Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study

Autores
Luna, Maria Alejandra; Silber, Juana J.; Moyano, Fernando; Correa, Nestor Mariano; Moyano, Fernando
Año de publicación
2016
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In this work we determine the permeability of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) vesicles in the presence of different cholesterol (Cho) contents, by using the enzymatic hydrolysis of N-benzoyl-l-tyrosine p-nitroanilide (Bz-Try-pNA) catalyzed by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media in a 4% p/p ethanol-water mixture and then in DOPC vesicles at different Cho content. In homogenous media, ethanol helps to solubilize the substrate, which is almost insoluble in water and therefore increases the effective concentrations. In DOPC vesicles, ethanol does not destroy the bilayer. In both cases, the enzymatic hydrolysis can be followed by UV-visible spectroscopy. The hydrolysis of Bz-Try-pNA catalyzed by α-CT follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM and kcat/KM were evaluated in both systems at the same solvent mixture compositions. To obtain the kinetic parameters and the permeability of the reactant in DOPC:Cho vesicles, we use a simple mathematical model and dynamic light scattering (DLS) measurements. The results show that the hydrolysis reaction takes place in the water entrapped in the interior of the DOPC vesicles and, that the enzyme encapsulated inside the vesicles, despite the significant differences in the permeability values of Bz-Try-pNA, has similar catalytic effects independently on the Cho composition used.
Fil: Luna, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina
Fil: Silber, Juana J.. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina
Fil: Moyano, Fernando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Moyano, Fernando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Materia
DOPC
PERMEABILITY
ENZYMATIC REACTION
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:11336/180077
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/180077
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic studyLuna, Maria AlejandraSilber, Juana J.Moyano, FernandoCorrea, Nestor MarianoMoyano, FernandoDOPCPERMEABILITYENZYMATIC REACTIONhttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1In this work we determine the permeability of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) vesicles in the presence of different cholesterol (Cho) contents, by using the enzymatic hydrolysis of N-benzoyl-l-tyrosine p-nitroanilide (Bz-Try-pNA) catalyzed by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media in a 4% p/p ethanol-water mixture and then in DOPC vesicles at different Cho content. In homogenous media, ethanol helps to solubilize the substrate, which is almost insoluble in water and therefore increases the effective concentrations. In DOPC vesicles, ethanol does not destroy the bilayer. In both cases, the enzymatic hydrolysis can be followed by UV-visible spectroscopy. The hydrolysis of Bz-Try-pNA catalyzed by α-CT follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM and kcat/KM were evaluated in both systems at the same solvent mixture compositions. To obtain the kinetic parameters and the permeability of the reactant in DOPC:Cho vesicles, we use a simple mathematical model and dynamic light scattering (DLS) measurements. The results show that the hydrolysis reaction takes place in the water entrapped in the interior of the DOPC vesicles and, that the enzyme encapsulated inside the vesicles, despite the significant differences in the permeability values of Bz-Try-pNA, has similar catalytic effects independently on the Cho composition used.Fil: Luna, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Silber, Juana J.. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Moyano, Fernando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Moyano, Fernando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaRoyal Society of Chemistry2016-06info: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/11336/180077Luna, Maria Alejandra; Silber, Juana J.; Moyano, Fernando; Correa, Nestor Mariano; Moyano, Fernando; Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study; Royal Society of Chemistry; RSC Advances; 6; 67; 6-2016; 62594-626012046-2069CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://pubs.rsc.org/en/content/articlelanding/2016/ra/c6ra12847einfo:eu-repo/semantics/altIdentifier/doi/10.1039/C6RA12847Einfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2026-02-06T12:03:58Zoai:ri.conicet.gov.ar:11336/180077instacron: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:34982026-02-06 12:03:58.694CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
title Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
spellingShingle Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
Luna, Maria Alejandra
DOPC
PERMEABILITY
ENZYMATIC REACTION
title_short Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
title_full Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
title_fullStr Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
title_full_unstemmed Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
title_sort Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study
dc.creator.none.fl_str_mv Luna, Maria Alejandra
Silber, Juana J.
Moyano, Fernando
Correa, Nestor Mariano
Moyano, Fernando
author Luna, Maria Alejandra
author_facet Luna, Maria Alejandra
Silber, Juana J.
Moyano, Fernando
Correa, Nestor Mariano
author_role author
author2 Silber, Juana J.
Moyano, Fernando
Correa, Nestor Mariano
author2_role author
author
author
dc.subject.none.fl_str_mv DOPC
PERMEABILITY
ENZYMATIC REACTION
topic DOPC
PERMEABILITY
ENZYMATIC REACTION
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.4
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv In this work we determine the permeability of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) vesicles in the presence of different cholesterol (Cho) contents, by using the enzymatic hydrolysis of N-benzoyl-l-tyrosine p-nitroanilide (Bz-Try-pNA) catalyzed by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media in a 4% p/p ethanol-water mixture and then in DOPC vesicles at different Cho content. In homogenous media, ethanol helps to solubilize the substrate, which is almost insoluble in water and therefore increases the effective concentrations. In DOPC vesicles, ethanol does not destroy the bilayer. In both cases, the enzymatic hydrolysis can be followed by UV-visible spectroscopy. The hydrolysis of Bz-Try-pNA catalyzed by α-CT follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM and kcat/KM were evaluated in both systems at the same solvent mixture compositions. To obtain the kinetic parameters and the permeability of the reactant in DOPC:Cho vesicles, we use a simple mathematical model and dynamic light scattering (DLS) measurements. The results show that the hydrolysis reaction takes place in the water entrapped in the interior of the DOPC vesicles and, that the enzyme encapsulated inside the vesicles, despite the significant differences in the permeability values of Bz-Try-pNA, has similar catalytic effects independently on the Cho composition used.
Fil: Luna, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina
Fil: Silber, Juana J.. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina
Fil: Moyano, Fernando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
Fil: Moyano, Fernando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina
description In this work we determine the permeability of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) vesicles in the presence of different cholesterol (Cho) contents, by using the enzymatic hydrolysis of N-benzoyl-l-tyrosine p-nitroanilide (Bz-Try-pNA) catalyzed by α-chymotrypsin (α-CT). The reaction was first studied in homogeneous media in a 4% p/p ethanol-water mixture and then in DOPC vesicles at different Cho content. In homogenous media, ethanol helps to solubilize the substrate, which is almost insoluble in water and therefore increases the effective concentrations. In DOPC vesicles, ethanol does not destroy the bilayer. In both cases, the enzymatic hydrolysis can be followed by UV-visible spectroscopy. The hydrolysis of Bz-Try-pNA catalyzed by α-CT follows the Michaelis-Menten mechanism and the kinetic parameters: kcat, KM and kcat/KM were evaluated in both systems at the same solvent mixture compositions. To obtain the kinetic parameters and the permeability of the reactant in DOPC:Cho vesicles, we use a simple mathematical model and dynamic light scattering (DLS) measurements. The results show that the hydrolysis reaction takes place in the water entrapped in the interior of the DOPC vesicles and, that the enzyme encapsulated inside the vesicles, despite the significant differences in the permeability values of Bz-Try-pNA, has similar catalytic effects independently on the Cho composition used.
publishDate 2016
dc.date.none.fl_str_mv 2016-06
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/11336/180077
Luna, Maria Alejandra; Silber, Juana J.; Moyano, Fernando; Correa, Nestor Mariano; Moyano, Fernando; Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study; Royal Society of Chemistry; RSC Advances; 6; 67; 6-2016; 62594-62601
2046-2069
CONICET Digital
CONICET
url http://hdl.handle.net/11336/180077
identifier_str_mv Luna, Maria Alejandra; Silber, Juana J.; Moyano, Fernando; Correa, Nestor Mariano; Moyano, Fernando; Determining the substrate permeability through the bilayer of large unilamellar vesicles of DOPC. A kinetic study; Royal Society of Chemistry; RSC Advances; 6; 67; 6-2016; 62594-62601
2046-2069
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://pubs.rsc.org/en/content/articlelanding/2016/ra/c6ra12847e
info:eu-repo/semantics/altIdentifier/doi/10.1039/C6RA12847E
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 Royal Society of Chemistry
publisher.none.fl_str_mv Royal Society of Chemistry
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 13.106097

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