Characterization of site-directed antisera against endothelin-converting enzymes

Site-directed antisera have been developed against the two endothelin-converting enzyme-1 (ECE-1) isoforms cloned to date in humans, ECE-1alpha and ECE-1beta. Antisera were raised in rabbits against synthetic peptides corresponding to the deduced amino acid sequences that differ between ECE-1alpha and ECE-1beta. Antisera were highly selective for their corresponding antigen (titer 1 x 10(4)) and did not detect ET-1 or big ET-1. Furthermore, no detectable crossreactivity was observed between the different site-specific antisera and the other immunizing peptides, suggesting that the antisera would be selective for ECE-1alpha and ECE-1beta. Standard displacement curves have been developed to determine the levels of immunoreactive ECE-1alpha and ECE-1beta in solubilized microsomal fractions of human tissue. In conclusion, we have described the first production and characterization of site-directed antisera raised against ECE-1alpha and ECE-1beta capable of discriminating between the two ECE-1 isoforms. Furthermore, using these antisera, we have found that ECE-1 a appears to be the predominant isoform in human tissue.