A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype

Autores
Carbajal, Agustin; Chesta, María Eugenia; Bisig, Carlos Gaston; Arce, Carlos Angel
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly– disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly–disassembly procedure. The effective inhibitory concentration of TSA was 3 μM in the homogenate and 1 μM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ∼64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly–disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly– disassembly kinetics were the same regardless of the degree of acetylation.
Fil: Carbajal, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
Fil: Chesta, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
Fil: Bisig, Carlos Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
Fil: Arce, Carlos Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
Materia
Acetylated Tubulin
Microtubules
Acetylation
Purification
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:11336/24990
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/24990
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A novel method for purification of polymerizable tubulin with a high content of the acetylated isotypeCarbajal, AgustinChesta, María EugeniaBisig, Carlos GastonArce, Carlos AngelAcetylated TubulinMicrotubulesAcetylationPurificationhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly– disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly–disassembly procedure. The effective inhibitory concentration of TSA was 3 μM in the homogenate and 1 μM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ∼64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly–disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly– disassembly kinetics were the same regardless of the degree of acetylation.Fil: Carbajal, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; ArgentinaFil: Chesta, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; ArgentinaFil: Bisig, Carlos Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; ArgentinaFil: Arce, Carlos Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; ArgentinaPortland Press2013-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/24990Carbajal, Agustin; Chesta, María Eugenia; Bisig, Carlos Gaston; Arce, Carlos Angel; A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype; Portland Press; Biochemical Journal; 449; 3; 2-2013; 643-6480264-6021CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1042/BJ20121439info:eu-repo/semantics/altIdentifier/url/http://www.biochemj.org/content/449/3/643info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:03:11Zoai:ri.conicet.gov.ar:11336/24990instacron: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:34982025-09-10 13:03:12.137CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
title A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
spellingShingle A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
Carbajal, Agustin
Acetylated Tubulin
Microtubules
Acetylation
Purification
title_short A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
title_full A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
title_fullStr A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
title_full_unstemmed A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
title_sort A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype
dc.creator.none.fl_str_mv Carbajal, Agustin
Chesta, María Eugenia
Bisig, Carlos Gaston
Arce, Carlos Angel
author Carbajal, Agustin
author_facet Carbajal, Agustin
Chesta, María Eugenia
Bisig, Carlos Gaston
Arce, Carlos Angel
author_role author
author2 Chesta, María Eugenia
Bisig, Carlos Gaston
Arce, Carlos Angel
author2_role author
author
author
dc.subject.none.fl_str_mv Acetylated Tubulin
Microtubules
Acetylation
Purification
topic Acetylated Tubulin
Microtubules
Acetylation
Purification
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly– disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly–disassembly procedure. The effective inhibitory concentration of TSA was 3 μM in the homogenate and 1 μM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ∼64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly–disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly– disassembly kinetics were the same regardless of the degree of acetylation.
Fil: Carbajal, Agustin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
Fil: Chesta, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
Fil: Bisig, Carlos Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
Fil: Arce, Carlos Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones En Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Cs.químicas. Centro de Investigaciones En Química Biológica de Córdoba; Argentina
description Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly– disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly–disassembly procedure. The effective inhibitory concentration of TSA was 3 μM in the homogenate and 1 μM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ∼64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly–disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly– disassembly kinetics were the same regardless of the degree of acetylation.
publishDate 2013
dc.date.none.fl_str_mv 2013-02
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/11336/24990
Carbajal, Agustin; Chesta, María Eugenia; Bisig, Carlos Gaston; Arce, Carlos Angel; A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype; Portland Press; Biochemical Journal; 449; 3; 2-2013; 643-648
0264-6021
CONICET Digital
CONICET
url http://hdl.handle.net/11336/24990
identifier_str_mv Carbajal, Agustin; Chesta, María Eugenia; Bisig, Carlos Gaston; Arce, Carlos Angel; A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype; Portland Press; Biochemical Journal; 449; 3; 2-2013; 643-648
0264-6021
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1042/BJ20121439
info:eu-repo/semantics/altIdentifier/url/http://www.biochemj.org/content/449/3/643
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
dc.publisher.none.fl_str_mv Portland Press
publisher.none.fl_str_mv Portland Press
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 12.993085

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