Palmitoylation of the three isoforms of human endothelin-converting enzyme-1

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endot helins. Here we have examined whether the three isoforms of human ECE-1 (EC E-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fat ty acid palmitate and have evaluated a potential functional role of this mo dification. To do this, wild-type and mutant enzymes were expressed and ana lysed by metabolic labelling with [H-3]palmitate, immunoprecipitation and S DS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydrox ylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys(46) in ECE-1a, Cys(58) in ECE-1b and Cys(42) in E CE-1c were identified as sites of palmitoylation and each of these cysteine s accounted for all the palmitoylation that occured in the corresponding is oform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations . Moreover, complete solubility of the three isoforms in Triton X-100 revea led that palmitoylation does not target ECE-1 to cholesterol and sphingolip id-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE -1b and ECE-1c were also not significantly affected by the absence of palmi toylation.