• Proceedings of the Zoological Society Korea Conference
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• 1998.10b
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• pp.291.1-291
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• 1998

No Abstract, See Full Text

• Molecules and Cells
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• v.24 no.3
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• pp.316-322
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• 2007

Glutaredoxins (Grxs), also known as thioltransferases (TTases), are thiol oxidoreductases that regulate cellular redox state in a variety of organisms. In the budding yeast Saccharomyces cerevisiae, Grx1 and 2 are cytosolic dithiol Grxs, while Grx3, 4 and 5 are monothiol Grxs. A gene encoding a new monothiol Grx, Grx6, was cloned from the genomic DNA of S. cerevisiae by PCR. Its DNA sequence contains 1,080 bp, and encodes a putative protein of 203 amino acid residues containing Cys-Phe-Tyr-Ser at the active site. Grx6 is similar to other monothiol Grxs in the same organism and to Grx3 in the fission yeast Schizosaccharomyces pombe. and its predicted three-dimensional structure resembles that of S. pombe Grx3. S. pombe cells harboring plasmid pFGRX6 containing the Grx6 gene had about 1.3-fold elevated Grx activity in the exponential phase, and grew better than the control cells under some stressful conditions. Synthesis of ${\beta}$-galactosidase from a Grx6-lacZ fusion gene in S. pombe was enhanced by potassium chloride, aluminum chloride and heat ($37^{\circ}C$) treatment. S. pombe cells harboring plasmid pFGRX6 had elevated ROS levels whereas S. pombe cells harboring extra copies of Grx3 had reduced ROS levels.

• Molecules and Cells
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• v.20 no.1
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• pp.74-82
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• 2005

Glutaredoxins (Grxs) are thioloxidoreductases which are required for maintaining thiol/disulfide equilibrium in living cells. The Grx3 gene, which encodes one of the three monothiol Grxs in the fission yeast Schizosaccharomyces pombe, was characterized, and its transcriptional regulation studied. Genomic DNA encoding Grx3 was isolated by PCR, and a plasmid pTT3 carrying this DNA was produced. The DNA sequence has 1,267 bp, which would encode a monothiol Grx of 166 amino acids with a molecular mass of 18.3 kDa. The putative protein has 27% homology with Grx5, and contains many hydrophobic amino acid residues in its N-terminal region. S. pombe cells harboring pTT3 had increased Grx activity and enhanced survival on minimal medium plates containing aluminum (5 mM), BSO (0.05 mM), menadione (0.01 mM) or cadmium (0.2 mM). The 568 bp upstream region of Grx3 was fused into the promoterless b-galactosidase gene of the shuttle vector YEp367R to generate fusion plasmid pMJS10. Potassium chloride (KCl) and metals including aluminum and cadmium enhanced the synthesis of ${\beta}$-galactosidase from the fusion gene. The synthesis of ${\beta}$-galactosidase was also enhanced, in a Pap1-dependent manner, by fermentable carbon sources such as glucose (at low concentrations) and sucrose, but not by non-fermentable carbon sources such as ethanol and acetate. Grx3 mRNA increased in response to treatment with BSO. These observations indicate that S. pombe Grx3 is involved in the response to stress, and is regulated by stress.

• BMB Reports
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• v.32 no.2
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• pp.133-139
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• 1999

Thioltransferase, also known as glutaredoxin, was previously purified and characterized from Chinese cabbage (Brassica campestris ssp. napus var. pekinensis). However, in the process of gel filtration on Sephadex G-75, there were two activity peaks. In this study, a second thioltransferase (TTase CC-2) in the minor peak of the Sephadex G-75 elution profile was further purified using affinity chromatography on an S-hexylglutathione-agarose column by eluting with buffer solution containing 2.5 mM S-hexylglutathione. It showed a single band on SDS-PAGE indicating that TTase CC-2 is electrophoretically homogeneous. The molecular weight of TTase CC-2 was estimated to be about 22,000 daltons, and its isoelectric point was determined to be 6.73. Its size appears to be atypical and much larger than that of the first thioltransferase (TTase CC-1) from Chinese cabbage, and it can utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates. S-sulfocysteine was found to be a superior substrate for TTase CC-2. TTase CC-2 also displayed the reducing activity for non-disulfides such as dehydroascorbic acid. Its optimum pH was 8.5, which was consistent with that of TTase CC-1. TTase CC-2 activity was greatly activated by L-cysteine and reduced glutathione, and was found to be less heat-stable compared with TTase CC-1. Molecular and physiological differences between TTase CC-1 and TTase CC-2 remain to be elucidated. Chinese cabbage is the first plant which is known to contain two kinds of thioltransferases.

• BMB Reports
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• v.35 no.4
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• pp.409-413
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• 2002

Thioltransferase, also known as glutaredoxin, is an enzyme that catalyzes the reduction of a variety of disulfide compounds. In Schizosaccharomyces pombe, two thioltransferases were reported and the cDNA of one of the thioltransferases (thioltransferase-1) was cloned. Using a Northern blot assay, we investigated the thioltransferase transcription in response to various stress conditions. When the culture was shifted to a high temperature, the thioltransferases transcription was not significantly changed compared to the unshifted $30^{\circ}C$ culture. Treatment of zinc chloride to exponentially-growing cells remarkably increased the thioltransferase transcription, whereas the treatment of mercury chloride greatly reduced the transcription. Treatment of hydrogen peroxide and cadmium chloride caused no significant effects on the transcription of the thioltransferase. These results suggest that the transcription of thioltransferase-1 in S. pombe is induced in response to metal stress that is caused by zinc chloride, but not in response to heat stress or oxidative stress that is caused by hydrogen peroxide.

