title: A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.
abstract: We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.
authors.affiliation: Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Matsumura
authors.forename: T
authors.initials: T
authors.lastname: Umekawa
authors.forename: H
authors.initials: H
authors.lastname: Kawamura
authors.forename: M
authors.initials: M
authors.lastname: Takaoka
authors.forename: P S
authors.initials: PS
authors.lastname: Robinson
authors.forename: N D
authors.initials: ND
authors.lastname: Cook
authors.forename: S
authors.initials: S
authors.lastname: Morimoto
mesh_terms: D000818:Animals; D001011:Aorta / Q000201:enzymology; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D008168:Lung / Q000201:enzymology; D008666:Metalloendopeptidases; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D013552:Swine; D013997:Time Factors
chemical_list: D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon