Preparation and Characterization of Latent α1-Antitrypsin
Autor: | David A. Lomas, Robin W. Carrell, Mark R. Wardell, Peter R. Elliott, Wun-Shaing W. Chang |
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Rok vydání: | 1995 |
Předmět: |
Guanidinium chloride
Protein Folding Ovalbumin Protein Conformation Stereochemistry Molecular Sequence Data Sodium Chloride Serpin Biochemistry Protein Structure Secondary chemistry.chemical_compound Protein structure Plasminogen Activator Inhibitor 1 Humans Urea Amino Acid Sequence Molecular Biology Polyacrylamide gel electrophoresis Reactive center Binding Sites biology Isoelectric focusing Cell Biology Molecular Weight chemistry alpha 1-Antitrypsin biology.protein Electrophoresis Polyacrylamide Gel Protein folding Isoelectric Focusing Oligopeptides |
Zdroj: | Journal of Biological Chemistry. 270:5282-5288 |
ISSN: | 0021-9258 |
Popis: | Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-pleated sheet and a mobile reactive center loop. The reactive center loop has been shown to adopt a range of conformations from the three turn alpha-helix of ovalbumin to the cleaved or latent inhibitor in which the reactive center loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation. We show here that the archetypal serpin, alpha 1-antitrypsin, can also be induced to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12 h. The resulting species elutes at a lower sodium chloride concentration on an anion-exchange column and has a more cathodal electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin. Latent antitrypsin is inactive as an inhibitor of bovine alpha-chymotrypsin, is stable to unfolding with 8 M urea, and is more resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet prevents the molecule accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from < 1% to approximately 35% by refolding from 6 M guanidinium chloride. |
Databáze: | OpenAIRE |
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