• BMB Reports
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• v.29 no.2
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• pp.116-121
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• 1996

In earlier studies, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C-3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3 (N to C-terminal) and C3-E. The hybrid thioredoxins were overexpressed in E. coli from the cloned chimeric thioredoxin genes by a T7 promoter/polymerase system. To investigate the structure-function relationship of thioredoxin, we purified the E-C3 hybrid thioredoxin through ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. Its purity was examined on SDS-polyacrylamide gel electrophoresis and the molecular weight of the purified E-C3 hybrid thioredoxin was estimated to be 12,000. On native polyacrylamide gels, the purified E-C3 hybrid thioredoxin shows a much lower mobility than E. coli thioredoxin. E-C3 hybrid thioredoxin exhibits a 40-fold lower catalytic efficiency with E. coli thioredoxin reductase than E. coli thioredoxin. It was shown to catalyze the reduction of insulin disulfide by dithiothreitol. The purified E-C3 hybrid thioredoxin was also characterized in other aspects.

• BMB Reports
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• v.28 no.1
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• pp.17-20
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• 1995

Thioredoxin reductase is a flavoprotein oxidoreductase catalyzing the reduction of a cystine disulfide in thioredoxin. Thioredoxin, in turn, can reduce disulfide bonds in other proteins and serves as a reducing agent in enzymatic reactions such as those of ribonucleotide reductase and methionine sulfoxide reductase. In this work thioredoxin reductase was found to directly reduce DTNB in the absence of thioredoxin. This new reactivity of E. coli thioredoxin reductase was produced by relatively high concentrations of univalent cations such as $Na^+$, $K^+$, $Li^+$, and ${NH_4}^+$, and it appeared with the oxidation of NADPH. These results indicate that E. coli thioredoxin reductase may be slightly modified by univalent cations, and the modified enzyme directly reacts with DTNB. This DTNB-reducing activity offers a new assay method for E. coli thioredoxin reductase.

• Tuberculosis and Respiratory Diseases
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• v.46 no.3
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• pp.327-337
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• 1999

Background: Reactive oxygen species are involved in multi-stage process of carcinogenesis. The moot of cancer cell lines and cancer cells in tumor tissue produce reactive oxygen species and on the other hand, the activities of catalase, Mn- and CuZn-superoxide dismutase in tumor cells are usually low. These persistent oxidative stress in tumor tissue facilitates tumor invasion and metastasis. 12-kDa thioredoxin, which regulates the intracellular redox potential with glutathione and glutaredoxin is involved in cell activation, proliferation, differentiation and redox-mediated apoptosis. It is also purified as 14-kDa and 10-kDa eooinophilic cytotoxic enhancing factor(ECEF) from human histiocytic cell(U937) and 10-kDa ECEF has more than 20 times eosinophilic stimulation activity than 14-kDa ECEF. It has been reported that adult T-cell leukemia, squamous cell carcinoma of uterine cervix, and hepatocellular carcinoma show increased amounts of human thioredoxin and thioredoxin mRNA is increased in lung cancer. In this study, we investigated the expression of conventional antioxidant enzymes such as catalase, CuZn-SOD, and glutathione peroxidase and thioredoxin in lung cancer tissue compared to adjacent normal lung tissue and the induction of thioredoxin in macrophage cells after treatment of oxidative stress and endotoxin Methods: We measured the amount of conventional antioxidant enzymes such as catalase, CuZn-SOD, and glutathione peroxidase and thioredoxin in lung cancer tissue compared to adjacent normal lung tissue by immunoblot analysis and the induction of thioredoxin in mouse monocyte-macrophage cells(RAW 264.7) by treatment of 5 ${\mu}M$ menadione and 1 ${\mu}g/ml$ endotoxin Results: On immunoblot analysis, the expression of 12-kDa thioredoxin was increased in lung cancer tissue compared to paired normal lung tissue. but the expression of catalase and CuZn-SOD were decreased in lung cancer tissue compared to paired normal tissue and the expression of glutathione peroxidase in lung cancer was variable. The expression of truncated thioredoxin was also increased in lung cancer. When mouse monocyte-macrophage cells were treated with 5 ${\mu}M$ menadione and 1 ${\mu}g/ml$ endotoxin, the expression of thioredoxin was peaked at 12 hrs and sustained to 48 hrs. Conclusion: In contrast with other conventional antioxidants, the expression of 12-kDa and truncated thioredoxin in lung cancer were increased and it is closely associated with persistent oxidative stress in tumor microenvironment. Considering especially the biological functions of truncated thioredoxin, the increased amount of truncated thioredoxin has significant role in tumor growth through cell proliferation.

• Microbiology and Biotechnology Letters
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• v.35 no.1
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• pp.36-40
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• 2007

Redox signaling is one of way to regulate growth and death of cell in response to change of redox of proteins. To search whether translation is regulated by redox, we attempted in vitro translation assay under condition with or without DTT. Interestingly in vitro translation activity was increased up to 40% In the presence of dithiothreitol (DTT). Then we checked whether this positive effect by DTT was further accelerated by addition of thioredoxin (Trx). When a Trx purified from Saccharomyces cerevisiae was added to the in vitro translation extract, we observed a dose-dependent increase in translational activity. These results suggest the possibility of translation factors being redox-regulated via Trx in vivo.

