Thapsigargin and Dimethyl Sulfoxide Activate Medium Pi ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca2+-ATPase
2001; Elsevier BV; Volume: 276; Issue: 50 Linguagem: Inglês
10.1074/jbc.m106320200
ISSN1083-351X
AutoresTshepo W Seekoe, Susan Peall, David B. McIntosh,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoThapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca2+-ATPase. It binds the Ca2+-free E2 conformation in the picomolar range, supposedly resulting in a largely catalytically inactive species. We now find that thapsigargin has little effect on medium Pi↔ HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me2SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me2SO on the four rate constants governing the reaction of Pi with Ca2+-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k −2), whereas that of Me2SO is to greatly retard Pi dissociation (k −1), accounting for its well known effect on increasing the apparent affinity for Pi. These effects persist when the agents are used in combination and substantially account for the activated oxygen exchange (v exchange =k −2[EP]). Kinetic simulations show that the overall rate constant for the formation of EP is very fast (∼300 s−1) when the exchange is maximal. Thapsigargin greatly stabilizes Ca2+-ATPase against denaturation in detergent in the absence of Ca2+, as revealed by glutaraldehyde cross-linking, suggesting that the membrane helices lock together. It seems that the reactions at the phosphorylation site, associated with the activated exchange reaction, are occurring without much movement of the transport site helices, and we suggest that they may be associated solely with an occluded H+ state. Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca2+-ATPase. It binds the Ca2+-free E2 conformation in the picomolar range, supposedly resulting in a largely catalytically inactive species. We now find that thapsigargin has little effect on medium Pi↔ HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me2SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me2SO on the four rate constants governing the reaction of Pi with Ca2+-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k −2), whereas that of Me2SO is to greatly retard Pi dissociation (k −1), accounting for its well known effect on increasing the apparent affinity for Pi. These effects persist when the agents are used in combination and substantially account for the activated oxygen exchange (v exchange =k −2[EP]). Kinetic simulations show that the overall rate constant for the formation of EP is very fast (∼300 s−1) when the exchange is maximal. Thapsigargin greatly stabilizes Ca2+-ATPase against denaturation in detergent in the absence of Ca2+, as revealed by glutaraldehyde cross-linking, suggesting that the membrane helices lock together. It seems that the reactions at the phosphorylation site, associated with the activated exchange reaction, are occurring without much movement of the transport site helices, and we suggest that they may be associated solely with an occluded H+ state. sarcoplasmic reticulum phosphoenzyme fluorescein 5′-isothiocyanate 3-(N-morpholino)propanesulfonic acid tetramethylammonium hydroxide 2-(N-morpholino)ethanesulfonic acid form of Ca2+-ATPase in the presence of Ca2+ form of Ca2+-ATPase in the absence of Ca2+ Sarcoplasmic reticulum (SR)1 Ca2+-ATPase pumps Ca2+ from the sarcoplasm to the reticular lumen to allow relaxation in skeletal muscle. Transport of Ca2+ is driven by ATP hydrolysis, and the catalytic cycle includes phosphorylation and dephosphorylation of an aspartic acid residue. The pump can operate in the reverse direction, coupling Piphosphorylation and phosphoryl transfer to ADP with Ca2+efflux down the concentration gradient. Chemical reactions at the active site depend on whether the transport sites are occupied with Ca2+ (E1 forms, reactive to ATP or ADP) or H+ (E2 forms, reactive to Pi and water) ions and their orientation with respect to the membrane.The reaction with Pi, in its simplest representation, can be described by two steps, shown in Scheme 1. Mg2+ and Pi either bind independently (1Punzengruber C. Prager R. Kolassa N. Winkler F. Suko J. Eur. J. Biochem. 1978; 92: 349-359Crossref PubMed Scopus (73) Google Scholar, 2Martin D.W. Tanford C. Biochemistry. 1981; 20: 4597-4602Crossref PubMed Scopus (29) Google Scholar) or as MgPi(3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar). E2+Mg2++Pi↔k−1k1E2·Mg·Pi↔k−2k2E2−P·Mg+H2O SCHEME1It is well known that the phosphorylation reaction is augmented by co-solvents like Me2SO and glycerol, by greatly increasing the apparent affinity for Pi (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar, 4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar, 5de Meis L. Inesi G. J. Biol. Chem. 1982; 257: 1289-1294Abstract Full Text PDF PubMed Google Scholar, 6Dupont Y. Pougeois R. FEBS Lett. 1983; 156: 93-98Crossref PubMed Scopus (71) Google Scholar, 7de Meis L. Inesi G. J. Biol. Chem. 1988; 263: 157-161Abstract Full Text PDF PubMed Google Scholar). The rate constants in Scheme 1 changed by Me2SO have not been fully elucidated. Although dephosphorylation has been found to be inhibited (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar, 4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar), thek obs for phosphorylation is activated in the 0–20% (v/v) Me2SO concentration range (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar) and strongly inhibited at 40% (4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar). de Meis et al. (4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar) consider that the main effect may be a true affinity change caused by decreased solubility of Pi in the medium and favored partitioning of Pi into the catalytic site. In contrast, Mintz et al. (8Mintz E. Forge V. Guillain F. Biochim. Biophys. Acta. 1993; 1162: 227-229Crossref PubMed Scopus (6) Google Scholar) consider that their competition studies of [45Ca]Ca2+ and Pi binding show that the apparent affinity change is due to an effect on the covalent phosphorylation reaction itself.Thapsigargin, a sesquiterpene lactone isolated from the plantThapsia garganica, is a specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase isoforms (9Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar, 10Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar, 11Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar, 12Kijima Y. Ogunbunmi E. Fleischer S. J. Biol. Chem. 1991; 266: 22912-22918Abstract Full Text PDF PubMed Google Scholar). It binds the E2 conformation of SR Ca+-ATPase extremely tightly in a 1:1 molar ratio (13Sagara Y. Fernandez-Belda F. de Meis L. Inesi G. J. Biol. Chem. 1992; 267: 12606-12613Abstract Full Text PDF PubMed Google Scholar, 14Wictome M. Henderson I. Lee A.G. East J.M. Biochem. J. 1992; 283: 525-529Crossref PubMed Scopus (79) Google Scholar, 15Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar, 16Davidson G.A. Varhol R.J. J. Biol. Chem. 1995; 270: 11731-11734Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Site- and segment-directed and natural mutagenesis has shown that thapsigargin binding is sensitive to amino acid changes in transmembrane helix 3, and Phe-256 and Gly-257 in this helix are especially critical (17Norregaard A. Vilsen B. Andersen J.P. FEBS Lett. 1993; 336: 248-254Crossref PubMed Scopus (16) Google Scholar, 18Yu M. Zhang L. Rishi A.K. Khadeer M. Inesi G. Hussain A. J. Biol. Chem. 1998; 273: 3542-3546Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Zhong L. Inesi G. J. Biol. Chem. 1998; 273: 12994-12998Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20Yu M. Lin J. Khadeer M. Yeh Y. Inesi G. Hussain A. Arch. Biochem. Biophys. 1999; 362: 225-232Crossref PubMed Scopus (27) Google Scholar, 21Ma H. Zhong L. Inesi G. Fortea I. Soler F. Fernandez-Belda F. Biochemistry. 1999; 38: 15522-15527Crossref PubMed Scopus (29) Google Scholar). Photolabeling with an azido analogue of thapsigargin located the site in the protein section between stalks S3 and S4 (22Hua S. Inesi G. Biochemistry. 1997; 36: 11865-11872Crossref PubMed Scopus (22) Google Scholar). More recently, three-dimensional reconstruction of images obtained by cryo-electron microscopy of tubular crystals suggests that thapsigargin binds to protein luminal loops between transmembrane segments M3/M4 and M7/M8 (23Young H.S. Xu C. Zhang P. Stokes D.L. J. Mol. Biol. 2001; 308: 231-240Crossref PubMed Scopus (20) Google Scholar). Thapsigargin binding inhibits Ca2+-dependent ATP phosphorylation as well as Pi phosphorylation and reportedly results in a largely catalytically inactive complex (13Sagara Y. Fernandez-Belda F. de Meis L. Inesi G. J. Biol. Chem. 1992; 267: 12606-12613Abstract Full Text PDF PubMed Google Scholar, 14Wictome M. Henderson I. Lee A.G. East J.M. Biochem. J. 1992; 283: 525-529Crossref PubMed Scopus (79) Google Scholar, 15Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar).During the course of the equilibrium phosphorylation reaction depicted in Scheme 1, the four oxygens of medium Pi are necessarily exchanged with those in water. If Pi enriched in18O is used, then the 18O atoms are gradually lost to water and 16O-substituted. In addition to the obligatory replacement of at least one oxygen atom per phosphorylation and dephosphorylation event, there may be more because of dynamic reversal of bound Pi to phosphoenzyme formation before the Pi is released to the medium. SR Ca2+-ATPase catalyzes a rapid medium 2The word “medium” is used here to contrast this exchange with intermediate Pi ↔ HOH oxygen exchange, where the Pi produced comes from a different chemical form, namely ATP. 2The word “medium” is used here to contrast this exchange with intermediate Pi ↔ HOH oxygen exchange, where the Pi produced comes from a different chemical form, namely ATP.Pi ↔ HOH oxygen exchange (24Kanazawa T. Boyer P.D. J. Biol. Chem. 1973; 248: 3163-3172Abstract Full Text PDF PubMed Google Scholar, 25Ariki M. Boyer P.D. Biochemistry. 1980; 19: 2001-2004Crossref PubMed Scopus (19) Google Scholar). Analysis of the development of the five Pi species with differing amounts of [18O]Pi allows, in combination with phosphoenzyme measurements, estimation of the average number of reversals of bound Pi and elucidation of all four rate constants of the reaction (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar, 26McIntosh D.B. Boyer P.D. Biochemistry. 1983; 22: 2867-2875Crossref PubMed Scopus (77) Google Scholar, 27Guillain F. Champeil P. Boyer P.D. Biochemistry. 1984; 23: 4754-4761Crossref PubMed Scopus (16) Google Scholar).In this study, we find that thapsigargin binding has little effect on Pi ↔ HOH oxygen exchange and that Me2SO greatly accelerates this activity. The basis for the effect is sought through evaluating the four rate constants of the Pireaction. The results describe the effects of thapsigargin and Me2SO, alone and in combination, on these rate constants at pH 6.0 and 7.0. Further, it is shown that thapsigargin greatly stabilizes the Ca2+-free Ca2+-ATPase against denaturation in detergent, suggesting that the membrane helices are locked in an immobilized state. The unexpected activation of catalytic events in the stable thapsigargin-Ca2+-ATPase complex has important implications for the coupling of these catalytic reactions with H+ transport events at this stage of the cycle. Sarcoplasmic reticulum (SR)1 Ca2+-ATPase pumps Ca2+ from the sarcoplasm to the reticular lumen to allow relaxation in skeletal muscle. Transport of Ca2+ is driven by ATP hydrolysis, and the catalytic cycle includes phosphorylation and dephosphorylation of an aspartic acid residue. The pump can operate in the reverse direction, coupling Piphosphorylation and phosphoryl transfer to ADP with Ca2+efflux down the concentration gradient. Chemical reactions at the active site depend on whether the transport sites are occupied with Ca2+ (E1 forms, reactive to ATP or ADP) or H+ (E2 forms, reactive to Pi and water) ions and their orientation with respect to the membrane. The reaction with Pi, in its simplest representation, can be described by two steps, shown in Scheme 1. Mg2+ and Pi either bind independently (1Punzengruber C. Prager R. Kolassa N. Winkler F. Suko J. Eur. J. Biochem. 