A rapid and selective endothelin-converting enzyme assay: characterization of a phosphoramidon-sensitive enzyme from guinea pig lung membrane.
Endothelin-1 (ET-1) is the most potent vasoconstrictor hormone known to date. Biosynthesis of ET-1 from its precursor big endothelin-1 (BET-1) is mediated by endothelin-converting enzyme (ECE), a phosphoramidon-sensitive metalloprotease. We have established a simple, rapid, and selective assay for the evaluation of ECE activity. This assay is based on the quantitative determination of [125I]ET-1 released from (3-[125I]idotyrosyl13)BET-1 by binding to the membrane-bound endothelin (ET) receptor. Using this assay we have discovered that guinea pig lung membrane (GPLGM) contains a phosphoramidon-sensitive ECE. Treatment of GPLGM with 0.06% lubrol increased ECE activity and ET binding of the membrane preparation. Lubrol-treated GPLGM (L-GPLGM) contains a high density of ET binding sites (Bmax = 2000 fmol/mg protein) and shows no proteolytic activity for degredation of ET-1. At protein concentrations suitable for measurement of ECE activity (0.2 mg/ml), L-GPLGM contains a high concentration of ET receptors and shows a rapid rate of ET-1 binding to the membrane preparation (binding equilibrium in < 5 min). Thus, we have utilized L-GPLGM preparation for both ECE activity and measurement of ET-1 binding in a single-step assay for the determination of enzyme activity. ECE activity of L-GPLGM was fully inhibited by phosphoramidon, 1,10-phenanthroline, and EDTA. Class-specific inhibitors of serine, cysteine, and aspartic proteases showed no significant effect on ECE activity in L-GPLGM. These results show that GPLGM contains exclusively a phosphoramidon-sensitive ECE.
Fawzi
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AB
Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539.
Cleven
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RM
Wright
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DL
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors; D001671:Biochemistry / Q000379:methods; D000072396:Endothelin-Converting Enzymes; D005069:Evaluation Studies as Topic; D006020:Glycopeptides / Q000494:pharmacology*; D006168:Guinea Pigs; D008168:Lung / Q000201:enzymology*; D008566:Membranes / Q000201:enzymology; D008666:Metalloendopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013997:Time Factors
D006020:Glycopeptides; D011480:Protease Inhibitors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon