Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i).
Carboxypeptidase N (kininase I, arginine carboxypeptidase; EC 3.4.17.3) cleaves COOH-terminal basic amino acids of kinins, anaphylatoxins, and other peptides. The tetrameric enzyme of Mr 280,000 was purified from human plasma by ion-exchange and arginine-Sepharose affinity chromatography. Treatment with 3 M guanidine dissociated the enzyme into subunits of 83,000 and 48,000 molecular weight, which were separated and purified by gel filtration or affinity chromatography. When tested with hippurylarginine, hippurylargininic acid, benzoylalanyllysine, or bradykinin, the Mr 48,000 subunit was as active as the intact enzyme and was easily distinguished from human pancreatic carboxypeptidase B (EC 3.4.17.2). However, the Mr 48,000 subunit was less stable at acid pH or at 37 degrees C than the intact enzyme was. The carbohydrate-containing Mr 83,000 subunit was enzymatically inactive but stabilized the Mr 48,000 subunit at 37 degrees C. Trypsin, plasmin, and plasma or urinary kallikrein cleaved carboxypeptidase N into lower molecular weight active fragments, which were unstable at 37 degrees C. Cleavage of the Mr 48,000 subunit with the same enzymes increased activity and yielded fragments of Mr 29,000 or less. Antibodies to the Mr 83,000 of Mr 48,000 subunits crossreacted with the intact enzyme, and antibody to carboxypeptidase N also recognized both subunits. However, antibody to the Mr 83,000 subunit did not recognize Mr 48,000 subunit and antibody to the Mr 48,000 subunit did not crossreact with the Mr 83,000 subunit. Thus, this study indicates that carboxypeptidase N is composed of two immunologically distinct subunits, a Mr 48,000 subunit that is responsible for the enzymatic activity and a Mr 83,000 subunit that may stabilize the enzyme in blood.
D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000097:blood* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D001121:Lysine Carboxypeptidase / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism; D011489:Protein Denaturation
D046911:Macromolecular Substances; D010446:Peptide Fragments; D002268:Carboxypeptidases; D010447:Peptide Hydrolases; D043424:Carboxypeptidase B; D001121:Lysine Carboxypeptidase