Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1.
Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound zinc-metallopeptidase that is related to neprilysin in amino acid sequence. A major in vivo function of ECE-1 is the proteolytic conversion of big endothelin-1 to endothelin-1, one of the most potent vasconstricting peptides known. Although ECE-1 was once thought to be specific for the processing of endothelin precursors, it is now known that the enzyme hydrolyzes a number of peptide hormones. We have incorporated knowledge gained from recent studies of ECE-1 substrate specificity to aid the design of internally-quenched fluorescent substrates derived from bradykinin. The best of these substrates, (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(2, 4-dinitrophenyl), is hydrolyzed by ECE-1 with a k(cat)/K(m) value of 1.9 x 10(7) M(-1) s(-1), making it the most sensitive substrate yet described for ECE-1. The substrate is suitable for the rapid, continuous assay of the enzyme using a microplate format in a fluorescence plate reader, thereby simplifying both the purification of ECE-1 and the characterization of its inhibitors. It is demonstrated that (7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(2, 4-dinitrophenyl) is also a substrate for neprilysin, but is hydrolyzed 10-fold more efficiently by ECE-1, making this substrate selective for ECE-1. Furthermore, this synthetic peptide is a poor substrate for the matrix metalloproteinases.
Johnson
G D
GD
Department of Biochemistry, Ann Arbor, Michigan, 48105, USA.
Ahn
K
K
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D001920:Bradykinin / Q000031:analogs & derivatives; D016466:CHO Cells; D002624:Chemistry, Clinical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D000072396:Endothelin-Converting Enzymes; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008666:Metalloendopeptidases; D015260:Neprilysin / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014162:Transfection
D005456:Fluorescent Dyes; D010455:Peptides; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D015260:Neprilysin; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D001920:Bradykinin