title: Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase.
abstract: 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 microS 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/K M 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 microM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 microM), 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 microM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4 degrees C and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.
authors.affiliation: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brasil.
authors.forename: A P
authors.initials: AP
authors.lastname: Abdala
authors.forename: L H
authors.initials: LH
authors.lastname: Takeda
authors.forename: J O
authors.initials: JO
authors.lastname: Freitas Junior
authors.forename: K B
authors.initials: KB
authors.lastname: Alves
mesh_terms: D000626:Aminopeptidases / Q000302:isolation & purification*; D007887:Fabaceae / Q000201:enzymology*; D010940:Plant Proteins / Q000302:isolation & purification*; D010946:Plants, Medicinal; D012639:Seeds / Q000201:enzymology*
chemical_list: D010940:Plant Proteins; D000626:Aminopeptidases