Partial purification and properties of rat urine arylamidases

RESUMO

Arylamidases were isolated from rat urine using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide as substrates to monitor the purification. Ion-exchange chromatography separated three peaks of activity (A, B and C). after gel filtration chromatography, the second and third peaks (B and C) were further purified to provide B1 and C1. Each behaved like a single active protein band on 7.5% polyacrylamide gel electrophoresis without SDS. he molecular weights of fractions B1 and C1 determined by SDS-PAGE were 440 and 270 kDa, respectively. The pH optimum for arylamidase activity was 7.5 for both forms on all substrate for both forms. The arylamidase activity of B1 and C1 was not affected by the presence of chloride ions and was increased in the presence of CaCl2 and MnCl2 only when L-Glu-2-naphthylamide was used as substrate. EDTA (3.3-33.0 micronM) and o-phenanthroline (0.1-1.0mM) but not -SH(0.08-0.67 mM) or -S-S-(0.42-3.3mM) group reagents inhibited the arylamidase activity. Hydrolysis of L-Leu-2-naphthylamide by fractions B1 and C1 was competitively inhibited by leucine (0.14-0.56 mM), indomethacin and puromycin (67-267 micronM) and bestatin (8.3-33.3 micronM). For each inhibitor, the Ki values were similar in the two fractions: 100 micronM for L-leucine, 10 micronM for indomethacin and puromycin and 1.0 micronM for bestatin. The enzymatic properties of fractions B1 and C1 were similar to those reported for fraction A1 by Alves et al. (Brazilian Journal of Medical and Biological...