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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.

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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 per cent) 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 per cent by 0.15 M NaCl, inhibited 94 per cent by 2.0 mM Zn2+, inhibited 91 per cent by sodium p-hydroxymercuribenzoate and inhibited 45 per cent 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 per cent the enzyme activity. The aminopeptidase activity in the seeds decayed 50 per cent after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

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We describe here two patients with angiographic diagnosis of intrastent restenosis and regional myocardial ischemia. One stent restenosis was located in a native coronary artery and the other in a vein graft. Both were treated with cutting balloon angioplasty (CBA), inflated at low pressures. Angiographic success was obtained and both patients were discharged in the day after the procedure. Cutting balloon angioplasty using low inflation pressures achieved important luminal gains, in these two cases of intrastent restenosis. Further studies are necessary before the effectiveness of this procedure can be precisely defined.

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The aminopeptidase activity of a homogenate of rabbit kidney treated with Triton X-100 was measured using L-aminoacyl-2-naphthylamides (AA-NA). After gradient elution ion-exchange chromatography, four peaks of aminopeptidase activity were eluted. The enzyme eluted at 450 microS containing 33.5% of the activity towards Arg-NA was applied to a Superdex 75 column and presented only one protein band on 10% SDS-polyacrylamide gel electrophoresis. This enzyme has an apparent molecular mass of 78 kDa, is five-fold activated by 0.15 M NaCl and the highest Vmax/KM ratio was obtained with Arg-NA. Enzyme activity was inhibited 100% by 0.13 mM sodium p-hydroxymercuribenzoate, 20% by 0.75 mM EDTA and 100% by 0.66 mM o-phenanthroline. Puromycin and bestatin behaved like competitive inhibitors with a Ki of 0.60 mM and 5.0 microM and 5.0 microM, respectively.

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The aminopeptidase activity of a homogenate of rabbit kidney treated with Triton X-100 was measured using L-aminoacyl-2-naphthylatmides (AA-NA). After gradient elution ion-exchange chromatography, four peaks of aminopeptidase activity were eluted. The enzyme eluted at 450 muS containing 33.5 per cent of the activity towards Arg-NA was applied to a Superdex 75 column and presented only one protein band on 10 per cent SDS-polyacrylamide gel electrophoresis. This enzyme has an apparent molecular mass of 78 kDa, is five-fold activated by 0.15 M NaCl and the highest Vmax/Km ratio was obtained with Arg-NA. Enzyme activity was inhibited 100 per cent by 0.13 mM sodium p-hydroxymercuribenzoate, 20 per cent by 0.75 mM EDTA and 100 per cent by 0.66 mM ophenanthroline. Puromycin and bestatin behaved like competitive inhibitors with a Ki of 0.60 mM and 5.0 muM, respectively.

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The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase.

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The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase

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The selective distribution of methionyl aminopeptidase (MAP) among rat liver mitochondria (heavy and light) and microsomes is reported. Several properties of MAP from the three subcellular fractions showed that the enzyme is a typical aminopeptidase able to remove N-terminal methionine from oligopeptides and methionyl-2-naphthylamide but not from Met-Ala-Ser. MAP is a membrane-bound enzyme sensitive to SH-group oxidants and inhibitable by L-methionine but not by usual arylaminopeptidase inhibitors. It is suggested that, MAP may play an important role during protein synthesis in rat liver.

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