Mode of cleavage of pig big endothelin-1 by chymotrypsin. Production and degradation of mature endothelin-1.
Pig endothelin-1 [ET-1-(1-21)] seems to be produced via proteolytic processing between Trp-21 and Val-22 of an intermediate form consisting of 39 amino acid residues, termed big ET-1-(1-39), by a chymotrypsin-like proteinase. We examined the chymotryptic-cleavage sites of big ET-1-(1-39) by reverse-phase h.p.l.c. and sequence analysis, and found that chymotrypsin cleaved initially the Tyr-31-Gly-32 bond of big ET-1-(1-39), followed by cleavage between Trp-21 and Val-22. Furthermore, chymotrypsin hydrolysed the generated ET-1-(1-21), producing a single major product that had the same amino acid sequence as ET-1-(1-21) with a cleavage between Tyr-13 and Phe-14. The disulphide bridge between Cys-1 and Cys-15 remained intact. These results indicate that the conversion of big ET-1-(1-39) into ET-1-(1-21) catalysed by chymotrypsin requires hydrolysis of the Tyr-31-Gly-32 bond before that of the Trp-21-Val-22 bond, an event followed by cleavage between Tyr-13 and Phe-14 within the loop of ET-1-(1-21). Thus a chymotrypsin-like proteinase might be involved not only in the production but also in the degradation of ET-1-(1-21) in vivo.
Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Japan.
M
M
Takaoka
Y
Y
Miyata
Y
Y
Takenobu
R
R
Ikegawa
Y
Y
Matsumura
S
S
Morimoto
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D016232:Endothelins; D004730:Endothelium, Vascular; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D013552:Swine
D016232:Endothelins; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; D002918:Chymotrypsin