Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media

Autores
Sanchez, Julieta Maria; Ciklic, Iván Francisco; Perillo, Maria Angelica
Año de publicación
2005
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
We had demonstrated that membrane adsorption or penetration differentially modulated β-Galactosidase (β-Gal) activity against soluble substrates (Coll. and Surf., 24, 21, 2002). In a heterogeneous media, not only the enzyme but also the rest of the chemical species taking part in a chemical reaction would eventually interact with the available surfaces. The aim of the present work was to investigate if, in addition to changes in the intrinsic mechanism of the reaction at the lipid-water interface, the kinetics of enzyme-catalyzed reactions could be significantly affected by the partitioning of the substrate (ortho-nitro-phenyl galactopyranoside (ONPG)), the product (ortho-nitro-phenol (ONP)) and the enzyme (E. coli β-Gal) towards the membrane. Multilamellar vesicles of sPC were used as model membranes. Membrane-water partition coefficients (P m/w) were determined according to the theory and methodology developed previously (J. Neurosci. Meth. 36, 203, 1991). The values of P m/w obtained (P ONPG = 0, P ONP = 50 and P β-Gal = 118) were applied to a two-compartment model, which assumed a free access of the substrate to the enzyme and a nucleophile-like activatory effect exerted, within the membrane compartment, by the lipid-water interface. This model: (i) reproduced the lipid concentration-dependence we had observed previously in V max, (ii) predicted the values of k 4 = 3.54 × 10 7 s - 1 and the extinction coefficient of the aglycone in the membrane phase, 4012 M - 1 cm - 1, with p < 0.0001 and p < 0.02, respectively, as well as for P β-Gal = 117 (which was poor (p = 0.6716) but gave a numerical value within the same order of magnitude that the experimental value) and (iii) emphasized the importance of the more efficient reaction mechanism in the membrane phase compared with that in the aqueous phase (k 4 ≫ k 3).
Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina
Fil: Ciklic, Iván Francisco. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina
Fil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina
Materia
Β-Galactosidase
Enzyme Kinetics
O-Nitrophenyl-Β-D-Galactopyranoside
Partition Coefficient
Phospholipids Vesicles
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:11336/58383
Acceder
id CONICETDig_60d887dbfb55dc7ebaffad275b926fc7
oai_identifier_str oai:ri.conicet.gov.ar:11336/58383
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous mediaSanchez, Julieta MariaCiklic, Iván FranciscoPerillo, Maria AngelicaΒ-GalactosidaseEnzyme KineticsO-Nitrophenyl-Β-D-GalactopyranosidePartition CoefficientPhospholipids Vesicleshttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1We had demonstrated that membrane adsorption or penetration differentially modulated β-Galactosidase (β-Gal) activity against soluble substrates (Coll. and Surf., 24, 21, 2002). In a heterogeneous media, not only the enzyme but also the rest of the chemical species taking part in a chemical reaction would eventually interact with the available surfaces. The aim of the present work was to investigate if, in addition to changes in the intrinsic mechanism of the reaction at the lipid-water interface, the kinetics of enzyme-catalyzed reactions could be significantly affected by the partitioning of the substrate (ortho-nitro-phenyl galactopyranoside (ONPG)), the product (ortho-nitro-phenol (ONP)) and the enzyme (E. coli β-Gal) towards the membrane. Multilamellar vesicles of sPC were used as model membranes. Membrane-water partition coefficients (P m/w) were determined according to the theory and methodology developed previously (J. Neurosci. Meth. 36, 203, 1991). The values of P m/w obtained (P ONPG = 0, P ONP = 50 and P β-Gal = 118) were applied to a two-compartment model, which assumed a free access of the substrate to the enzyme and a nucleophile-like activatory effect exerted, within the membrane compartment, by the lipid-water interface. This model: (i) reproduced the lipid concentration-dependence we had observed previously in V max, (ii) predicted the values of k 4 = 3.54 × 10 7 s - 1 and the extinction coefficient of the aglycone in the membrane phase, 4012 M - 1 cm - 1, with p < 0.0001 and p < 0.02, respectively, as well as for P β-Gal = 117 (which was poor (p = 0.6716) but gave a numerical value within the same order of magnitude that the experimental value) and (iii) emphasized the importance of the more efficient reaction mechanism in the membrane phase compared with that in the aqueous phase (k 4 ≫ k 3).Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; ArgentinaFil: Ciklic, Iván Francisco. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; ArgentinaFil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; ArgentinaElsevier Science2005-12-01info: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/11336/58383Sanchez, Julieta Maria; Ciklic, Iván Francisco; Perillo, Maria Angelica; Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media; Elsevier Science; Biophysical Chemistry; 118; 2-3; 1-12-2005; 69-770301-4622CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0301462205001250info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bpc.2005.05.009info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:15:03Zoai:ri.conicet.gov.ar:11336/58383instacron: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 10:15:03.379CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
title Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
spellingShingle Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
Sanchez, Julieta Maria
Β-Galactosidase
Enzyme Kinetics
O-Nitrophenyl-Β-D-Galactopyranoside
Partition Coefficient
Phospholipids Vesicles
title_short Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
title_full Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
title_fullStr Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
title_full_unstemmed Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
title_sort Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media
dc.creator.none.fl_str_mv Sanchez, Julieta Maria
Ciklic, Iván Francisco
Perillo, Maria Angelica
author Sanchez, Julieta Maria
author_facet Sanchez, Julieta Maria
Ciklic, Iván Francisco
Perillo, Maria Angelica
author_role author
author2 Ciklic, Iván Francisco
Perillo, Maria Angelica
author2_role author
author
dc.subject.none.fl_str_mv Β-Galactosidase
Enzyme Kinetics
O-Nitrophenyl-Β-D-Galactopyranoside
Partition Coefficient
Phospholipids Vesicles
topic Β-Galactosidase
Enzyme Kinetics
O-Nitrophenyl-Β-D-Galactopyranoside
Partition Coefficient
Phospholipids Vesicles
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv We had demonstrated that membrane adsorption or penetration differentially modulated β-Galactosidase (β-Gal) activity against soluble substrates (Coll. and Surf., 24, 21, 2002). In a heterogeneous media, not only the enzyme but also the rest of the chemical species taking part in a chemical reaction would eventually interact with the available surfaces. The aim of the present work was to investigate if, in addition to changes in the intrinsic mechanism of the reaction at the lipid-water interface, the kinetics of enzyme-catalyzed reactions could be significantly affected by the partitioning of the substrate (ortho-nitro-phenyl galactopyranoside (ONPG)), the product (ortho-nitro-phenol (ONP)) and the enzyme (E. coli β-Gal) towards the membrane. Multilamellar vesicles of sPC were used as model membranes. Membrane-water partition coefficients (P m/w) were determined according to the theory and methodology developed previously (J. Neurosci. Meth. 36, 203, 1991). The values of P m/w obtained (P ONPG = 0, P ONP = 50 and P β-Gal = 118) were applied to a two-compartment model, which assumed a free access of the substrate to the enzyme and a nucleophile-like activatory effect exerted, within the membrane compartment, by the lipid-water interface. This model: (i) reproduced the lipid concentration-dependence we had observed previously in V max, (ii) predicted the values of k 4 = 3.54 × 10 7 s - 1 and the extinction coefficient of the aglycone in the membrane phase, 4012 M - 1 cm - 1, with p < 0.0001 and p < 0.02, respectively, as well as for P β-Gal = 117 (which was poor (p = 0.6716) but gave a numerical value within the same order of magnitude that the experimental value) and (iii) emphasized the importance of the more efficient reaction mechanism in the membrane phase compared with that in the aqueous phase (k 4 ≫ k 3).
Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina
Fil: Ciklic, Iván Francisco. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina
Fil: Perillo, Maria Angelica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina
description We had demonstrated that membrane adsorption or penetration differentially modulated β-Galactosidase (β-Gal) activity against soluble substrates (Coll. and Surf., 24, 21, 2002). In a heterogeneous media, not only the enzyme but also the rest of the chemical species taking part in a chemical reaction would eventually interact with the available surfaces. The aim of the present work was to investigate if, in addition to changes in the intrinsic mechanism of the reaction at the lipid-water interface, the kinetics of enzyme-catalyzed reactions could be significantly affected by the partitioning of the substrate (ortho-nitro-phenyl galactopyranoside (ONPG)), the product (ortho-nitro-phenol (ONP)) and the enzyme (E. coli β-Gal) towards the membrane. Multilamellar vesicles of sPC were used as model membranes. Membrane-water partition coefficients (P m/w) were determined according to the theory and methodology developed previously (J. Neurosci. Meth. 36, 203, 1991). The values of P m/w obtained (P ONPG = 0, P ONP = 50 and P β-Gal = 118) were applied to a two-compartment model, which assumed a free access of the substrate to the enzyme and a nucleophile-like activatory effect exerted, within the membrane compartment, by the lipid-water interface. This model: (i) reproduced the lipid concentration-dependence we had observed previously in V max, (ii) predicted the values of k 4 = 3.54 × 10 7 s - 1 and the extinction coefficient of the aglycone in the membrane phase, 4012 M - 1 cm - 1, with p < 0.0001 and p < 0.02, respectively, as well as for P β-Gal = 117 (which was poor (p = 0.6716) but gave a numerical value within the same order of magnitude that the experimental value) and (iii) emphasized the importance of the more efficient reaction mechanism in the membrane phase compared with that in the aqueous phase (k 4 ≫ k 3).
publishDate 2005
dc.date.none.fl_str_mv 2005-12-01
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/11336/58383
Sanchez, Julieta Maria; Ciklic, Iván Francisco; Perillo, Maria Angelica; Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media; Elsevier Science; Biophysical Chemistry; 118; 2-3; 1-12-2005; 69-77
0301-4622
CONICET Digital
CONICET
url http://hdl.handle.net/11336/58383
identifier_str_mv Sanchez, Julieta Maria; Ciklic, Iván Francisco; Perillo, Maria Angelica; Effect of partitioning equilibria on the activity of β-galactosidase in heterogeneous media; Elsevier Science; Biophysical Chemistry; 118; 2-3; 1-12-2005; 69-77
0301-4622
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://www.sciencedirect.com/science/article/pii/S0301462205001250
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bpc.2005.05.009
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
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_ 1844614084115300352
score 13.070432

Publicaciones similares