Kinin-converting aminopeptidase from human urine. Further purification and characterization through kinetic and inhibitory studies.
An aminopeptidase from human urine (HUA) able to hydrolyze L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and to convert both MLBK and LBK to BK has been further purified and characterized. The preparation now obtained showed a 3-fold higher specific activity than the previously described one and a single active protein band in 7% polyacrylamide gel electrophoresis accounting for 86% of total protein. Kinetic constants for this kinin-converting enzyme were determined using L-aminoacyl-2-naphthylamides, L-Leu-p-nitroanilide and LBK. The Km values for different naphthylamides were in the 10(-5) M range while that for L-Leu-p-nitroanilide was 3.6 X 10(-4) M. With LBK as substrate the aminopeptidase activity showed the highest catalytic efficiency in spite of a Km in the mM range. The enzyme was poorly inhibited by -SH and -S-S- group reagents. Some L-aminoacids, as well as mono- and diamines, indomethacin, puromycin and bestatin were equipotent competitive inhibitors of both arylamidase and aminopeptidase activities. Results obtained in this paper are compatible with our conclusion that human urine, unlike other enzyme sources, contains only one aminopeptidase, and that this enzyme displays both arylamidase and kinin-converting activities. The enzyme's action may be important in the metabolism of kinins, yielding peptides which could interact with both B-1 and B-2 kinin receptors in the kidney.
D000588:Amines / Q000494:pharmacology; D000596:Amino Acids / Q000494:pharmacology; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000652:urine*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006801:Humans; D007700:Kinetics; D013379:Substrate Specificity
D000588:Amines; D000596:Amino Acids; D000900:Anti-Bacterial Agents; D000893:Anti-Inflammatory Agents; D000626:Aminopeptidases; C023946:kinin-converting enzyme