Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.

Autores
Liggieri, Constanza Silvina; Arribére, María Cecilia; Trejo, Sebastián Alejandro; Canals, Francesc; Avilés, Francesc X.; Priolo de Lufrano, Nora Silvia
Año de publicación
2004
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.
Centro de Investigación de Proteínas Vegetales
Materia
Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by/4.0/
Repositorio
SEDICI (UNLP)
Institución
Universidad Nacional de La Plata
OAI Identificador
oai:sedici.unlp.edu.ar:10915/135515
Acceder
id SEDICI_7eda9a11509ab03968b70ee6ca2c0335
oai_identifier_str oai:sedici.unlp.edu.ar:10915/135515
network_acronym_str SEDICI
repository_id_str 1329
network_name_str SEDICI (UNLP)
spelling Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.Liggieri, Constanza SilvinaArribére, María CeciliaTrejo, Sebastián AlejandroCanals, FrancescAvilés, Francesc X.Priolo de Lufrano, Nora SilviaBiologíaAsclepias curassavicaAsclepiadaceaecysteine proteinaselatexlaticifersIn this work we report the isolation, purification and characterization of a new protease from latex of <i>Asclepias curassavica</i> L. Crude extract (CE) was obtained by gathering latex on 0.1M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (k<sub>m</sub> and K<sub>cat</sub>) were performed with PFLNA.Centro de Investigación de Proteínas Vegetales2004-08info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionArticulohttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdf403-411http://sedici.unlp.edu.ar/handle/10915/135515enginfo:eu-repo/semantics/altIdentifier/issn/1572-3887info:eu-repo/semantics/altIdentifier/doi/10.1023/b:jopc.0000039554.18157.69info:eu-repo/semantics/altIdentifier/pmid/15517987info:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International (CC BY 4.0)reponame:SEDICI (UNLP)instname:Universidad Nacional de La Platainstacron:UNLP2025-10-15T11:23:48Zoai:sedici.unlp.edu.ar:10915/135515Institucionalhttp://sedici.unlp.edu.ar/Universidad públicaNo correspondehttp://sedici.unlp.edu.ar/oai/snrdalira@sedici.unlp.edu.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:13292025-10-15 11:23:48.953SEDICI (UNLP) - Universidad Nacional de La Platafalse
dc.title.none.fl_str_mv Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
title Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
spellingShingle Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
Liggieri, Constanza Silvina
Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
title_short Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
title_full Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
title_fullStr Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
title_full_unstemmed Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
title_sort Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
dc.creator.none.fl_str_mv Liggieri, Constanza Silvina
Arribére, María Cecilia
Trejo, Sebastián Alejandro
Canals, Francesc
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author Liggieri, Constanza Silvina
author_facet Liggieri, Constanza Silvina
Arribére, María Cecilia
Trejo, Sebastián Alejandro
Canals, Francesc
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author_role author
author2 Arribére, María Cecilia
Trejo, Sebastián Alejandro
Canals, Francesc
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
topic Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
dc.description.none.fl_txt_mv In this work we report the isolation, purification and characterization of a new protease from latex of <i>Asclepias curassavica</i> L. Crude extract (CE) was obtained by gathering latex on 0.1M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (k<sub>m</sub> and K<sub>cat</sub>) were performed with PFLNA.
Centro de Investigación de Proteínas Vegetales
description In this work we report the isolation, purification and characterization of a new protease from latex of <i>Asclepias curassavica</i> L. Crude extract (CE) was obtained by gathering latex on 0.1M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (k<sub>m</sub> and K<sub>cat</sub>) were performed with PFLNA.
publishDate 2004
dc.date.none.fl_str_mv 2004-08
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
Articulo
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://sedici.unlp.edu.ar/handle/10915/135515
url http://sedici.unlp.edu.ar/handle/10915/135515
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/issn/1572-3887
info:eu-repo/semantics/altIdentifier/doi/10.1023/b:jopc.0000039554.18157.69
info:eu-repo/semantics/altIdentifier/pmid/15517987
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by/4.0/
Creative Commons Attribution 4.0 International (CC BY 4.0)
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
Creative Commons Attribution 4.0 International (CC BY 4.0)
dc.format.none.fl_str_mv application/pdf
403-411
dc.source.none.fl_str_mv reponame:SEDICI (UNLP)
instname:Universidad Nacional de La Plata
instacron:UNLP
reponame_str SEDICI (UNLP)
collection SEDICI (UNLP)
instname_str Universidad Nacional de La Plata
instacron_str UNLP
institution UNLP
repository.name.fl_str_mv SEDICI (UNLP) - Universidad Nacional de La Plata
repository.mail.fl_str_mv alira@sedici.unlp.edu.ar
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