Characterization of serine protease-derived metabolites of big endothelin in the cytosolic fraction from human polymorphonuclear leukocytes.
We have characterized by bioassay and reversed-phase high-performance liquid chromatography analysis (rp-HPLC) the conversion of human big endothelin-1 (bET-1) to endothelin-1 (ET-1) by the cytosolic fraction from human polymorphonuclear leukocytes (PMNs). Either the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the selective elastase inhibitor ONO-5046 (100 microM) blocked the formation of ET-1 from bET-1. Interestingly, human leukocyte elastase formed some of the same products from bET-1 as the PMN cytosol, but generated negligible amounts of ET-1. However, coincubation of the elastase-derived fragments of bET-1 with the PMN cytosol in the presence of ONO-5046 resulted in a 17-fold increase in the formation of ET-1, indicating that an elastase-derived intermediate of bET-1 was subsequently cleaved by a soluble protease(s) to form mature ET-1. We have identified by electrospray-mass spectrometry (ESMS) analysis this intermediate as bET-1(1-22). Analysis of bET-1 digestion by human leukocyte cathepsin G revealed the formation of a biologically active metabolite chromatographically distinct from ET-1, identified as bET-1(1-31) by ESMS. These findings indicate the presence of complex enzyme systems in human PMNs capable of activating bET-1.
D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D019332:Endothelin-1; D016232:Endothelins / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D049934:Isocoumarins; D019272:Leukocyte Elastase; D013058:Mass Spectrometry; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology
D003374:Coumarins; D019332:Endothelin-1; D016232:Endothelins; D049934:Isocoumarins; D011498:Protein Precursors; D013449:Sulfonamides; C046235:3,4-dichloroisocoumarin; C069195:sivelestat; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D005998:Glycine