• BMB Reports
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• v.28 no.1
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• pp.51-56
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• 1995

Thioredoxin is a small redox protein with an active-site disulfide/dithiol, and is ubiquitous in bacteria, plants, and animals. To investigate the structure-function relationship of thioredoxin, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3(N to C-terminal) and C3-E. The hybrid thioredoxin genes were put under the T7 promoter and their productions were confirmed. The two hybrid thioredoxins complemented phenotypes of a thioredoxin-deficient E. coli strain. A strain containing the C3-E hybrid thioredoxin supported growth of the T7 phage, whereas a strain expressing the E-C3 hybrid thioredoxin did not. However, both hybrids supported growth of M13 phages. The two hybrid thioredoxins were also characterized in other aspects. Differences in activity between the hybrid thioredoxins were attributed to altered interactions of the N- and C-terminal domains of the molecule, which produced changes in the three-dimensional structure of the active site region.

• Journal of Microbiology
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• v.44 no.5
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• pp.556-561
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• 2006

A differential display for the expression profiles of wild-type Cryphonectria parasitica and its virally-infected isogenic hypovirulent strain revealed several transcripts of interest, which evidenced significant matches with fungal genes of known function. Among which, we have further analyzed an amplified PCR product with significant sequence similarity to the known fungal stress-responsive thioredoxin gene from Neurospora crassa. The product of the cloned thioredoxin gene, CpTrx1, consists of 117 amino acids, with a predicted molecular mass of 13.0 kDa and a pI of 5.4. Sequence comparisons demonstrated that the deduced protein sequence of the CpTrx1 gene evidenced a high degree of homology to all known thioredoxins, with the highest degree of homology with trx1, a thioredoxin gene from Saccharomyces cerevisiae, and evidenced a preservation of the conserved hall markresidues (Trp-Cys-Gly-Pro-Cys) at the active site of thioredoxin. The E. coli-generated CpTRX1 manifested thioredoxin activity, according to the insulin reduction assay, which indicates that the cloned gene does indeed encode for the C. parasitica thioredoxin.

• BMB Reports
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• v.34 no.2
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• pp.139-143
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• 2001

Thioredoxin (Trx) is a redox protein possessing conserved sequence Cys-Gly-Pro-Cys in ail organisms. Trx acts as an electron donor of many proteins including thioredoxin peroxidase (TPx). Yeast Trx 2 has two redox active cysteine residues at positions 31 and 34. To investigate the redox activity of each cysteine, we generated mutants C31S, C34S, and C31S/C34S using site directed mutagenesis and examined the redox activity of Trx variants as an electron donor for yeast TPx enzymes. None of the three Cysmutated Trx proteins was active as a redox protein in the 5', 5'-dithiobis-(2-dinitrobenzoic acid) reduction under the condition of the presence of NADPH and thioredoxin reductase, and in the thioredoxin dependent peroxidase activity of yeast TPx II. C34S enhanced the glutamine synthetase protection activity of yeast TPx I, even though 100 times more protein was needed to exhibit the same activity to WT. The formation of a mixed disulfide intermediate between Trx and TPx II subunits was analyzed by SDS-PAGE. The mixed dieter form of TPx II was found only for C34S. These results suggest that Cys-31 more effectively acts as an electron donor for TPx enzymes.

• BMB Reports
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• v.31 no.6
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• pp.572-577
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• 1998

A soluble protein from Saccharomyces cerevisiae provides protection against a thiol-containing oxidation system but not against an oxidation system without thiol. This 25-kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was thus named thioredoxin peroxidase. The protective role of thioredoxin peroxidase against ionizing radiation, which generates reactive oxygen species harmful tocellular function, was investigated in wild-type and mutant yeast strains in which the tsa gene encoding thioredoxin peroxidase was disrupted by homologous recombination. Upon exposure to ionizing radiation, there was a distinct difference between these two strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein. Activities of other antioxidant enzymes, such as catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase were increased at 200-600 Gy of irradiation in wild-type cells. However, the activities of antioxidant enzymes were not significantly changed by ionizing radiation in thioredoxin peroxidase-deficient mutant cells. These results suggest that thioredoxin peroxidase acts as an antioxidant enzyme in cellular defense against ionizing radiation through the removal of reactive oxygen species as well as in the protection of antioxidant enzymes.

• BMB Reports
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• v.44 no.10
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• pp.692-697
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• 2011

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.

• BMB Reports
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• v.30 no.5
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• pp.320-325
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• 1997

Thioredoxin is a multi-functional protein which is ubiquitous in microorganisms, animals and plants. Previously, expression of the E. coli thioredoxin gene was found to be negatively regulated by cAMP. In the present study, the effect of cAMP on two separate promoters of the E. coli thioredoxin gene was investigated. Cyclic AMP had a repressible effect on P1 and P1P2 promoter activity of the constructs. This effect was also observed in the cya strain. The P2 promoter construct gave very high -galactosidase activity, and its expression was not affected by exogenous cAMP. It was assumed that a cis-acting negative element, probably the cAMP-CRP binding site, might have been deleted in the P1 promoter construct. Repression of the thioredoxin gene expression by cAMP appeared to be independent of ppGpp.