Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC

Autores
Montes, Monica Raquel; Spiaggi, Alejandro Javier; Monti, José Luis Eugenio; Cornelius, Flemming; Olesen, Claus; Garrahan, Patricio Jose; Rossi, Rolando Carlos
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Despite its similarity with the Na+/K+-ATPase, it has not been possible so far to isolate a K+-occluded state in the H+/K+-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb+ (as surrogate for K+) in H+/K+-ATPase from gastric vesicles at 25 °C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb+ ions from the intravesicular medium than C12E8 (a non-ionic detergent). In the presence of vanadate and Mg2+, the time course of [86Rb]Rb+ uptake displayed a fast phase due to Rb+ occlusion. The specific inhibitor of the H+/K+-ATPase SCH28080 significantly reduces the amount of Rb+ occluded in the vanadate–H+/K+-ATPase complex. Occluded Rb+ varies with [Rb+] according to a hyperbolic function with K0.5 = 0.29 ± 0.06 mM. The complex between the Rb+-occluded state and vanadate proved to be very stable even after removal of free Mg2+ with EDTA. Our results yield a stoichiometry lower than one occluded Rb+ per phosphorylation site, which might be explained assuming that, unlike for the Na+/K+-ATPase, Mg2+-vanadate is unable to recruit all the Rb+-bound to the Rb+-occluded form of the Rb+–vanadate–H+/K+-ATPase complex.
Fil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Spiaggi, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Monti, José Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Cornelius, Flemming. University Aarhus; Dinamarca
Fil: Olesen, Claus. University Aarhus; Dinamarca
Fil: Garrahan, Patricio Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Materia
H+/K+-Atpase
Rb+-Occlusion
Gastric Vesicles
Alamethicin
Vanadate
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/18186
Acceder
id CONICETDig_2fbfef425e6c3f543da31e831e721179
oai_identifier_str oai:ri.conicet.gov.ar:11336/18186
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºCMontes, Monica RaquelSpiaggi, Alejandro JavierMonti, José Luis EugenioCornelius, FlemmingOlesen, ClausGarrahan, Patricio JoseRossi, Rolando CarlosH+/K+-AtpaseRb+-OcclusionGastric VesiclesAlamethicinVanadatehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Despite its similarity with the Na+/K+-ATPase, it has not been possible so far to isolate a K+-occluded state in the H+/K+-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb+ (as surrogate for K+) in H+/K+-ATPase from gastric vesicles at 25 °C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb+ ions from the intravesicular medium than C12E8 (a non-ionic detergent). In the presence of vanadate and Mg2+, the time course of [86Rb]Rb+ uptake displayed a fast phase due to Rb+ occlusion. The specific inhibitor of the H+/K+-ATPase SCH28080 significantly reduces the amount of Rb+ occluded in the vanadate–H+/K+-ATPase complex. Occluded Rb+ varies with [Rb+] according to a hyperbolic function with K0.5 = 0.29 ± 0.06 mM. The complex between the Rb+-occluded state and vanadate proved to be very stable even after removal of free Mg2+ with EDTA. Our results yield a stoichiometry lower than one occluded Rb+ per phosphorylation site, which might be explained assuming that, unlike for the Na+/K+-ATPase, Mg2+-vanadate is unable to recruit all the Rb+-bound to the Rb+-occluded form of the Rb+–vanadate–H+/K+-ATPase complex.Fil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Spiaggi, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Monti, José Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Cornelius, Flemming. University Aarhus; DinamarcaFil: Olesen, Claus. University Aarhus; DinamarcaFil: Garrahan, Patricio Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaElsevier Science2011-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/18186Montes, Monica Raquel; Spiaggi, Alejandro Javier; Monti, José Luis Eugenio; Cornelius, Flemming; Olesen, Claus; et al.; Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1808; 1; 1-2011; 316-3220005-2736CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0005273610003032info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2010.08.022info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:38:14Zoai:ri.conicet.gov.ar:11336/18186instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:38:14.357CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
title Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
spellingShingle Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
Montes, Monica Raquel
H+/K+-Atpase
Rb+-Occlusion
Gastric Vesicles
Alamethicin
Vanadate
