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Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system

Title
Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system
Authors
Kim J.-A.Park S.Kim K.Rhee S.G.Kang S.W.
Ewha Authors
이서구강상원
SCOPUS Author ID
이서구scopusscopus; 강상원scopus
Issue Date
2005
Journal Title
Analytical Biochemistry
ISSN
0003-2697JCR Link
Citation
Analytical Biochemistry vol. 338, no. 2, pp. 216 - 236
Indexed
SCI; SCIE; SCOPUS WOS scopus
Document Type
Article
Abstract
2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H 2O2-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 μM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx. © 2004 Elsevier Inc. All rights reserved.
DOI
10.1016/j.ab.2004.12.008
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일반대학원 > 생명·약학부 > Journal papers
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