Partial purification and properties of rat urine arylamidases.

1. Arylamidases were isolated from rat urine using L-aminoacyl-2-naphthylamides and L-Leu-p-nitroanilide as substrates to monitor the purification. 2. 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. The molecular weights of fractions B1 and C1 determined by SDS-PAGE were 440 and 270 kDa, respectively. 3. The pH optimum for arylamidase activity was 7.5 for both forms on all substrates tested. The maximum value for the Vmax/Km ratio was obtained using L-Leu-2-naphthylamide as substrate for both forms. 4. 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 microM) and o-phenanthroline (0.1-1.0 mM) but not -SH (0.08-0.67 mM) or -S-S-(0.42-3.3 mM) 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 microM) and bestatin (8.3-33.3 microM). For each inhibitor, the Ki values were similar in the two fractions: 100 microM for L-leucine, 10 microM for indomethacin and puromycin and 1.0 microM for bestatin.(ABSTRACT TRUNCATED AT 250 WORDS)