Disulfide Bonds in Big ET-1 Are Essential for the Specific Cleavage at the Trp21–Val22 Bond by Soluble Endothelin Converting Enzyme-1 from Baculovirus/Insect Cells
| Autor: | Kyunghye Ahn, Douglass C. Fahnoe, Sarah B. Herman, Gary D. Johnson |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
medicine.hormone
Glycosylation Endothelin converting enzyme 1 Stereochemistry Molecular Sequence Data Biophysics Peptide Endothelin-Converting Enzymes Spodoptera Bradykinin Cleavage (embryo) Biochemistry Endothelins medicine Animals Aspartic Acid Endopeptidases Humans Amino Acid Sequence Disulfides Enzyme Inhibitors Protein Precursors education Molecular Biology Peptide sequence Integral membrane protein Chromatography High Pressure Liquid chemistry.chemical_classification education.field_of_study Endothelin-1 Chemistry Glycopeptides Metalloendopeptidases Peptide Fragments Recombinant Proteins Protein tertiary structure Kinetics Spectrometry Fluorescence Quinazolines Metalloendopeptidase Baculoviridae |
| Zdroj: | Archives of Biochemistry and Biophysics. 373:385-393 |
| ISSN: | 0003-9861 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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