Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Sep 15;310(Pt 3):859–868. doi: 10.1042/bj3100859

Binding of sesquiterpene lactone inhibitors to the Ca(2+)-ATPase.

M Wictome 1, Y M Khan 1, J M East 1, A G Lee 1
PMCID: PMC1135975  PMID: 7575419

Abstract

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin, trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I), consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+, inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential, with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA, and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA < trilobolide < thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I, and that binding constants are very similar for E1Ca2, E1 and E2.

Full text

PDF
859

Selected References

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

  1. Baker K. J., East J. M., Lee A. G. Localization of the hinge region of the Ca(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer. Biochim Biophys Acta. 1994 Jun 1;1192(1):53–60. doi: 10.1016/0005-2736(94)90142-2. [DOI] [PubMed] [Google Scholar]
  2. Barrabin H., Scofano H. M., Inesi G. Adenosinetriphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme. Biochemistry. 1984 Mar 27;23(7):1542–1548. doi: 10.1021/bi00302a031. [DOI] [PubMed] [Google Scholar]
  3. Cha S. A simple method for derivation of rate equations for enzyme-catalyzed reactions under the rapid equilibrium assumption or combined assumptions of equilibrium and steady state. J Biol Chem. 1968 Feb 25;243(4):820–825. [PubMed] [Google Scholar]
  4. Christensen S. B., Andersen A., Poulsen J. C., Treiman M. Derivatives of thapsigargin as probes of its binding site on endoplasmic reticulum Ca2+ ATPase. Stereoselectivity and important functional groups. FEBS Lett. 1993 Dec 13;335(3):345–348. doi: 10.1016/0014-5793(93)80416-r. [DOI] [PubMed] [Google Scholar]
  5. Coll R. J., Murphy A. J. Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography. J Biol Chem. 1984 Nov 25;259(22):14249–14254. [PubMed] [Google Scholar]
  6. Dupont Y., Leigh J. B. Transient kinetics of sarcoplasmic reticulum CA2+ + Mg2+ ATPase studied by fluorescence. Nature. 1978 Jun 1;273(5661):396–398. doi: 10.1038/273396a0. [DOI] [PubMed] [Google Scholar]
  7. East J. M., Lee A. G. Lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid. Biochemistry. 1982 Aug 17;21(17):4144–4151. doi: 10.1021/bi00260a035. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Froud R. J., Lee A. G. Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions. Biochem J. 1986 Jul 1;237(1):197–206. doi: 10.1042/bj2370197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Godt R. E. Calcium-activated tension of skinned muscle fibers of the frog. Dependence on magnesium adenosine triphosphate concentration. J Gen Physiol. 1974 Jun;63(6):722–739. doi: 10.1085/jgp.63.6.722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hardwicke P. M., Green N. M. The effect of delipidation on the adenosine triphosphatase of sarcoplasmic reticulum. Electron microscopy and physical properties. Eur J Biochem. 1974 Feb 15;42(1):183–193. doi: 10.1111/j.1432-1033.1974.tb03328.x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Henderson I. M., Starling A. P., Wictome M., East J. M., Lee A. G. Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: kinetic studies. Biochem J. 1994 Feb 1;297(Pt 3):625–636. doi: 10.1042/bj2970625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee A. G., Baker K., Khan Y. M., East J. M. Effects of K+ on the binding of Ca2+ to the Ca(2+)-ATPase of sarcoplasmic reticulum. Biochem J. 1995 Jan 1;305(Pt 1):225–231. doi: 10.1042/bj3050225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lytton J., Westlin M., Hanley M. R. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J Biol Chem. 1991 Sep 15;266(26):17067–17071. [PubMed] [Google Scholar]
  16. Michelangeli F., Grimes E. A., East J. M., Lee A. G. Effects of phospholipids on the function of (Ca2(+)-Mg2+)-ATPase. Biochemistry. 1991 Jan 15;30(2):342–351. doi: 10.1021/bi00216a006. [DOI] [PubMed] [Google Scholar]
  17. Papp B., Enyedi A., Kovács T., Sarkadi B., Wuytack F., Thastrup O., Gárdos G., Bredoux R., Levy-Toledano S., Enouf J. Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles. J Biol Chem. 1991 Aug 5;266(22):14593–14596. [PubMed] [Google Scholar]
  18. Pick U., Karlish S. J. Regulation of the conformation transition in the Ca-ATPase from sarcoplasmic reticulum by pH, temperature, and calcium ions. J Biol Chem. 1982 Jun 10;257(11):6120–6126. [PubMed] [Google Scholar]
  19. 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]
  20. Rasmussen U., Christensen S. B., Sandberg F. Phytochemistry of the genus Thapsia. Planta Med. 1981 Dec;43(4):336–341. doi: 10.1055/s-2007-971521. [DOI] [PubMed] [Google Scholar]
  21. Sagara Y., Inesi G. Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations. J Biol Chem. 1991 Jul 25;266(21):13503–13506. [PubMed] [Google Scholar]
  22. 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]
  23. Stahl N., Jencks W. P. Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca2+ that are not satisfactorily described by an E1-E2 model. Biochemistry. 1987 Dec 1;26(24):7654–7667. doi: 10.1021/bi00398a019. [DOI] [PubMed] [Google Scholar]
  24. Starling A. P., East J. M., Lee A. G. Effects of phosphatidylcholine fatty acyl chain length on calcium binding and other functions of the (Ca(2+)-Mg2+)-ATPase. Biochemistry. 1993 Feb 16;32(6):1593–1600. doi: 10.1021/bi00057a025. [DOI] [PubMed] [Google Scholar]
  25. Stefanova H. I., East J. M., Gore M. G., Lee A. G. Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes. Biochemistry. 1992 Jul 7;31(26):6023–6031. doi: 10.1021/bi00141a010. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Wakabayashi S., Imagawa T., Shigekawa M. Does fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole incorporated into sarcoplasmic reticulum ATPase monitor putative E1-E2 conformational transition? J Biochem. 1990 Apr;107(4):563–571. doi: 10.1093/oxfordjournals.jbchem.a123087. [DOI] [PubMed] [Google Scholar]
  28. Wictome M., Henderson I., Lee A. G., East J. M. Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin. Biochem J. 1992 Apr 15;283(Pt 2):525–529. doi: 10.1042/bj2830525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wictome M., Holub M., East J. M., Lee A. G. The importance of the hydroxyl moieties for inhibition of the Ca(2+)-ATPase by trilobolide and 2,5-di(tert-butyl)-1,4-benzohydroquinone. Biochem Biophys Res Commun. 1994 Mar 15;199(2):916–921. doi: 10.1006/bbrc.1994.1316. [DOI] [PubMed] [Google Scholar]
  30. Wictome M., Michelangeli F., Lee A. G., East J. M. The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca2+,Mg(2+)-ATPase. FEBS Lett. 1992 Jun 15;304(2-3):109–113. doi: 10.1016/0014-5793(92)80599-c. [DOI] [PubMed] [Google Scholar]
  31. Wictome M., Starling A. P., Sharma R. P., East J. M., Lee A. G. Inhibition of the Ca(2+)-ATPase by sesquiterpene lactones. Biochem Soc Trans. 1993 Nov;21(4):348S–348S. doi: 10.1042/bst021348s. [DOI] [PubMed] [Google Scholar]

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

RESOURCES