Isolation and characterization of thioredoxin and NADPH-dependent thioredoxin reductase from tomato (Solanum lycopersicum)
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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 ∼290nm, 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. [BMB reports 2011; 44(10): 692-697]
Abstract
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 ∼290nm, 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. [BMB reports 2011; 44(10): 692-697]
참고문헌 (17)
- Arner, E. S, "Physiological functions of thioredoxin and thioredoxin reductase", Eur. J. Biochem, 267, pp.6102~6109 (2000)
- Serrato, A. J, "Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat", Biochem. J, 367, pp.491~497 (2002)
- Obiero, J, "Thioredoxin system from Deinococcus radiodurans", J. Bacteriol, 192, pp.494~501 (2010)
- Bashandy, T, "Interplay between the NADP-linked thioredoxin and glutathione systems in Arabidopsis auxin signaling", Plant Cell, 22, pp.376~391 (2010)
- Jacquot, J. P, "Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli", J. Mol. Biol, 235, pp.1357~1363 (1994)
- Tarrago, L, "Regeneration mechanisms of Arabidopsis thaliana methionine sulfoxide reductases B by glutaredoxins and thioredoxins", J. Biol. Chem, 284, pp.18963~18971 (2009)
- Rouhier, N, "Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases", J. Biol. Chem, 282, pp.3367~3378 (2007)
- Lemaire, S. D, "Primary structure determinants of the pH- and temperature- dependent aggregation of thioredoxin", Biochim. Biophys. Acta, 1476, pp.311~323 (2000)
- Holmgren, A, "Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide", J. Biol. Chem, 254, pp.9627~9632 (1979)
- Juttner, J, "Cloning and expression of a distinct subclass of plant thioredoxins", Eur. J. Biochem, 267, pp.7109~7117 (2000)
- Luthman, M, "Rat liver thioredoxin and thioredoxin reductase: purification and characterization", Biochemistry, 21, pp.6628~6633 (1982)
- Vieira Dos Santos, C, "Plant thioredoxins are key actors in the oxidative stress response", Trends. Plant. Sci, 11, pp.329~334 (2006)
- Laloi, C, "The Arabidopsis cytosolic thioredoxin h5 gene induction by oxidative stress and its Wbox- mediated response to pathogen elicitor", Plant Physiol, 134, pp.1006~1016 (2004)
- Carmel-Harel, O, "Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress", Annu. Rev. Microbiol, 54, pp.439~461 (2000)
- Lin, G. -Z, "Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli", PROTEIN EXPRESSION AND PURIFICATION, 55(2), pp.287~292 (2007), ACADEMIC PRESS INC ELSEVIER SCIENCE
- Bradford, M. M, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding", Anal. Biochem, 72, pp.248~254 (1976)
- Changbo Dai, "Expression Pattern of a Peptide Methionine Sulfoxide Reductase Gene from Tomato (Solanum lycopersicum) in Response to Abiotic and Oxidative Stresses", Journal of the Korean Society for Applied Biological Chemistry, 53(2), pp.127~132 (2010), 한국응용생명화학회