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KMID : 0624620110440100692
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BMB Reports
2011 Volume.44 No. 10 p.692 ~ p.697
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Isolation and characterization of thioredoxin and NADPH-dependent thioredoxin reductase from tomato (Solanum lycopersicum)
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Dai Changbo
Wang Myeong-Hyeon
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Abstract
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To investigate the pathways of oxidoreductases in plants, 2 key components in thioredox systems i.e. thioredoxin h (Trx h) and NADPH-dependent thioredoxin reductase (NTR) genes were first isolated from tomatoes (Solanum lycopersicum). Subsequently, the coding sequences of Trx h and NTR were inserted into pET expression vectors, and overexpressed in Escherichia coli. In the UV-Visible spectra of the purified proteins, tomato Trx h was shown to have a characteristic 'shoulder' at -290 nm, while the NTR protein had the 3 typical peaks unique to flavoenzymes. The activities of both proteins were demonstrated by following insulin reduction, as well as DTNB reduction. Moreover, both NADPH and NADH could serve as substrates in the NTR reduction system, but the catalytic efficiency of NTR with NADPH was 2500-fold higher than with NADH. Additionally, our results reveal that the tomato Trx system might be involved in oxidative stress, but not in cold damage.
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KEYWORD
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Kinetic parameters, Oxidative stress, Thioredoxin, Thioredoxin reductase, Tomato
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