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.
Highlights
Presented at the XIV Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, Brasil, August 25-28, 1999
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
Aminopeptidases (EC 3.4.11.XX) are enzymes that participate in the final stages of protein degradation and hydrolyze peptide bonds yielding amino acids from N-terminal peptides and proteins and act on some artificial substrates like aminoacyl-ß-naphthylamides (AA-NA) and aminoacyl-p-nitroanilide (AA-Nan)
Summary
Presented at the XIV Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, Brasil, August 25-28, 1999. 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. In the present study we describe the purification and characterization of one aminopeptidase from Phaseolus vulgaris seed crude extract which presented arylamidase activity on the L-Leu-p-nitroanilide (LeuNan) and AA-NA of Leu, Ala, Arg and Met. The hydrolysis of AA-NA was measured by the method of Höpsu et al [6] and that of Leu-Nan by the method of Tuppy at al.
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