A comparison of endothelin-converting enzyme activity from primary porcine aortic endothelial cells with activities in cultured human cell lines.
We examined four commercially available human cell lines for endothelin-converting-enzyme-(ECE) like activity and compared the results with primary porcine aortic endothelial cell enzymes. The cells that were investigated were 293 (transformed primary human embryonal kidney), Hep G2 (human hepatocellular carcinoma), HUVECs (human umbilical vein endothelial cells), and U937 (human histiocytic lymphoma). The relative ECE-like activities were determined in cytosolic and particulate extracts of each cell type. Enzyme activity against pro-ET-1 was measured at pH 4 and 7, using a C-terminal Trp-specific antibody to ET-1 radioimmunoassay and by high-performance liquid chromatography analysis of the enzyme hydrolysis products of pro-ET-1. Inhibition by EDTA at pH 7 or pepstatin at pH 4 was used to classify the pro-ET-1 processing enzymes from the human cell lines as either metallo- or aspartylproteinases. The particulate extract of the primary porcine aortic endothelial cells contained both aspartyl and metallo ECE-like enzymes. No ECE-like activity was present in either the cytosolic or particulate extracts of the U937 nor 293 cells. Neither the particulate extract of the Hep G2 cells nor the cytosolic extract of the HUVECs had any ECE-like activity. The cytosolic extract of the Hep G2 cells and the particulate extract of the HUVECs had an ECE-like activity at pH 7 that was inhibited by 10 mM EDTA, qualifying these enzymes as members of the metalloproteinase family.
Knap
A K
AK
Research Department, Ciba-Geigy Corp., Summit, New Jersey 07901.
Wigg
A M
AM
D000818:Animals; D001011:Aorta / Q000201:enzymology; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid / Q000494:pharmacology; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000302:isolation & purification; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000378:metabolism; D010436:Pepstatins / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification; D011498:Protein Precursors / Q000302:isolation & purification; D011863:Radioimmunoassay; D013552:Swine; D014407:Tumor Cells, Cultured
D019332:Endothelin-1; D016232:Endothelins; D010436:Pepstatins; D010446:Peptide Fragments; D011498:Protein Precursors; C093240:proendothelin 1; C014195:Streptomyces pepsin inhibitor; D004492:Edetic Acid; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C031375:pepstatin