9687–9698. [PubMed] [Google Scholar]
  10. Fernández Belda F., García de Ancos J., Inesi G. Fluorometric titration of the sarcoplasmic reticulum adenosinetriphosphatase calcium sites in the presence of vanadate. Biochim Biophys Acta. 1986 Jan 29;854(2):257–264. doi: 10.1016/0005-2736(86)90118-5. [DOI] [PubMed] [Google Scholar]
  11. Green N. M. ATP-driven cation pumps: alignment of sequences. Biochem Soc Trans. 1989 Dec;17(6):972–972. doi: 10.1042/bst0170972. [DOI] [PubMed] [Google Scholar]
  12. Green N. M., Stokes D. L. Structural modelling of P-type ion pumps. Acta Physiol Scand Suppl. 1992;607:59–68. [PubMed] [Google Scholar]
  13. 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]
  14. Henderson I. M., Khan Y. M., East J. M., Lee A. G. Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies. Biochem J. 1994 Feb 1;297(Pt 3):615–624. doi: 10.1042/bj2970615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hua S., Inesi G. Synthesis of a radioactive azido derivative of thapsigargin and photolabeling of the sarcoplasmic reticulum ATPase. Biochemistry. 1997 Sep 30;36(39):11865–11872. doi: 10.1021/bi970105n. [DOI] [PubMed] [Google Scholar]
  16. Inesi G., Cantilina T., Yu X., Nikic D., Sagara Y., Kirtley M. E. Long-range intramolecular linked functions in activation and inhibition of SERCA ATPases. Ann N Y Acad Sci. 1992 Nov 30;671:32–48. doi: 10.1111/j.1749-6632.1992.tb43782.x. [DOI] [PubMed] [Google Scholar]
  17. Inesi G., Lewis D., Murphy A. J. Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase. J Biol Chem. 1984 Jan 25;259(2):996–1003. [PubMed] [Google Scholar]
  18. 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]
  19. Lanzetta P. A., Alvarez L. J., Reinach P. S., Candia O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979 Nov 15;100(1):95–97. doi: 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  20. MacLennan D. H., Rice W. J., Green N. M. The mechanism of Ca2+ transport by sarco(endo)plasmic reticulum Ca2+-ATPases. J Biol Chem. 1997 Nov 14;272(46):28815–28818. doi: 10.1074/jbc.272.46.28815. [DOI] [PubMed] [Google Scholar]
  21. Masuda H., de Meis L. Phosphorylation of the sarcoplasmic reticulum membrane by orthophosphate. Inhibition by calcium ions. Biochemistry. 1973 Nov 6;12(23):4581–4585. doi: 10.1021/bi00747a006. [DOI] [PubMed] [Google Scholar]
  22. McIntosh D. B., Woolley D. G., Berman M. C. 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-AMP and -ATP photolabel Lys-492 at the active site of sarcoplasmic reticulum Ca(2+)-ATPase. J Biol Chem. 1992 Mar 15;267(8):5301–5309. [PubMed] [Google Scholar]
  23. Mitchinson C., Wilderspin A. F., Trinnaman B. J., Green N. M. Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+ -dependent adenosine triphosphatase of sarcoplasmic reticulum. FEBS Lett. 1982 Sep 6;146(1):87–92. doi: 10.1016/0014-5793(82)80710-2. [DOI] [PubMed] [Google Scholar]
  24. Murphy A. J. Effects of divalent cations and nucleotides on the reactivity of the sulfhydryl groups of sarcoplasmic reticulum membranes. Evidence for structural changes occurring during the calcium transport cycle. J Biol Chem. 1978 Jan 25;253(2):385–389. [PubMed] [Google Scholar]
  25. Murphy A. J. Sarcoplasmic reticulum adenosine triphosphatase: labeling of an essential lysyl residue with pyridoxal-5'-phosphate. Arch Biochem Biophys. 1977 Apr 15;180(1):114–120. doi: 10.1016/0003-9861(77)90014-5. [DOI] [PubMed] [Google Scholar]
  26. Murphy A. J. Sulfhydryl group modification of sarcoplasmic reticulum membranes. Biochemistry. 1976 Oct 5;15(20):4492–4496. doi: 10.1021/bi00665a025. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Ogawa H., Stokes D. L., Sasabe H., Toyoshima C. Structure of the Ca2+ pump of sarcoplasmic reticulum: a view along the lipid bilayer at 9-A resolution. Biophys J. 1998 Jul;75(1):41–52. doi: 10.1016/S0006-3495(98)77493-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Pick U. The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum. J Biol Chem. 1982 Jun 10;257(11):6111–6119. [PubMed] [Google Scholar]
  31. Ross D. C., McIntosh D. B. Intramolecular cross-linking at the active site of the Ca2+-ATPase of sarcoplasmic reticulum. High and low affinity nucleotide binding and evidence of active site closure in E2-P. J Biol Chem. 1987 Sep 25;262(27):12977–12983. [PubMed] [Google Scholar]
  32. Ross D. C., McIntosh D. B. Intramolecular cross-linking of domains at the active site links A1 and B subfragments of the Ca2+-ATPase of sarcoplasmic reticulum. J Biol Chem. 1987 Feb 15;262(5):2042–2049. [PubMed] [Google Scholar]
  33. Sagara Y., Wade J. B., Inesi G. A conformational mechanism for formation of a dead-end complex by the sarcoplasmic reticulum ATPase with thapsigargin. J Biol Chem. 1992 Jan 15;267(2):1286–1292. [PubMed] [Google Scholar]
  34. Saito-Nakatsuka K., Yamashita T., Kubota I., Kawakita M. Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide. J Biochem. 1987 Feb;101(2):365–376. doi: 10.1093/oxfordjournals.jbchem.a121921. [DOI] [PubMed] [Google Scholar]
  35. Schwarz F. P., Inesi G. Entropic drive in the sarcoplasmic reticulum ATPase interaction with Mg2+ and Pi. Biophys J. 1997 Oct;73(4):2179–2182. doi: 10.1016/S0006-3495(97)78249-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Seebregts C. J., McIntosh D. B. 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency. J Biol Chem. 1989 Feb 5;264(4):2043–2052. [PubMed] [Google Scholar]
  37. Squier T. C., Bigelow D. J., Garcia de Ancos J., Inesi G. Localization of site-specific probes on the Ca-ATPase of sarcoplasmic reticulum using fluorescence energy transfer. J Biol Chem. 1987 Apr 5;262(10):4748–4754. [PubMed] [Google Scholar]
  38. Varga S., Csermely P., Martonosi A. The binding of vanadium (V) oligoanions to sarcoplasmic reticulum. Eur J Biochem. 1985 Apr 1;148(1):119–126. doi: 10.1111/j.1432-1033.1985.tb08815.x. [DOI] [PubMed] [Google Scholar]
  39. Watanabe T., Inesi G. Structural effects of substrate utilization on the adenosinetriphosphatase chains of sarcoplasmic reticulum. Biochemistry. 1982 Jul 6;21(14):3254–3259. doi: 10.1021/bi00257a001. [DOI] [PubMed] [Google Scholar]
  40. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  41. Wictome M., Khan Y. M., East J. M., Lee A. G. Binding of sesquiterpene lactone inhibitors to the Ca(2+)-ATPase. Biochem J. 1995 Sep 15;310(Pt 3):859–868. doi: 10.1042/bj3100859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yamagata K., Daiho T., Kanazawa T. Labeling of lysine 492 with pyridoxal 5'-phosphate in the sarcoplasmic reticulum Ca(2+)-ATPase. Lysine 492 residue is located outside the fluorescein 5-isothiocyanate-binding region in or near the ATP binding site. J Biol Chem. 1993 Oct 5;268(28):20930–20936. [PubMed] [Google Scholar]
  43. Yamamoto H., Tagaya M., Fukui T., Kawakita M. Affinity labeling of the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by adenosine triphosphopyridoxal: identification of the reactive lysyl residue. J Biochem. 1988 Mar;103(3):452–457. doi: 10.1093/oxfordjournals.jbchem.a122291. [DOI] [PubMed] [Google Scholar]
  44. Yamasaki K., Sano N., Ohe M., Yamamoto T. Determination of the primary structure of intermolecular cross-linking sites on the Ca2(+)-ATPase of sarcoplasmic reticulum using 14C-labeled N,N'-(1,4-phenylene)bismaleimide or N-ethylmaleimide. J Biochem. 1990 Dec;108(6):918–925. doi: 10.1093/oxfordjournals.jbchem.a123315. [DOI] [PubMed] [Google Scholar]
  45. Yonekura K., Stokes D. L., Sasabe H., Toyoshima C. The ATP-binding site of Ca(2+)-ATPase revealed by electron image analysis. Biophys J. 1997 Mar;72(3):997–1005. doi: 10.1016/S0006-3495(97)78752-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang P., Toyoshima C., Yonekura K., Green N. M., Stokes D. L. Structure of the calcium pump from sarcoplasmic reticulum at 8-A resolution. Nature. 1998 Apr 23;392(6678):835–839. doi: 10.1038/33959. [DOI] [PubMed] [Google Scholar]
  47. de Meis L., Vianna A. L. Energy interconversion by the Ca2+-dependent ATPase of the sarcoplasmic reticulum. Annu Rev Biochem. 1979;48:275–292. doi: 10.1146/annurev.bi.48.070179.001423. [DOI] [PubMed] [Google Scholar]

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

RESOURCES