title_short Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
title_full Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
title_fullStr Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
title_full_unstemmed Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
title_sort Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC
dc.creator.none.fl_str_mv Montes, Monica Raquel
Spiaggi, Alejandro Javier
Monti, José Luis Eugenio
Cornelius, Flemming
Olesen, Claus
Garrahan, Patricio Jose
Rossi, Rolando Carlos
author Montes, Monica Raquel
author_facet Montes, Monica Raquel
Spiaggi, Alejandro Javier
Monti, José Luis Eugenio
Cornelius, Flemming
Olesen, Claus
Garrahan, Patricio Jose
Rossi, Rolando Carlos
author_role author
author2 Spiaggi, Alejandro Javier
Monti, José Luis Eugenio
Cornelius, Flemming
Olesen, Claus
Garrahan, Patricio Jose
Rossi, Rolando Carlos
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv H+/K+-Atpase
Rb+-Occlusion
Gastric Vesicles
Alamethicin
Vanadate
topic H+/K+-Atpase
Rb+-Occlusion
Gastric Vesicles
Alamethicin
Vanadate
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Despite its similarity with the Na+/K+-ATPase, it has not been possible so far to isolate a K+-occluded state in the H+/K+-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb+ (as surrogate for K+) in H+/K+-ATPase from gastric vesicles at 25 °C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb+ ions from the intravesicular medium than C12E8 (a non-ionic detergent). In the presence of vanadate and Mg2+, the time course of [86Rb]Rb+ uptake displayed a fast phase due to Rb+ occlusion. The specific inhibitor of the H+/K+-ATPase SCH28080 significantly reduces the amount of Rb+ occluded in the vanadate–H+/K+-ATPase complex. Occluded Rb+ varies with [Rb+] according to a hyperbolic function with K0.5 = 0.29 ± 0.06 mM. The complex between the Rb+-occluded state and vanadate proved to be very stable even after removal of free Mg2+ with EDTA. Our results yield a stoichiometry lower than one occluded Rb+ per phosphorylation site, which might be explained assuming that, unlike for the Na+/K+-ATPase, Mg2+-vanadate is unable to recruit all the Rb+-bound to the Rb+-occluded form of the Rb+–vanadate–H+/K+-ATPase complex.
Fil: Montes, Monica Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Spiaggi, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Monti, José Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Cornelius, Flemming. University Aarhus; Dinamarca
Fil: Olesen, Claus. University Aarhus; Dinamarca
Fil: Garrahan, Patricio Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Rossi, Rolando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
description Despite its similarity with the Na+/K+-ATPase, it has not been possible so far to isolate a K+-occluded state in the H+/K+-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb+ (as surrogate for K+) in H+/K+-ATPase from gastric vesicles at 25 °C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb+ ions from the intravesicular medium than C12E8 (a non-ionic detergent). In the presence of vanadate and Mg2+, the time course of [86Rb]Rb+ uptake displayed a fast phase due to Rb+ occlusion. The specific inhibitor of the H+/K+-ATPase SCH28080 significantly reduces the amount of Rb+ occluded in the vanadate–H+/K+-ATPase complex. Occluded Rb+ varies with [Rb+] according to a hyperbolic function with K0.5 = 0.29 ± 0.06 mM. The complex between the Rb+-occluded state and vanadate proved to be very stable even after removal of free Mg2+ with EDTA. Our results yield a stoichiometry lower than one occluded Rb+ per phosphorylation site, which might be explained assuming that, unlike for the Na+/K+-ATPase, Mg2+-vanadate is unable to recruit all the Rb+-bound to the Rb+-occluded form of the Rb+–vanadate–H+/K+-ATPase complex.
publishDate 2011
dc.date.none.fl_str_mv 2011-01
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/18186
Montes, Monica Raquel; Spiaggi, Alejandro Javier; Monti, José Luis Eugenio; Cornelius, Flemming; Olesen, Claus; et al.; Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1808; 1; 1-2011; 316-322
0005-2736
CONICET Digital
CONICET
url http://hdl.handle.net/11336/18186
identifier_str_mv Montes, Monica Raquel; Spiaggi, Alejandro Javier; Monti, José Luis Eugenio; Cornelius, Flemming; Olesen, Claus; et al.; Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1808; 1; 1-2011; 316-322
0005-2736
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S0005273610003032
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2010.08.022
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.069144

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