Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum

Autores
Arias, Diego Gustavo; Márquez, Vanina Elizabet; Beccaria, Alejandro José; Guerrero, Sergio Adrian; Iglesias, Alberto Alvaro
Año de publicación
2010
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg-1 with an optimal activity at pH 8.0 and 32 °C. We determined Km values of 14 μM and 60 μM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.
Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Márquez, Vanina Elizabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Beccaria, Alejandro José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Materia
GLUTATHIONE
GLUTATHIONE REDUCTASE
PHAEODACTYLUM TRICORNUTUM
REDOX METABOLISM
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:11336/84784
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/84784
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutumArias, Diego GustavoMárquez, Vanina ElizabetBeccaria, Alejandro JoséGuerrero, Sergio AdrianIglesias, Alberto AlvaroGLUTATHIONEGLUTATHIONE REDUCTASEPHAEODACTYLUM TRICORNUTUMREDOX METABOLISMhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg-1 with an optimal activity at pH 8.0 and 32 °C. We determined Km values of 14 μM and 60 μM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Márquez, Vanina Elizabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Beccaria, Alejandro José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaElsevier Gmbh2010-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/84784Arias, Diego Gustavo; Márquez, Vanina Elizabet; Beccaria, Alejandro José; Guerrero, Sergio Adrian; Iglesias, Alberto Alvaro; Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum; Elsevier Gmbh; Protist; 161; 1; 1-2010; 91-1011434-4610CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.protis.2009.06.001info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:04:00Zoai:ri.conicet.gov.ar:11336/84784instacron: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:34982025-09-03 10:04:00.419CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
title Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
spellingShingle Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
Arias, Diego Gustavo
GLUTATHIONE
GLUTATHIONE REDUCTASE
PHAEODACTYLUM TRICORNUTUM
REDOX METABOLISM
title_short Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
title_full Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
title_fullStr Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
title_full_unstemmed Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
title_sort Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
dc.creator.none.fl_str_mv Arias, Diego Gustavo
Márquez, Vanina Elizabet
Beccaria, Alejandro José
Guerrero, Sergio Adrian
Iglesias, Alberto Alvaro
author Arias, Diego Gustavo
author_facet Arias, Diego Gustavo
Márquez, Vanina Elizabet
Beccaria, Alejandro José
Guerrero, Sergio Adrian
Iglesias, Alberto Alvaro
author_role author
author2 Márquez, Vanina Elizabet
Beccaria, Alejandro José
Guerrero, Sergio Adrian
Iglesias, Alberto Alvaro
author2_role author
author
author
author
dc.subject.none.fl_str_mv GLUTATHIONE
GLUTATHIONE REDUCTASE
PHAEODACTYLUM TRICORNUTUM
REDOX METABOLISM
topic GLUTATHIONE
GLUTATHIONE REDUCTASE
PHAEODACTYLUM TRICORNUTUM
REDOX METABOLISM
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg-1 with an optimal activity at pH 8.0 and 32 °C. We determined Km values of 14 μM and 60 μM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.
Fil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Márquez, Vanina Elizabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Beccaria, Alejandro José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
description Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg-1 with an optimal activity at pH 8.0 and 32 °C. We determined Km values of 14 μM and 60 μM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.
publishDate 2010
dc.date.none.fl_str_mv 2010-01
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/11336/84784
Arias, Diego Gustavo; Márquez, Vanina Elizabet; Beccaria, Alejandro José; Guerrero, Sergio Adrian; Iglesias, Alberto Alvaro; Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum; Elsevier Gmbh; Protist; 161; 1; 1-2010; 91-101
1434-4610
CONICET Digital
CONICET
url http://hdl.handle.net/11336/84784
identifier_str_mv Arias, Diego Gustavo; Márquez, Vanina Elizabet; Beccaria, Alejandro José; Guerrero, Sergio Adrian; Iglesias, Alberto Alvaro; Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum; Elsevier Gmbh; Protist; 161; 1; 1-2010; 91-101
1434-4610
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.protis.2009.06.001
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
application/pdf
dc.publisher.none.fl_str_mv Elsevier Gmbh
publisher.none.fl_str_mv Elsevier Gmbh
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 13.13397

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