• 식품기술
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• 제17권4호
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• pp.98-106
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• 2004

Two mutant enzymes of $\gamma$-glutamylcysteine synthetase ($\gamma$-GCS) which catalyzed the synthesis of $\gamma$-glutamylcysteine from L-glutamic acid and L-cysteine in the presence of ATP, were prepared bypoint mutation of $\gamma$-GCS gene with site-directed mutagensis in E. coli. Conformational structuresand catalytic reaction kinetics of mutant enzymes were compared with wild type $\gamma$-GCS afterpurification. The S495F mutant enzyme (serine at 495 residue was substituted with phenylalanine),which had no catalytic activity for $\gamma$-glutamylcysteine synthesis, rarely folded even in neutral pH.However, the mutant A494V (alanine of 494 residue was replaced by valnine) which showed 50 %increase of activity, had a high folding structure. The folding structure of A494V also more stable athigh temperature and extreme pH compared to wild type and S495F. Reaction kinetics of wild typeand A494V were also investigated, Km value of A494V was smaller than that of wild type, while itshowed a little difference at Vmax values. This result evolved that alanine at 494 may be involved inbinding site of substrate rather than catalytic site. In addition, change of catalytic activity by onepoint mutation was highly correlated with the folding structure of enzyme.

• Journal of Microbiology
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• 제41권3호
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• pp.248-251
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• 2003

Glutathione (GSH) is an important factor in determining tolerance against oxidative stress in living organisms. It is synthesized in two sequential reactions catalyzed by ${\gamma}$-glutamylcysteine synthetase (GCS) and glutathione synthetase (GS) in the presence of ATP. In this work, the effects of three different oxidative stresses were examined on GSH content and GSH-related enzyme activities in the fission yeast Schizosaccharomyces pombe. GSH content in S. pombe was significantly enhanced by treatment with hydrogen peroxide, ${\beta}$-naphthoflavone (BNF) and tert-butylhydroquinone (BHQ). Simultaneously, they greatly induced GCS and GS activity. However, they did not have any effects on glutathione reductase activity. These results suggest that GCS and GS activities in S. pombe are up-regulated by oxidative stress.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.61-61
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• 2003

We have cloned the CGR1 gene encoding glutathione reductase (GR) which catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) from Candida albicans. The cgr1/cgr1 mutants were not viable when CaMAL2 promoter repressed the CGR1 expression. The growth of the mutants could be partially overcome by thiol compounds such as GSH, dithiothreitol, cysteine, N-acetylcysteine and GSSG. Interestingly, C. albicans with CGR1 overexpressed showed defective hyphal growth on solid medium and attenuated virulence. We have also cloned the GCS1 gene encoding ${\gamma}$-glutamylcysteine synthetase which catalyzes the first step of glutathione biosynthesis. The gcs1/gcs1 mutants were nonviable in minimal defined medium. The growth of the mutants could be resumed by supplementing with GSH, GSSG and ${\gamma}$-glutamylcysteine in the medium. The mutants had increased intracellular D-erythroascorbic acid level up to 2.25-fold when transferred to GSH-free medium. When the mutants were depleted of GSH, they showed typical markers of apoptosis. In conclusion, these results suggest that glutathione is an essential metabolite, and involved in hyphal growth, virulence and apoptosis in C. albicans.

• Journal of Microbiology
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• 제42권3호
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• pp.233-238
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• 2004

