Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides.
A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004355:Drug Stability; D000882:Haplorhini; D006868:Hydrolysis; D007700:Kinetics; D008251:Macaca; D008623:Mercaptoethanol / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D013379:Substrate Specificity
D000596:Amino Acids; D010455:Peptides; D008623:Mercaptoethanol; D000626:Aminopeptidases; D004229:Dithiothreitol