Peptide-based assay for the identification of endothelin-converting enzyme inhibitors.
A simple, solid-phase assay usable for the detection of endothelin-converting enzyme activity and inhibitors has been developed. It uses a multimeric peptide immobilized on microtiter plates that is able to specifically recognize the Big Endothelin fragment 16-32 derivatized with biotin. This fragment is cleaved between residues 21-22 by alpha-chymotrypsin with almost the same proteolysis rate as Big Endothelin, and after enzyme treatment it does not bind to the multimeric peptide adsorbed on the microtiter plates. The amount of uncleaved peptide bound to the plate is detected by subsequent treatment with streptavidin conjugated to peroxidase, followed by a chromogenic reaction. Model inhibitor profiles generated for alpha-chymotrypsin, a protease known to convert Big Endothelin in endothelin, demonstrated the utility of this assay as a rapid high-throughput aid in the study of Big Endothelin enzymatic processing and possibly in the identification of putative enzyme inhibitors.
Fassina
G
G
Protein Engineering Unit, Tecnogen S.c.p.A., Milano, Italy.
Cassani
G
G
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors*; D001426:Bacterial Proteins; D001710:Biotin; D002918:Chymotrypsin / Q000378:metabolism; D003124:Colorimetry / Q000379:methods*; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides; D019809:Streptavidin
D001426:Bacterial Proteins; D016232:Endothelins; D010446:Peptide Fragments; D010455:Peptides; C081090:big-endothelin (16-32); C061533:endothelin (16-21); D001710:Biotin; D019809:Streptavidin; D002918:Chymotrypsin; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes