Big endothelin-1 structure important for specific processing by endothelin-converting enzyme of bovine endothelial cells.
Phosphoramidon-sensitive endothelin-converting enzyme of bovine endothelial cells showed substrate selectivity for big endothelin-1 (ET-1) when compared to big ET-1(1-38), big ET-2(1-37), big ET-2(1-38) and big ET-3(1-41). To investigate the big ET-1 structure important for specific conversion by the endothelin-converting enzyme, we synthesized a series of truncated analogues of big ET-1, measured the hydrolysis of their Trp21-Val22 bonds, and found that a 16-residue peptide, big ET-1(19-34), is the minimal peptide sequence. This suggests that an unusually long carboxy-terminal sequence is required for big ET-1 conversion. Alanine substitution for individual amino acids in the carboxy-terminal region of big ET-1(19-34) demonstrated that His27, Val29, Pro30, Tyr31, Gly32, Leu33 and Gly34 are more important than Asn23, Thr24, Pro25, Glu26 and Val28 for eliciting efficient hydrolysis of the Trp21-Val22 bond, even though the former residues are located at more distant positions from the cleavage sites than are the latter. These results, together with the fact that big ET-2 and big ET-3 show heterogeneity in the big ET-1 residues His27, Val28, Val29 and Gly34, suggest that the His27-Val-Val-Pro-Tyr-Gly-Leu-Gly34 sequence in the carboxy-terminal region of big ET-1 plays the most important role in selective conversion by endothelin converting enzyme.
Okada
K
K
New Drug Discovery Research Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Japan.
Arai
Y
Y
Hata
M
M
Matsuyama
K
K
Yano
M
M
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000737:chemistry / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D019332:Endothelin-1; D016232:Endothelins; D010446:Peptide Fragments; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes