Identification and partial purification of a thiol endothelin-converting enzyme from porcine aortic endothelial cells.
Endothelin is a potent peptide vasoconstrictor. The final step in the processing of endothelin has been postulated to be the cleavage of the Trp21-Val22 peptide bond in proendothelin by a putative endothelin-converting enzyme. A soluble extract of primary porcine aortic endothelial cells was found to contain an enzyme activity that converted proendothelin-1 (proET-1) to an endothelin-1 (ET-1)-like peptide as determined by the rabbit aortic ring contraction assay. This enzyme was partially purified by DE52 ion-exchange chromatography. Incubation of proET-1 with the partially purified enzyme generated a product which had a retention time on HPLC identical to that of authentic ET-1. Further analysis of the product showed that it caused contraction of rabbit aortic rings, had a molecular weight identical to ET-1 as measured by fast atom bombardment mass spectrometry, and competed for [125I]ET-1 binding in an RIA using specific antibodies which recognize the carboxy terminal tryptophan of ET-1. The enzyme activity could be inhibited by thiol protease inhibitors such as Z-phe-pheCHN2 and p-hydroxymercuribenzoate, but not by serine- or metalloprotease inhibitors. The optimal pH for the enzymatic activity was between 7.0 and 7.5, and no activity was detected at pH 4.0. These results demonstrate that this thiol protease is a potential endothelin-converting enzyme.
Deng
Y
Y
Pharmaceuticals Division, CIBA-GEIGY Corp., Summit, NJ 07901.
Savage
P
P
Shetty
S S
SS
Martin
L L
LL
Jeng
A Y
AY
D000818:Animals; D001011:Aorta / Q000201:enzymology; D001013:Aorta, Thoracic / Q000187:drug effects; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D008666:Metalloendopeptidases; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013552:Swine; D014661:Vasoconstriction / Q000187:drug effects
D016232:Endothelins; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes