Characterization of two 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 were identified in solubilized membranes prepared from rat lung. The endothelin-converting activities were partially purified by using A80227 ((2S,3R,4S)-2-([N-acetylcyclohexylalanyl-isoleucyl]amino)-1-(2-nap hthyl)-3,4-dihydroxy-6-methylheptane) coupled to an affinity-gel column (Affigel), and subsequently by concanavalin-A immobilized gel chromatography. An endothelin-converting activity was identified in the fraction containing proteins that did not bind to A80227-Affigel. This protease was sensitive to phosphoramidon, soybean trypsin inhibitor, and chymostatin, and preferred big ET-1 or big ET-2 as its substrate over bit ET-3. A second endothelin-converting activity was identified in the fraction containing proteins that bound to the A80227-coupled gel and was eluted 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 also preferred big ET-1 or big ET-2 over big ET-3 as its substrate. Both enzymes were glycoproteins based on their binding to concanavalin-A immobilized gel and were readily eluted by a buffer containing 0.5 M manopyranoside. 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 reported endothelin-converting enzymes.
Chiou
W J
WJ
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064.
Shiosaki
K
K
Tasker
A S
AS
Wu-Wong
J R
JR
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008666:Metalloendopeptidases; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes