title: Purification of a novel endothelin-converting enzyme specific for big endothelin-3.
abstract: Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a Km value of 0.14 microM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET(B) receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.
authors.affiliation: Department of Pharmacology, Kyoto University, Faculty of Medicine, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Hasegawa
authors.forename: K
authors.initials: K
authors.lastname: Hiki
authors.forename: T
authors.initials: T
authors.lastname: Sawamura
authors.forename: T
authors.initials: T
authors.lastname: Aoyama
authors.forename: Y
authors.initials: Y
authors.lastname: Okamoto
authors.forename: S
authors.initials: S
authors.lastname: Miwa
authors.forename: S
authors.initials: S
authors.lastname: Shimohama
authors.forename: J
authors.initials: J
authors.lastname: Kimura
authors.forename: T
authors.initials: T
authors.lastname: Masaki
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D002829:Choroid / Q000201:enzymology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019334:Endothelin-3; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D005123:Eye / Q000201:enzymology*; D006801:Humans; D007498:Iris / Q000201:enzymology; D007700:Kinetics; D008666:Metalloendopeptidases; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism*; D012160:Retina / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D019334:Endothelin-3; D016232:Endothelins; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; D000072396:Endothelin-Converting Enzymes