A fluorogenic assay for endothelin-converting enzyme.
The potent vasoconstrictor peptide endothelin-1 is proposed to arise via proteolysis of a precursor molecule, "big endothelin," at a unique cleavage site. To aid in the identification of a putative endothelin-converting enzyme, we have developed an assay that mimics the relevant cleavage reaction. The assay takes advantage of the intramolecular fluorescence energy transfer between the scissile-site tryptophan and a dansyl moiety present in the same synthetic substrate. Cleavage of the peptide chain separates the fluorophore and quencher, resulting in an increase in fluorescence. The assay has been validated using chymotrypsin as a model protease and has been employed in the identification of novel endothelin-converting enzyme activities.
von Geldern
T W
TW
Cardiovascular Research Division, Abbott Laboratories, Abbott Park, IL 60064.
Holleman
W H
WH
Opgenorth
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000032:analysis*; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D008168:Lung / Q000201:enzymology; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D019332:Endothelin-1; D016232:Endothelins; D005456:Fluorescent Dyes; D010455:Peptides; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes