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A rapid and versatile method for screening endothelin converting enzyme activity.

D K Little·D M Floyd·A A Tymiak
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Abstract

A flexible two-step protocol has been developed for high-throughput assays of endothelin converting enzyme (ECE) activity. The method is illustrated using endothelial cell monolayers and crude cell extracts as enzyme sources for conversion of exogenous substrates (big endothelin isopeptides) to their respective vasoactive products (endothelin isopeptides). Cleavage conditions have been optimized to minimize substrate utilization (reducing cost) while maintaining sensitivity and selectivity for mature endothelin detection in receptor- and antibody-based assays. Endothelins are quantified in a separate step and comparable estimates of mature endothelin formation are obtained using both EIA and radioreceptor assays in 96-well formats. Human umbilical vein (HUVEC) and human aorta (HAEC) endothelial cells characterized by this method are found to preferentially convert the big endothelin-1 (big ET-1) isopeptide through a membrane-bound, thiorphan-insensitive, and phosphoramidon-sensitive zinc metalloendopeptidase. Both intact cells and membrane preparations used as the enzyme source predict similar IC50 values for phosphoramidon inhibition of ECE (ca 1 microM). The procedure described is simple, rapid, and suitable for high-volume screening.

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AnimalsAspartic Acid EndopeptidasesCells, CulturedEndothelin-Converting EnzymesEndothelium, VascularHumansMetalloendopeptidasesNeprilysinRatsJournal Article

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D K Little, D M Floyd, A A Tymiak. 1994. A rapid and versatile method for screening endothelin converting enzyme activity.. https://doi.org/10.1016/1056-8719(94)90004-3

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