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

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. Proteolyt...

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Detalles Bibliográficos
Autores principales: Liggieri, Constanza Silvina, Arribére, María Cecilia, Trejo, Sebastián Alejandro, Canals, Francesc, Avilés, Francesc X., Priolo de Lufrano, Nora Silvia
Formato: Articulo
Lenguaje:Inglés
Publicado: 2004
Materias:
Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
Acceso en línea:http://sedici.unlp.edu.ar/handle/10915/135515
Aporte de:
SEDICI (UNLP) de Universidad Nacional de La Plata
id I19-R120-10915-135515
record_format dspace
institution Universidad Nacional de La Plata
institution_str I-19
repository_str R-120
collection SEDICI (UNLP)
language Inglés
topic Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
spellingShingle Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
Liggieri, Constanza Silvina
Arribére, María Cecilia
Trejo, Sebastián Alejandro
Canals, Francesc
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
topic_facet Biología
Asclepias curassavica
Asclepiadaceae
cysteine proteinase
latex
laticifers
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.
format Articulo
Articulo
author Liggieri, Constanza Silvina
Arribére, María Cecilia
Trejo, Sebastián Alejandro
Canals, Francesc
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author_facet Liggieri, Constanza Silvina
Arribére, María Cecilia
Trejo, Sebastián Alejandro
Canals, Francesc
Avilés, Francesc X.
Priolo de Lufrano, Nora Silvia
author_sort Liggieri, Constanza Silvina
title Purification and Biochemical Characterization of Asclepain c I from the Latex of <i>Asclepias curassavica</i> L.
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.
publishDate 2004
url http://sedici.unlp.edu.ar/handle/10915/135515
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