A simple method for isolating human endothelin converting enzyme free from contamination by neutral endopeptidase 24.11.
Subcellular fractionation of the phosphoramidon sensitive membrane-bound endothelin converting enzyme (ECE-1) activity from homogenates of bovine aortic endothelial cells and the human endothelial cell line EA.hy 926, combined with studies of intact cells, shows ECE-1 to be localised primarily to the plasma membrane with the topology of an ectoenzyme. To overcome the problem of neutral endopeptidase 24.11 contaminating the human ECE-1 activity solubilised from the plasma membrane fractions of EA.hy 926, we have used isoelectric focusing to simultaneously solubilise and separate these activities. The metallopeptidase ECE-1 obtained displayed a neutral pH optimum, a molecular weight of 250 kDa on gel filtration chromatography and was inhibited by phosphoramidon with an IC50 of 0.8 microM.
William Harvey Research Institute, St. Bartholomew's Hospital Medical College, London, UK.
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Corder
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Khan
V J
VJ
Harrison
D000818:Animals; D001011:Aorta; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003429:Cross Reactions; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases; D015260:Neprilysin / Q000302:isolation & purification*; D007703:Peptidyl-Dipeptidase A / Q000302:isolation & purification* / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011863:Radioimmunoassay; D013347:Subcellular Fractions / Q000201:enzymology; D014471:Umbilical Veins
D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D011498:Protein Precursors; D007703:Peptidyl-Dipeptidase A; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D015260:Neprilysin; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon