Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

Detalhes bibliográficos
Autor(a) principal: Abdala, Ana Paula Lima [UNIFESP]
Data de Publicação: 1999
Outros Autores: Takeda, Lincoln Haruki [UNIFESP], Freitas Junior, Jose Olavo [UNIFESP], Alves, Kaethy Bisan [UNIFESP]
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNIFESP
Texto Completo: http://dx.doi.org/10.1590/S0100-879X1999001200006
http://repositorio.unifesp.br/handle/11600/850
Resumo: The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.
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spelling Purification and partial characterization of Phaseolus vulgaris seed aminopeptidasePhaseolus vulgaris aminopeptidasebean seed aminopeptidaseleucyl aminopeptidaseThe aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.Universidade Federal de São Paulo (UNIFESP)UNIFESPSciELOAssociação Brasileira de Divulgação CientíficaUniversidade Federal de São Paulo (UNIFESP)Abdala, Ana Paula Lima [UNIFESP]Takeda, Lincoln Haruki [UNIFESP]Freitas Junior, Jose Olavo [UNIFESP]Alves, Kaethy Bisan [UNIFESP]2015-06-14T13:24:57Z2015-06-14T13:24:57Z1999-12-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion1489-1492application/pdfhttp://dx.doi.org/10.1590/S0100-879X1999001200006Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 12, p. 1489-1492, 1999.10.1590/S0100-879X1999001200006S0100-879X1999001200006.pdf0100-879XS0100-879X1999001200006http://repositorio.unifesp.br/handle/11600/850WOS:000084453600005engBrazilian Journal of Medical and Biological Researchinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-08-05T22:29:53Zoai:repositorio.unifesp.br/:11600/850Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-08-05T22:29:53Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false
dc.title.none.fl_str_mv Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
title Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
spellingShingle Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
Abdala, Ana Paula Lima [UNIFESP]
Phaseolus vulgaris aminopeptidase
bean seed aminopeptidase
leucyl aminopeptidase
title_short Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
title_full Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
title_fullStr Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
title_full_unstemmed Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
title_sort Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
author Abdala, Ana Paula Lima [UNIFESP]
author_facet Abdala, Ana Paula Lima [UNIFESP]
Takeda, Lincoln Haruki [UNIFESP]
Freitas Junior, Jose Olavo [UNIFESP]
Alves, Kaethy Bisan [UNIFESP]
author_role author
author2 Takeda, Lincoln Haruki [UNIFESP]
Freitas Junior, Jose Olavo [UNIFESP]
Alves, Kaethy Bisan [UNIFESP]
author2_role author
author
author
dc.contributor.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
dc.contributor.author.fl_str_mv Abdala, Ana Paula Lima [UNIFESP]
Takeda, Lincoln Haruki [UNIFESP]
Freitas Junior, Jose Olavo [UNIFESP]
Alves, Kaethy Bisan [UNIFESP]
dc.subject.por.fl_str_mv Phaseolus vulgaris aminopeptidase
bean seed aminopeptidase
leucyl aminopeptidase
topic Phaseolus vulgaris aminopeptidase
bean seed aminopeptidase
leucyl aminopeptidase
description The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.
publishDate 1999
dc.date.none.fl_str_mv 1999-12-01
2015-06-14T13:24:57Z
2015-06-14T13:24:57Z
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.1590/S0100-879X1999001200006
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 12, p. 1489-1492, 1999.
10.1590/S0100-879X1999001200006
S0100-879X1999001200006.pdf
0100-879X
S0100-879X1999001200006
http://repositorio.unifesp.br/handle/11600/850
WOS:000084453600005
url http://dx.doi.org/10.1590/S0100-879X1999001200006
http://repositorio.unifesp.br/handle/11600/850
identifier_str_mv Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 12, p. 1489-1492, 1999.
10.1590/S0100-879X1999001200006
S0100-879X1999001200006.pdf
0100-879X
S0100-879X1999001200006
WOS:000084453600005
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Brazilian Journal of Medical and Biological Research
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 1489-1492
application/pdf
dc.publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
dc.source.none.fl_str_mv reponame:Repositório Institucional da UNIFESP
instname:Universidade Federal de São Paulo (UNIFESP)
instacron:UNIFESP
instname_str Universidade Federal de São Paulo (UNIFESP)
instacron_str UNIFESP
institution UNIFESP
reponame_str Repositório Institucional da UNIFESP
collection Repositório Institucional da UNIFESP
repository.name.fl_str_mv Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)
repository.mail.fl_str_mv biblioteca.csp@unifesp.br
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