CHARACTERIZATION OF 2 ENDOTHELIN-CONVERTING ENZYMES AND THEIR PREFERENCE FOR BIG ENDOTHELIN-1 AND -2 AS SUBSTRATES

Two proteolytic activities that convert big ET to ET at neutral pH wer e identified in solubilized membranes prepared from rat lung. The endo thelin-converting activities were partially purified by using A80227 ( 2S,3R,4S)-2-{[N-acetylcyclohexylalanyl-isoleucyl] ino}-1-(2-naphthyl)- 3,4-dihydroxy-6-methylheptane) coupled to an affinity-gel column (Affi gel), and subsequently by concanavalin-A immobilized gel chromatograph y. An endothelin-converting activity was identified in the fraction co ntaining proteins that did not bind to A80227-Affigel. This protease w as sensitive to phosphoramidon, soybean trypsin inhibitor, and chymost atin, and preferred big ET-1 or big ET-2 as its substrate over big ET- 3. A second endothelin-converting activity was identified in the fract ion containing proteins that bound to the A80227-coupled gel and was e luted by raising the pH. This protease exhibited activities throughout a range of pH 5.5-9.5, was inhibited by pepstatin A and A80227, and a lso preferred big ET-1 or big ET-2 over big ET-3 as its substrate. Bot h enzymes were glycoproteins based on their binding to concanavalin-A immobilized gel and were readily eluted by a buffer containing 0.5 M m anopyranoside. The results from the pH and protease inhibitor profiles suggesting that these two ET-converting activities extracted from rat lung membranes are distinct and are different from the previously rep orted endothelin-converting enzymes.