Identification of endothelin converting enzyme in bovine lung membranes using a new fluorogenic substrate.
An enzyme partially purified from bovine lung membranes appears to be endothelin converting enzyme (ECE). This enzyme specifically cleaves big endothelin-1 (big ET-1) at the proper site, between Trp21 and Val22, with maximum activity at pH 7.5 and with a Km of roughly 3 microM, to produce endothelin-1 (ET-1) and C-terminal peptide (CTP). This same enzyme hydrolyzes the fluorogenic substrate succinyl-Ile-Ile-Trp-methylcoumarinamide to release the highly fluorescent 7-amino-4-methylcoumarin. The peptide derivative has the same amino acid sequence as big ET-1 and is a good substrate with a Km of about 27 microM. This enzyme is a metalloproteinase. It is not inhibited by five common proteinase inhibitors (pepstatin A, PMSF, NEM, E-64 and thiorphan) but it is inhibited by phosphoramidon and chelating compounds. The apoenzyme is restored to nearly full activity by a zinc-EDTA buffer with pZn = 13.
Kundu
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GC
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Wilson
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IB
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000737:chemistry*; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000737:chemistry; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008666:Metalloendopeptidases; D009842:Oligopeptides / Q000737:chemistry*; D011498:Protein Precursors / Q000737:chemistry; D013379:Substrate Specificity
D003374:Coumarins; D019332:Endothelin-1; D016232:Endothelins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009842:Oligopeptides; D011498:Protein Precursors; C073581:succinyl-isoleucyl-isoleucyl-tryptophyl-methylcoumarinamide; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C028743:7-amino-4-methylcoumarin