A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.
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.
Matsumura
Y
Y
Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Japan.
Umekawa
T
T
Kawamura
H
H
Takaoka
M
M
Robinson
P S
PS
Cook
N D
ND
Morimoto
S
S
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
D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon