Disulfide bonds in big ET-1 are essential for the specific cleavage at the Trp(21)-Val(22) bond by soluble endothelin converting enzyme-1 from baculovirus/insect cells.
Endothelin converting enzyme-1 (ECE-1) is a type II integral membrane protein and a zinc metalloendopeptidase. ECE-1 generates endothelin-1 (ET-1), the most potent vasoconstrictor yet discovered, by specific proteolytic processing of a precursor peptide, big ET-1. An insect cell expression system, which generates up to 4.3 mg of a secreted, soluble form of ECE-1 (solECE-1) per liter culture medium, has been established and solECE-1 was purified to homogeneity using five chromatographic steps. SolECE-1 expressed in insect cells could be suitable for X-ray structure determination as it is much less glycosylated than solECE-1 from mammalian cells. SolECE-1 from both sources, nonetheless, has comparable enzymatic properties. Despite apparent structural similarities, ECE-1 cleaves big ET-1 exclusively between Trp(21) and Val(22), in contrast to neprilysin, which cleaves big ET-1 at various sites. However, when linear big ET-1, in which the formation of disulfide bonds has been prevented by alkylation of the four cysteines, was used as substrate, it was cleaved by solECE-1 at multiple sites. This result indicates that secondary/tertiary structure of big ET-1 induced by disulfide bonds is essential for the specific cleavage of the Trp(21)-Val(22) bond by ECE-1. A continuous, fluorescent ECE-1 assay has been developed using a novel substrate, 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-(p-nitro-Phe(8))-Arg. This simple and rapid assay can greatly facilitate discovery of novel ECE inhibitors useful as pharmaceutical agents.
Fahnoe
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Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105, USA.
Johnson
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Herman
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Ahn
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K
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001920:Bradykinin / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006020:Glycopeptides / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry*; D011799:Quinazolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000235:genetics
D004220:Disulfides; D019332:Endothelin-1; D016232:Endothelins; D004791:Enzyme Inhibitors; D006020:Glycopeptides; C110777:PD 069185; D010446:Peptide Fragments; D011498:Protein Precursors; D011799:Quinazolines; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D001920:Bradykinin; C008890:phosphoramidon