Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase
Autor(a) principal: | |
---|---|
Data de Publicação: | 1999 |
Outros Autores: | , , |
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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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 |
_version_ |
1814268404796751872 |