• Horticultural Science & Technology
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• v.30 no.6
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• pp.734-742
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• 2012

To compare RNA interference-mediated gene silencing technique and T-DNA integration for gene function analysis in Chinese cabbage, BrSAMS-knockout (KO) line and BrSAMS-knockdown (KD) line were used. The KO line had lost the function of a Brassica rapa S-adenosylmethionine synthetase (BrSAMS) gene by T-DNA insertion and the KD line had shown down-regulated BrSAMS genes' expression by dsRNA cleavage. From microarray results of the KO and KD lines, genes linked to SAMS such as sterol, sucrose, homogalacturonan biosynthesis and glutaredoxin-related protein, serine/threonine protein kinase, and gibberellin-responsive protein showed distinct differences in their expression levels. Even though one BrSAMS gene in the KO line was broken by T-DNA insertion, gene expression pattern of that line did not show remarkable differences compared to wild type control. However, the KD line obtained by RNAi technique showed prominent difference in its gene expression. Besides, change of polyamine and ethylene synthesis genes directly associated with BrSAMS was displayed much more in the KD line. In the microarray analysis of the KO line, BrSAMS function could not be clearly defined because of BrSAMS redundancy due to the genome triplication events in Brassicaceae. In conclusion, we supposed that gene knock-down method by RNAi silencing is more effective than knock-out method by T-DNA insertion for gene function analysis of polyploidy crops such as Chinese cabbage.

• BMB Reports
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• v.33 no.2
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• pp.179-183
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• 2000

The glutathione S-transferase (GST) activities of S-type and L-type thioltransferases (TTases), which are purified from the seeds and leaves of Arabidopsis thaliana, respectively, were identified and compared. The S-type and L-type TTases showed $K_m$ values of 9.72 mM and 3.18mM on 1-chloro-2,4-dinitrobenzene (CDNB), respectively, indicating the L-type TTase has higher affinity for CDNB. The GST activity of the L-type TTase was rapidly inactivated after being heated at $70^{\circ}C$ or higher. The GST activity of the S-type TTase remains active in a range of $30-90^{\circ}C$. $Hg^{2+}$ inhibited the GST activity of the S-type TTase, whereas $Ca^{2+}$ and $Cd^{2+}$ inhibited the GST activity of the L-type TTase. Our results suggest that the GST activities of two TTases of Arabidopsis thaliana may have different catalytic mechanisms. The importance of the co-existence of TTAse and GST activities in one protein remains to be elucidated.

• BMB Reports
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• v.34 no.3
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• pp.278-283
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• 2001

Antioxidant and redox enzyme activities are known to be involved in the cellular responses to various stresses. Their variations were observed according to the growth cycle of the fission yeast Schizosaccharomyces pombe. Peroxidase activity appeared to be notably higher in the early exponential phase than in the mid-exponential and stationary phases. However, catalase activity showed a variation pattern resembling the growth curve. Glutathione S-transferase activity was higher in the early exponential and late stationary phases. Activities of the two redox enzymes, thioredoxin and thioltransferase (glutaredoxin), were high in the stationary phase. However, their activities appeared to increase from the early exponential to mid-exponential phase. Total glutathione content had a varying pattern similar to that of thioredoxin and thioltransferase. However, its content in the early exponential phase was high. These results propose that antioxidant and redox enzymes tested are also involved in the mechanism of cell growth.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1364-1367
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• 2008

Oxidative stress damages all cellular constituents, and therefore, cell has to possess various defense mechanisms to cope. Saccharomyces cerevisiae, widely used as a model organism for studying cellular responses to oxidative stress, contains three glutathione peroxidase (Gpx) proteins. Among them, Gpx3 plays a major defense role against oxidative stress in S. cerevisiae. In this study, in order to identity the new interaction proteins of Gpx3, we carried out two-dimensional gel electrophoresis after immunoprecipitation (IP-2DE), and MALDI-TOF mass spectrometry. The results showed that several proteins including protein disulfide isomerase, glutaredoxin 2, and SSY protein 3 specifically interact with Gpx3. These findings led us to suggest the possibility that Gpx3, known as a redox sensor and ROS scavenger, has another functional role by interacting with several proteins with various cellular functions.

• BMB Reports
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• v.31 no.1
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• pp.20-24
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• 1998

Thioltransferase, also known as glutaredoxin, is an enzyme that catalyzes the reduction of a variety of disulfides, including protein disulfides, in the presence of reduced glutathione. Thioltransferase was purified from kale through ammonium sulfate fractionation, DE-52 ion-exchange chromatography, Sephadex G-75 gel filtration, and Q-Sepharose ion-exchange chromatography. Its molecular size was estimated to be about 31,000 daltons on SDS-PAGE. The purified enzyme has an optimum pH of about 8.0 with 2-hydroxyethyl disulfide as a substrate. The enzyme also utilizes L-sulfocysteine, L-cystine, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme has $K_m$ values of 0.24-0.67 mM for these substrates. The enzyme was partly inactivated after heating at $80^{\circ}C$ or higher temperature for 30 min. The enzyme was stimulated by various thiol compounds such as reduced glutathione, dithiothreitol, L-cysteine, and $\beta$-mercaptoethanol. This is a second example of a plant thioltransferase which was purified and characterized.