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 enzyme (ECE). In the study reported here we determined the minimal peptide sequence of big ET necessary for enzyme recognition and cleavage at the P1-P1' 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 bovine ECE preparation and after analyzing the cleavage pattern by high-performance liquid chromatography (HPLC), we were able to identify big ET(18-23) amide as the minimal peptide core recognized and cleaved by the enzyme. This hydrolysis was inhibited by phosphoramidon but not by thiorphan, a characteristic 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 18 is a key residue for the recognition phenomenon. The delineation of the minimal structure will be a useful tool to further characterize ECE.
Létourneau
M
M
Centre of Research for Human Health, INRS/Armand-Frappier Institute, University of Québec, Pointe-Claire, Canada.
Roby
P
P
Tremblay
F
F
Carette
J
J
Fournier
A
A
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002417:Cattle; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000737:chemistry / Q000378:metabolism*; D008666:Metalloendopeptidases; D010446:Peptide Fragments / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D019332:Endothelin-1; D016232:Endothelins; D010446:Peptide Fragments; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes