Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Aorta
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Aspartic Acid Endopeptidases / isolation & purification*
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Aspartic Acid Endopeptidases / metabolism
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Cattle
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Cell Fractionation / methods
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Cell Line
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Cells, Cultured
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Centrifugation, Density Gradient / methods
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Chromatography, Gel / methods
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Chromatography, Ion Exchange / methods
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Cross Reactions
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Endothelin-1
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Endothelin-Converting Enzymes
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Endothelins / metabolism
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Endothelium, Vascular / enzymology*
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Glycopeptides / pharmacology
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Humans
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Hydrogen-Ion Concentration
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Kinetics
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Metalloendopeptidases
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Neprilysin / isolation & purification*
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Peptidyl-Dipeptidase A / isolation & purification*
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Peptidyl-Dipeptidase A / metabolism
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Protein Precursors / metabolism
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Radioimmunoassay
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Subcellular Fractions / enzymology
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Umbilical Veins
Substances
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Endothelin-1
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Endothelins
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Glycopeptides
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Protein Precursors
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Peptidyl-Dipeptidase A
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Aspartic Acid Endopeptidases
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Metalloendopeptidases
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Neprilysin
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ECE1 protein, human
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Endothelin-Converting Enzymes
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phosphoramidon