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.
Skidgel
R A
RA
Department of Anesthesiology, College of Medicine, University of Illinois, Chicago 60680.
Davis
R M
RM
Tan
F
F
D002268:Carboxypeptidases / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005260:Female; D058851:GPI-Linked Proteins; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology*; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013379:Substrate Specificity
D058851:GPI-Linked Proteins; D006023:Glycoproteins; D002268:Carboxypeptidases; C058479:carboxypeptidase M; D008666:Metalloendopeptidases