E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2

Autores
Montes, Monica Raquel; Monti, José Luis Eugenio; Rossi, Rolando Carlos
Año de publicación
2012
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na+ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na+-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+.
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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina
Materia
Na+/K+-Atpase
Conformational Transition
Rb+-Deocclusion
Vanadate
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/18197
Acceder
id CONICETDig_f55e6586bc913b85d07a79b350c5d7c0
oai_identifier_str oai:ri.conicet.gov.ar:11336/18197
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2Montes, Monica RaquelMonti, José Luis EugenioRossi, Rolando CarlosNa+/K+-AtpaseConformational TransitionRb+-DeocclusionVanadatehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na+ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na+-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+.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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; 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; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaElsevier Science2012-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/18197Montes, Monica Raquel; Monti, José Luis Eugenio; Rossi, Rolando Carlos; E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1818; 9; 9-2012; 2087-20930005-2736CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/ark/http://www.sciencedirect.com/science/article/pii/S000527361200123Xinfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2012.04.001info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:33:38Zoai:ri.conicet.gov.ar:11336/18197instacron: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 09:33:38.855CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
title E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
spellingShingle E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
Montes, Monica Raquel
Na+/K+-Atpase
Conformational Transition
Rb+-Deocclusion
Vanadate
title_short E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
title_full E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
title_fullStr E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
title_full_unstemmed E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
title_sort E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2
dc.creator.none.fl_str_mv Montes, Monica Raquel
Monti, José Luis Eugenio
Rossi, Rolando Carlos
author Montes, Monica Raquel
author_facet Montes, Monica Raquel
Monti, José Luis Eugenio
Rossi, Rolando Carlos
author_role author
author2 Monti, José Luis Eugenio
Rossi, Rolando Carlos
author2_role author
author
dc.subject.none.fl_str_mv Na+/K+-Atpase
Conformational Transition
Rb+-Deocclusion
Vanadate
topic Na+/K+-Atpase
Conformational Transition
Rb+-Deocclusion
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 This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na+ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na+-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+.
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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; 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. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina
description This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb+ deocclusion stimulated by Na+ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb+ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb+ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb+]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb+-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na+-stimulated Rb+ deocclusion would require the release of at least one Rb+ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb+ in equilibrium conditions. Results show that, while Mg2 + decreases the affinity for Rb+, addition of vanadate offsets this effect, increasing the affinity for Rb+. In transient experiments, we investigated the exchange of Rb+ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na+ without significantly affecting the rate of Rb+ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb+ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb+.
publishDate 2012
dc.date.none.fl_str_mv 2012-09
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/11336/18197
Montes, Monica Raquel; Monti, José Luis Eugenio; Rossi, Rolando Carlos; E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1818; 9; 9-2012; 2087-2093
0005-2736
CONICET Digital
CONICET
url http://hdl.handle.net/11336/18197
identifier_str_mv Montes, Monica Raquel; Monti, José Luis Eugenio; Rossi, Rolando Carlos; E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1818; 9; 9-2012; 2087-2093
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/ark/http://www.sciencedirect.com/science/article/pii/S000527361200123X
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2012.04.001
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv 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
_version_ 1844613035115675648
score 13.070432

Publicaciones similares