1978; 92: 349-359Crossref PubMed Scopus (73) Google Scholar, 2Martin D.W. Tanford C. Biochemistry. 1981; 20: 4597-4602Crossref PubMed Scopus (29) Google Scholar) or as MgPi(3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar). E2+Mg2++Pi↔k−1k1E2·Mg·Pi↔k−2k2E2−P·Mg+H2O SCHEME1It is well known that the phosphorylation reaction is augmented by co-solvents like Me2SO and glycerol, by greatly increasing the apparent affinity for Pi (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar, 4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar, 5de Meis L. Inesi G. J. Biol. Chem. 1982; 257: 1289-1294Abstract Full Text PDF PubMed Google Scholar, 6Dupont Y. Pougeois R. FEBS Lett. 1983; 156: 93-98Crossref PubMed Scopus (71) Google Scholar, 7de Meis L. Inesi G. J. Biol. Chem. 1988; 263: 157-161Abstract Full Text PDF PubMed Google Scholar). The rate constants in Scheme 1 changed by Me2SO have not been fully elucidated. Although dephosphorylation has been found to be inhibited (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar, 4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar), thek obs for phosphorylation is activated in the 0–20% (v/v) Me2SO concentration range (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar) and strongly inhibited at 40% (4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar). de Meis et al. (4de Meis L. Martins O.B. Alves E.W. Biochemistry. 1980; 19: 4252-4261Crossref PubMed Scopus (181) Google Scholar) consider that the main effect may be a true affinity change caused by decreased solubility of Pi in the medium and favored partitioning of Pi into the catalytic site. In contrast, Mintz et al. (8Mintz E. Forge V. Guillain F. Biochim. Biophys. Acta. 1993; 1162: 227-229Crossref PubMed Scopus (6) Google Scholar) consider that their competition studies of [45Ca]Ca2+ and Pi binding show that the apparent affinity change is due to an effect on the covalent phosphorylation reaction itself. Thapsigargin, a sesquiterpene lactone isolated from the plantThapsia garganica, is a specific inhibitor of sarco-endoplasmic reticulum Ca2+-ATPase isoforms (9Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar, 10Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar, 11Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar, 12Kijima Y. Ogunbunmi E. Fleischer S. J. Biol. Chem. 1991; 266: 22912-22918Abstract Full Text PDF PubMed Google Scholar). It binds the E2 conformation of SR Ca+-ATPase extremely tightly in a 1:1 molar ratio (13Sagara Y. Fernandez-Belda F. de Meis L. Inesi G. J. Biol. Chem. 1992; 267: 12606-12613Abstract Full Text PDF PubMed Google Scholar, 14Wictome M. Henderson I. Lee A.G. East J.M. Biochem. J. 1992; 283: 525-529Crossref PubMed Scopus (79) Google Scholar, 15Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar, 16Davidson G.A. Varhol R.J. J. Biol. Chem. 1995; 270: 11731-11734Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Site- and segment-directed and natural mutagenesis has shown that thapsigargin binding is sensitive to amino acid changes in transmembrane helix 3, and Phe-256 and Gly-257 in this helix are especially critical (17Norregaard A. Vilsen B. Andersen J.P. FEBS Lett. 1993; 336: 248-254Crossref PubMed Scopus (16) Google Scholar, 18Yu M. Zhang L. Rishi A.K. Khadeer M. Inesi G. Hussain A. J. Biol. Chem. 1998; 273: 3542-3546Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 19Zhong L. Inesi G. J. Biol. Chem. 1998; 273: 12994-12998Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20Yu M. Lin J. Khadeer M. Yeh Y. Inesi G. Hussain A. Arch. Biochem. Biophys. 1999; 362: 225-232Crossref PubMed Scopus (27) Google Scholar, 21Ma H. Zhong L. Inesi G. Fortea I. Soler F. Fernandez-Belda F. Biochemistry. 1999; 38: 15522-15527Crossref PubMed Scopus (29) Google Scholar). Photolabeling with an azido analogue of thapsigargin located the site in the protein section between stalks S3 and S4 (22Hua S. Inesi G. Biochemistry. 1997; 36: 11865-11872Crossref PubMed Scopus (22) Google Scholar). More recently, three-dimensional reconstruction of images obtained by cryo-electron microscopy of tubular crystals suggests that thapsigargin binds to protein luminal loops between transmembrane segments M3/M4 and M7/M8 (23Young H.S. Xu C. Zhang P. Stokes D.L. J. Mol. Biol. 2001; 308: 231-240Crossref PubMed Scopus (20) Google Scholar). Thapsigargin binding inhibits Ca2+-dependent ATP phosphorylation as well as Pi phosphorylation and reportedly results in a largely catalytically inactive complex (13Sagara Y. Fernandez-Belda F. de Meis L. Inesi G. J. Biol. Chem. 1992; 267: 12606-12613Abstract Full Text PDF PubMed Google Scholar, 14Wictome M. Henderson I. Lee A.G. East J.M. Biochem. J. 1992; 283: 525-529Crossref PubMed Scopus (79) Google Scholar, 15Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar). During the course of the equilibrium phosphorylation reaction depicted in Scheme 1, the four oxygens of medium Pi are necessarily exchanged with those in water. If Pi enriched in18O is used, then the 18O atoms are gradually lost to water and 16O-substituted. In addition to the obligatory replacement of at least one oxygen atom per phosphorylation and dephosphorylation event, there may be more because of dynamic reversal of bound Pi to phosphoenzyme formation before the Pi is released to the medium. SR Ca2+-ATPase catalyzes a rapid medium 2The word “medium” is used here to contrast this exchange with intermediate Pi ↔ HOH oxygen exchange, where the Pi produced comes from a different chemical form, namely ATP. 2The word “medium” is used here to contrast this exchange with intermediate Pi ↔ HOH oxygen exchange, where the Pi produced comes from a different chemical form, namely ATP.Pi ↔ HOH oxygen exchange (24Kanazawa T. Boyer P.D. J. Biol. Chem. 1973; 248: 3163-3172Abstract Full Text PDF PubMed Google Scholar, 25Ariki M. Boyer P.D. Biochemistry. 1980; 19: 2001-2004Crossref PubMed Scopus (19) Google Scholar). Analysis of the development of the five Pi species with differing amounts of [18O]Pi allows, in combination with phosphoenzyme measurements, estimation of the average number of reversals of bound Pi and elucidation of all four rate constants of the reaction (3Champeil P. Guillain F. Venien C. Gingold M.P. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar, 26McIntosh D.B. Boyer P.D. Biochemistry. 1983; 22: 2867-2875Crossref PubMed Scopus (77) Google Scholar, 27Guillain F. Champeil P. Boyer P.D. Biochemistry. 1984; 23: 4754-4761Crossref PubMed Scopus (16) Google Scholar). In this study, we find that thapsigargin binding has little effect on Pi ↔ HOH oxygen exchange and that Me2SO greatly accelerates this activity. The basis for the effect is sought through evaluating the four rate constants of the Pireaction. The results describe the effects of thapsigargin and Me2SO, alone and in combination, on these rate constants at pH 6.0 and 7.0. Further, it is shown that thapsigargin greatly stabilizes the Ca2+-free Ca2+-ATPase against denaturation in detergent, suggesting that the membrane helices are locked in an immobilized state. The unexpected activation of catalytic events in the stable thapsigargin-Ca2+-ATPase complex has important implications for the coupling of these catalytic reactions with H+ transport events at this stage of the cycle. We thank David Woolley for expert technical assistance, Gordon McIntosh for help with the computer simulations, Philippe Champeil for comments on the manuscript, and Chikashi Toyoshima for information on unpublished work.
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