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.
Corder
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William Harvey Research Institute, St. Bartholomew's Hospital Medical College, London, UK.
Khan
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N
Harrison
V J
VJ
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