endothelin-1 and 7-amino-4-methylcoumarin

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.

Topics: Animals; Aspartic Acid Endopeptidases; Cattle; Cell Membrane; Chromatography, Affinity; Chromatography, High Pressure Liquid; Coumarins; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Enzyme Inhibitors; Fluorescent Dyes; Hydrogen-Ion Concentration; Hydrolysis; Indicators and Reagents; Kinetics; Lung; Metalloendopeptidases; Oligopeptides; Protein Precursors; Substrate Specificity