New short peptide substrates of endothelin-converting enzyme and characterization of the enzyme

Endothelin (ET) is a 21 amino acid peptide produced following the cleavage of its precursor, big ET, by a metalloprotease, the endothelin-converting e nzyme (ECE). In the study reported here we determined the minimal peptide s equence of big ET necessary for enzyme recognition and cleavage at the P1-P 1' site. Furthermore, we have explored the role of the amino acids found at the boundaries of the cleavage site. To reach these goals, we synthesized a series of fragments, all containing the P1-P1' cleavage site, Trp21-Val22 . Following the incubation of peptide fragments with a partly purified bovi ne ECE preparation and after analyzing the cleavage pattern by high-perform ance liquid chromatography (HPLC), we were able to identify big ET18-23 ami de as the minimal peptide core recognized and cleaved by the enzyme. This h ydrolysis was inhibited by phosphoramidon but not by thiorphan, a character istic of the ECE metalloprotease. However, none of the shorter peptides was able to inhibit the cleavage of big ET-1 by ECE, suggesting that they are not recognized by the enzyme. Particularly, it appears that aspartic acid 1 8 is a key residue for the recognition phenomenon. The delineation of the m inimal structure will be a useful tool to further characterize ECE.