Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

Autor Abdala, Ana Paula Lima Autor UNIFESP Google Scholar
Takeda, Lincoln Haruki Autor UNIFESP Google Scholar
Freitas Junior, Jose Olavo Autor UNIFESP Google Scholar
Alves, Kaethy Bisan Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
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.
Palavra-chave Phaseolus vulgaris aminopeptidase
bean seed aminopeptidase
leucyl aminopeptidase
Idioma Inglês
Data de publicação 1999-12-01
Publicado em Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 12, p. 1489-1492, 1999.
ISSN 0100-879X (Sherpa/Romeo, fator de impacto)
Publicador Associação Brasileira de Divulgação Científica
Extensão 1489-1492
Fonte http://dx.doi.org/10.1590/S0100-879X1999001200006
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000084453600005
SciELO S0100-879X1999001200006 (estatísticas na SciELO)
Endereço permanente http://repositorio.unifesp.br/handle/11600/850

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