title: Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells.
abstract: A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.
authors.affiliation: Department of Dermatology, University of California, San Francisco 94143-0536.
authors.forename: M
authors.initials: M
authors.lastname: Kikuchi
authors.forename: K
authors.initials: K
authors.lastname: Fukuyama
authors.forename: K
authors.initials: K
authors.lastname: Hirayama
authors.forename: W L
authors.initials: WL
authors.lastname: Epstein
mesh_terms: D000803:Angiotensin I / Q000378:metabolism; D000818:Animals; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D043422:Carboxypeptidases A; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004745:Enkephalins / Q000378:metabolism; D004817:Epidermis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
chemical_list: D004745:Enkephalins; D000803:Angiotensin I; D002268:Carboxypeptidases; D043422:Carboxypeptidases A