Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase

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dc.contributor.authorSeekoe, Tshepo
dc.contributor.authorPeall, Susan
dc.contributor.authorMcIntosh, David B
dc.date.accessioned2021-10-08T07:20:47Z
dc.date.available2021-10-08T07:20:47Z
dc.date.issued2001
dc.description.abstractThapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase. It binds the Ca(2+)-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 P(i) <--> HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me(2)SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me(2)SO on the four rate constants governing the reaction of P(i) with Ca(2+)-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k(-2)), whereas that of Me(2)SO is to greatly retard P(i) dissociation (k(-1)), accounting for its well known effect on increasing the apparent affinity for P(i). 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 (approximately 300 s(-1)) when the exchange is maximal. Thapsigargin greatly stabilizes Ca(2+)-ATPase against denaturation in detergent in the absence of Ca(2+), 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.
dc.identifier.apacitationSeekoe, T., Peall, S., & McIntosh, D. B. (2001). Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase. <i>The Journal of Biological Chemistry</i>, 276(50), 46737 - 46744. http://hdl.handle.net/11427/35006en_ZA
dc.identifier.chicagocitationSeekoe, Tshepo, Susan Peall, and David B McIntosh "Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase." <i>The Journal of Biological Chemistry</i> 276, 50. (2001): 46737 - 46744. http://hdl.handle.net/11427/35006en_ZA
dc.identifier.citationSeekoe, T., Peall, S. & McIntosh, D.B. 2001. Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase. <i>The Journal of Biological Chemistry.</i> 276(50):46737 - 46744. http://hdl.handle.net/11427/35006en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - Seekoe, Tshepo AU - Peall, Susan AU - McIntosh, David B AB - Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase. It binds the Ca(2+)-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 P(i) <--> HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me(2)SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me(2)SO on the four rate constants governing the reaction of P(i) with Ca(2+)-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k(-2)), whereas that of Me(2)SO is to greatly retard P(i) dissociation (k(-1)), accounting for its well known effect on increasing the apparent affinity for P(i). 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 (approximately 300 s(-1)) when the exchange is maximal. Thapsigargin greatly stabilizes Ca(2+)-ATPase against denaturation in detergent in the absence of Ca(2+), 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. DA - 2001 DB - OpenUCT DP - University of Cape Town IS - 50 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 2001 SM - 0021-9258 SM - 1083-351X T1 - Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase TI - Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase UR - http://hdl.handle.net/11427/35006 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/35006
dc.identifier.vancouvercitationSeekoe T, Peall S, McIntosh DB. Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase. The Journal of Biological Chemistry. 2001;276(50):46737 - 46744. http://hdl.handle.net/11427/35006.en_ZA
dc.language.isoeng
dc.publisher.departmentDivision of Chemical Pathology
dc.publisher.facultyFaculty of Health Sciences
dc.sourceThe Journal of Biological Chemistry
dc.source.journalissue50
dc.source.journalvolume276
dc.source.pagination46737 - 46744
dc.source.urihttps://dx.doi.org/10.1074/jbc.M106320200
dc.subject.otherAdenosine Triphosphatases
dc.subject.otherAnimals
dc.subject.otherCalcium-Transporting ATPases
dc.subject.otherCatalysis
dc.subject.otherCatalytic Domain
dc.subject.otherCross-Linking Reagents
dc.subject.otherDimethyl Sulfoxide
dc.subject.otherDose-Response Relationship, Drug
dc.subject.otherEnzyme Inhibitors
dc.subject.otherFree Radical Scavengers
dc.subject.otherGlutaral
dc.subject.otherHydrogen
dc.subject.otherHydrogen-Ion Concentration
dc.subject.otherHydrolysis
dc.subject.otherKinetics
dc.titleThapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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