title: Characterization of site-directed antisera against endothelin-converting enzymes.
abstract: Site-directed antisera have been developed against the two endothelin-converting enzyme-1 (ECE-1) isoforms cloned to date in humans, ECE-1 alpha and ECE-1 beta. Antisera were raised in rabbits against synthetic peptides corresponding to the deduced amino acid sequences that differ between ECE-1 alpha and ECE-1 beta. 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-1 alpha and ECE-1 beta. Standard displacement curves have been developed to determine the levels of immunoreactive ECE-1 alpha and ECE-1 beta in solubilized microsomal fractions of human tissue. In conclusion, we have described the first production and characterization of site-directed antisera raised against ECE-1 alpha and ECE-1 beta capable of discriminating between the two ECE-1 isoforms. Furthermore, using these antisera, we have found that ECE-1 alpha appears to be the predominant isoform in human tissue.
authors.affiliation: Clinical Pharmacology Unit, University of Cambridge, Addenbrooke's Hospital, England.
authors.forename: J W
authors.initials: JW
authors.lastname: Mockridge
authors.forename: R E
authors.initials: RE
authors.lastname: Kuc
authors.forename: N S
authors.initials: NS
authors.lastname: Huskisson
authors.forename: P J
authors.initials: PJ
authors.lastname: Barker
authors.forename: A P
authors.initials: AP
authors.lastname: Davenport
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology*; D000918:Antibody Specificity; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000276:immunology*; D003429:Cross Reactions; D000072396:Endothelin-Converting Enzymes; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000276:immunology*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits
chemical_list: D019138:Antibodies, Blocking; D007527:Isoenzymes; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes