A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles

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dc.contributor.authorChampeil, Philippe
dc.contributor.authorHenao, Fernando
dc.contributor.authorLacapère, Jean-Jacques
dc.contributor.authorMcIntosh, David B
dc.date.accessioned2021-10-08T07:20:46Z
dc.date.available2021-10-08T07:20:46Z
dc.date.issued2001
dc.description.abstractAfter the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.
dc.identifier.apacitationChampeil, P., Henao, F., Lacapère, J., & McIntosh, D. B. (2001). A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles. <i>The Journal of Biological Chemistry</i>, 276(8), 5795 - 5803. http://hdl.handle.net/11427/35002en_ZA
dc.identifier.chicagocitationChampeil, Philippe, Fernando Henao, Jean-Jacques Lacapère, and David B McIntosh "A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles." <i>The Journal of Biological Chemistry</i> 276, 8. (2001): 5795 - 5803. http://hdl.handle.net/11427/35002en_ZA
dc.identifier.citationChampeil, P., Henao, F., Lacapère, J. & McIntosh, D.B. 2001. A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles. <i>The Journal of Biological Chemistry.</i> 276(8):5795 - 5803. http://hdl.handle.net/11427/35002en_ZA
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.ris TY - Journal Article AU - Champeil, Philippe AU - Henao, Fernando AU - Lacapère, Jean-Jacques AU - McIntosh, David B AB - After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism. DA - 2001 DB - OpenUCT DP - University of Cape Town IS - 8 J1 - The Journal of Biological Chemistry LK - https://open.uct.ac.za PY - 2001 SM - 0021-9258 SM - 1083-351X T1 - A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles TI - A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles UR - http://hdl.handle.net/11427/35002 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/35002
dc.identifier.vancouvercitationChampeil P, Henao F, Lacapère J, McIntosh DB. A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles. The Journal of Biological Chemistry. 2001;276(8):5795 - 5803. http://hdl.handle.net/11427/35002.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.journalissue8
dc.source.journalvolume276
dc.source.pagination5795 - 5803
dc.source.urihttps://dx.doi.org/10.1074/jbc.M006980200
dc.subject.otherBiological Transport, Active
dc.subject.otherCalcium
dc.subject.otherCalcium-Transporting ATPases
dc.subject.otherCell Polarity
dc.subject.otherCytosol
dc.subject.otherEnzyme Stability
dc.subject.otherFluorescein-5-isothiocyanate
dc.subject.otherFluorescence
dc.subject.otherModels, Chemical
dc.subject.otherOrganophosphates
dc.subject.otherPhosphates
dc.subject.otherPhosphoproteins
dc.subject.otherSarcoplasmic Reticulum
dc.subject.otherSpectrophotometry
dc.subject.otherOrganophosphates
dc.subject.otherPhosphates
dc.titleA Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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