• BMB Reports
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• 제29권2호
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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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• 제28권1호
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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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• 제46권3호
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• pp.327-337
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• 1999

연구배경: 활성산소종(reactive oxygen species)은 발암 기전의 여러 단계 과정에 관여한다. 대부분의 종양 세포주 및 종양 조직내의 종양 세포는 활성산소종을 생성하는 반면 종양 세포의 catalase, Mn- 및 CuZn-SOD등 기존 항산화 단백의 활성도는 대부분 저하되어 있다. 이로 인한 종양 조직내의 지속적인 산화 스트레스는 종양의 국소 침습 및 전이를 촉진한다. 12-kDa thioredoxin은 glutathione 및 glutaredoxin과 함께 세포내 산화-환원 전위를 조절하여 세포 활성, 증식, 분화 및 산화-환원에 의한 아포토시스 조절에 관여하는 것으로 알려져 있다. 한편 histiocytic lymphoma 세포 (U937, human)에서 14-kDa 및 10-kDa의 eosinophilic cytotoxic enhancing factor(ECEF)로 정제되었으며 호산구 자극의 생물학적 기능은 10-kDa에서 20배 이상 높은 것으로 알려져 있다. 성인 T-세포백혈병, 자궁경부상피세포암 및 간세포암에서 thioredoxin 양이 증가 되어 있고 폐암에서는 thioredoxin mRNA가 증가되어 있는 것으로 알려져 있다. 이에 폐암 조직과 주위 정상 조직을 비교하여 catalase, CuZn-SOD 및 glutathione peroxidase 등 기존 항산화 단백과 thioredoxin 발현 변화를 비교 관찰하고 대식세포에서 산화 스트레스 및 내독소에 의한 thioredoxin 발현 변화를 관찰하고자 하였다. 방 법: 동일한 환자의 폐암 조직과 주변의 정상 폐 조직을 immunoblot 분석으로 catalase, CuZn-SOD, glutathione peroxidase 및 thioredoxin 발현을 비교 관찰하였으며 대식세포인 mouse monocyte-macrophage 세포 (RAW 264.7)에 5 ${\mu}M$ menadione 및 1 ${\mu}g/ml$ endotoxin을 처치하여 thioredoxin 발현을 관찰하였다. 결 과: Immunoblot 분석상 12-kDa의 thioredoxin 발현은 폐암 조직에서 정상 폐조직과 비교하여 의미있는 증가를 보였으나 catalase 및 CuZn-SOD의 발현은 폐암 조직에서 정상 폐조직과 비교하여 감소하였고 glutathione peroxidase의 발현은 일정 하지 않은 변화를 보였다. 절단형(truncated) thioredoxin 역시 폐암에서 증가하였다. Mouse monocyte-macrophage cells에 5 ${\mu}M$ menadione 및 1 ${\mu}g/ml$ endotoxin을 처치하였을때 thioredoxin 발현은 12시간에 최고로 증가하여 48 시간까지 지속되었다. 결 론: 폐암에서 기존의 항산화 단백과는 달리 12-kDa 및 절단형 thioredoxin 발현이 증가하며 이는 종양 조직내의 지속적인 산화 스트레스와 밀접한 연관이 있다. 특히 절단형 thioredoxin의 생물학적 기능을 고려할 때 절단형 thioredoxin 발현 증가는 종양 세포 증식을 통한 종양 성장에 더욱 의미있는 역할하리라고 생각된다.

• 한국미생물·생명공학회지
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• 제35권1호
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• pp.36-40
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• 2007

Redox signaling은 단백질을 산화환원 시키는 세포의 중요 신호가 전달되어, 그 단백질의 기능이 변화함으로써 세포의 성장 및 사멸을 조절하게 되는 과정이다. 단백질 합성 구성원의 산화, 환원 과정에 의한 단백질 합성 조절을 알아보기 위해 환원제인 DTT 존재 하에 단백질 합성 활성을 관찰한 결과 DTT가 존재하지 않는 것에 비해 단백질합성이 1.4배 정도 증가됨이 관찰되어 redox potential을 보이는 것으로 보아 환원제가 단백질 합성을 좀 더 증진시키는 것으로 사료된다. DTT에 의한 이러한 현상은 산화환원 조절 단백질인 thioredoxin를 첨가한다면 thiol기에 환원력이 전달되어 단백질합성이 더욱 촉진되기 때문에 효모에서 thioredoxin유전자를 cloning하고 이로부터 효모에서 GST-thioredoxin을 분리하였다. DTT 존재 하에 산화환원 조절 단백질인 thioredoxin을 농도별로 첨가하였을 때의 단백질 합성이 어떻게 조절되는지 알아보았다. 반응 액에 DTT를 넣은 것과 넣지 않은 것을 사용하여 thioredoxin을 0ng, 18ng, 90ng, 460ng, 2,300 ng의 농도로 각각 넣어서 반응시켜 보았다. 이렇게 반응시킨 반응물에서 만들어진 단백질 활성을 측정하였는데 thioredoxin의 농도가 높아질수록 그 활성이 높게 나타났으며, thioredoxin을 넣은 것이 넣지 않은 것에 비해 활성이 약 4배 이상 높게 나왔다 이 결과는 산화환원 조절 단백질인 thioredoxin이 환원력을 단백질합성구성원에 효율적으로 전달하는데 관여함을 보여주는 것이며, 산화환원이 단백질 합성 시 중요한 신호전달 과정임을 암시한다.

• BMB Reports
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• 제28권1호
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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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• 제44권5호
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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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• 제34권2호
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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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• 제31권6호
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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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• 제44권10호
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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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• 제30권5호
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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.