PURIFICATION OF A NOVEL ENDOTHELIN-CONVERTING ENZYME SPECIFIC FOR BIGENDOTHELIN-3

Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes, The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big E T-1, with a K-m value of 0.14 mu M for big ET-3. The conversion to ET- 3 nas confirmed with sandwich EEA by monoclonal antibodies, the elutio n profile of HPLC, and intracellular calcium mobilization in CHO-K1 ce lls expressing recombinant human ETB receptors, The conversion activit y was inhibited by an inhibitor of neutral endopeptidase 23.11 (NEP) p hosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3. (C) 1998 Federation o f European Biochemical Societies.