The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements

Autores
Faraj, Santiago Enrique; Valsecchi, Wanda Mariela; Cerf, Nicole Talia; Fedosova, N. U.; Rossi, Rolando Carlos; Montes, Monica Raquel
Año de publicación
2021
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1−E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K → E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that—during dephosphorylation—the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of kobs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.
Fil: Faraj, Santiago Enrique. 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: Valsecchi, Wanda Mariela. 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: Cerf, Nicole Talia. 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: Fedosova, N. U.. Aarhus University. Department of Bioscience; Dinamarca
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
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
Materia
CONFORMATIONAL CHANGES
ENZYME KINETICS
ENZYME MECHANISM
H,K-ATPASE
LIGAND BINDING
NA,K-ATPASE
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:11336/156330
Acceder
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network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurementsFaraj, Santiago EnriqueValsecchi, Wanda MarielaCerf, Nicole TaliaFedosova, N. U.Rossi, Rolando CarlosMontes, Monica RaquelCONFORMATIONAL CHANGESENZYME KINETICSENZYME MECHANISMH,K-ATPASELIGAND BINDINGNA,K-ATPASEhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1−E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K → E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that—during dephosphorylation—the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of kobs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.Fil: Faraj, Santiago Enrique. 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: Valsecchi, Wanda Mariela. 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: Cerf, Nicole Talia. 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: Fedosova, N. U.. Aarhus University. Department of Bioscience; DinamarcaFil: 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; ArgentinaFil: 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; ArgentinaElsevier Science2021-01info: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/11336/156330Faraj, Santiago Enrique; Valsecchi, Wanda Mariela; Cerf, Nicole Talia; Fedosova, N. U.; Rossi, Rolando Carlos; et al.; The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1863; 1; 1-2021; 1-90005-2736CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S0005273620303205info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2020.183477info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:06:48Zoai:ri.conicet.gov.ar:11336/156330instacron: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:34982025-10-15 15:06:48.91CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
title The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
spellingShingle The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
Faraj, Santiago Enrique
CONFORMATIONAL CHANGES
ENZYME KINETICS
ENZYME MECHANISM
H,K-ATPASE
LIGAND BINDING
NA,K-ATPASE
title_short The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
title_full The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
title_fullStr The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
title_full_unstemmed The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
title_sort The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements
dc.creator.none.fl_str_mv Faraj, Santiago Enrique
Valsecchi, Wanda Mariela
Cerf, Nicole Talia
Fedosova, N. U.
Rossi, Rolando Carlos
Montes, Monica Raquel
author Faraj, Santiago Enrique
author_facet Faraj, Santiago Enrique
Valsecchi, Wanda Mariela
Cerf, Nicole Talia
Fedosova, N. U.
Rossi, Rolando Carlos
Montes, Monica Raquel
author_role author
author2 Valsecchi, Wanda Mariela
Cerf, Nicole Talia
Fedosova, N. U.
Rossi, Rolando Carlos
Montes, Monica Raquel
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv CONFORMATIONAL CHANGES
ENZYME KINETICS
ENZYME MECHANISM
H,K-ATPASE
LIGAND BINDING
NA,K-ATPASE
topic CONFORMATIONAL CHANGES
ENZYME KINETICS
ENZYME MECHANISM
H,K-ATPASE
LIGAND BINDING
NA,K-ATPASE
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1−E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K → E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that—during dephosphorylation—the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of kobs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.
Fil: Faraj, Santiago Enrique. 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: Valsecchi, Wanda Mariela. 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: Cerf, Nicole Talia. 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: Fedosova, N. U.. Aarhus University. Department of Bioscience; Dinamarca
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
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
description H,K-ATPase and Na,K-ATPase show the highest degree of sequence similarity among all other members of the P-type ATPases family. To explore their common features in terms of ligand binding, we evaluated conformational transitions due to the binding of Na+, K+ and Pi in the H,K-ATPase, and compared the results with those obtained for the Na,K-ATPase. This work shows that eosin fluorescence time courses provide a reasonably precise method to study the kinetics of the E1−E2 conformational changes in the H,K-ATPase. We found that, although Na+ shifts the equilibrium toward the E1 conformation and seems to compete with H+ in ATPase activity assays, it was neither possible to isolate a Na+-occluded state, nor to reveal an influx of Na+ related to H,K-ATPase activity. The high rate of the E2K → E1 transition found for the H,K-ATPase, which is not compatible with the presence of a K+-occluded form, agrees with the negligible level of occluded Rb+ (used as a K+ congener) found in the absence of added ligands. The use of vanadate and fluorinated metals to induce E2P-like states increased the level of occluded Rb+ and suggests that—during dephosphorylation—the probability of K+ to remain occluded increases from the E2P-ground to the E2P-product state. From kinetic experiments we found an unexpected increase in the values of kobs for E2P formation with [Pi]; consequently, to obey the Albers-Post model, the binding of Pi to the E2 state cannot be a rapid-equilibrium reaction.
publishDate 2021
dc.date.none.fl_str_mv 2021-01
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/11336/156330
Faraj, Santiago Enrique; Valsecchi, Wanda Mariela; Cerf, Nicole Talia; Fedosova, N. U.; Rossi, Rolando Carlos; et al.; The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1863; 1; 1-2021; 1-9
0005-2736
CONICET Digital
CONICET
url http://hdl.handle.net/11336/156330
identifier_str_mv Faraj, Santiago Enrique; Valsecchi, Wanda Mariela; Cerf, Nicole Talia; Fedosova, N. U.; Rossi, Rolando Carlos; et al.; The interaction of Na+, K+, and phosphate with the gastric H,K-ATPase. Kinetics of E1–E2 conformational changes assessed by eosin fluorescence measurements; Elsevier Science; Biochimica et Biophysica Acta - Biomembranes; 1863; 1; 1-2021; 1-9
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/https://linkinghub.elsevier.com/retrieve/pii/S0005273620303205
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2020.183477
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
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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