Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral Carboxypeptidase B-like Enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The Enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The Enzyme cleaves arginine or lysine from the COOH terminus of synthetic Peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the Enzyme shares some properties with other Carboxypeptidase B-like Enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic Carboxypeptidase A or B, human plasma Carboxypeptidase N, and Carboxypeptidase H ("enkephalin convertase"). To denote that the Enzyme is membrane-bound, and to distinguish it from Other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, Carboxypeptidase M could inactivate or modulate the activity of peptide Hormones either before or after their interaction with plasma membrane receptors.