Transcriptional regulation of the Schizosaccharomyces pombe y-glutamylcysteine synthetase (GCS) gene was examined using the two GCS-lacZ fusion plasmids pUGCS101 and pUGCS102, which harbor 607 bp and 447 bp upstream regions, respectively. The negatively-acting sequence was located in the -607 - -447 bp upstream region of the GCS gene. The upstream sequence responsible for induction by menadione(MD) and L-buthionine-(S, R)-sulfoximine (BSO) resides in the -607 - -447 bp region, whereas the sequence which codes for nitric oxide induction is located within the -447 bp region, measured from the translational initiation point. Carbon source-dependent regulation of the GCS gene appeared to be dependent on the nucleotide sequence within -447 bp region. The transcription factor Papl is involved in the induction of the GCS gene by MD and BSO, but not by nitric oxide. Induction of the GCS gene occurring due to low glucose concentration does not depend on the presence of Pap1. These data imply that induction by MD and BSO may be mediated by the Pap1 binding site, probably located in the -607 - -447 region, and also that the nitric oxide-mediated regulation of the S. pombe GCS gene may share a similar mechanism with its carbon-dependent induction.

• Toxicological Research
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• 제23권2호
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• pp.127-133
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• 2007

Metformin is a drug used to lower blood sugar levels in patients with type 2 diabetes via activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK). The primary objective of this study was to investigate whether metformin at the pharmacologically effective concentrations affects the expressions of ${\gamma}$-glutamylcysteine synthetase and phase II antioxidant genes in the H4IIE cell. Treatment of the cells with either metformin or 5-aminoimidazole-4-carboxamide riboside (AICAR) abrogated tert-butylhydroxyquinone (t-BHQ) induction of ${\gamma}$-glutamylcysteine synthetase, a rate limiting enzyme of GSH synthesis. The ability of t-BHQ to induce glutathione S-transferases (GSTs), a major class of phase II detoxifying enzymes that playa critical role in protecting cells from oxidative stress or electrophiles, was also inhibited by the agents. Transcriptional gene repression by metformin was verified by the GSTA2 promoter luciferase assay. Moreover, either metformin or AICAR treatment significantly decreased t-BHQ-dependent induction of other GSTs (i.e., $GST{\mu}$ and $GST{\pi}$ forms). Taken together, our data indicate that metformin treatment may result in the repression of ${\gamma}$-glutamylcysteine synthetase and glutathione S-transferase genes possibly via AMPK activation.

• 약학회지
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• 제53권6호
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• pp.314-320
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• 2009

We examined metabolic conversion of cysteine into glutathione (GSH) and taurine in rat liver under oxidative stress. Administration of tert-butylhydroperoxide (t-BHP) into the portal vein of male rats resulted in a rapid elevation of serum sorbitol dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities, which decreased gradually in 24 hr. Hepatic cysteine concentration was reduced in 3 hr, and recovered progressively, reaching a level greater than 200% of the normal value in 24 hr. GSH was increased both in liver and blood at 9 hr after t-BHP challenge, whereas hypotaurine or taurine was not altered. $\gamma$-Glutamylcysteine synthetase (GCS) activity was increased from 9 hr after t-BHP treatment, but protein expression of the GCS-heavy subunit was not changed in liver. Activity or expression of cysteine dioxygenase was not affected by t-BHP treatment. Taken together, these data show that an acute oxidant challenge to the rats may induce upregulation of cysteine availability and GCS activity, resulting in an enhancement of hepatic GSH synthesis, but the increased cysteine level does not stimulate taurine synthesis via cysteine sulfinate pathway. It is indicated that the regulation of GSH and taurine biosynthesis from cysteine is not solely dependent on the cysteine concentration in rat liver under oxidative stress.

• Journal of Yeungnam Medical Science
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• 제12권1호
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• pp.32-47
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• 1995

생쥐의 백혈병세포 L1210과 항암제에 대하여 내성이 유도된 L1210AdR, L1210VcR과 L1210Cis에서 glutathione의 농도와 glutathione의 합성 조절에 관여하는 ${\gamma}$-glutamylcysteine synthetase(GCS)와 ${\gamma}$-glutamyl transpeptidase (GGT), 세포 이물질을 축합하는데 촉매하는 glutathione S-transferase(GST)의 효소 활성도와 유전자의 발현 여부를 관찰하였다. 세포내 glutathione농도(${\mu}M/mg$ protein)는 L1210이 $0.41{\pm}0.003$, L1210AdR가 $0.73{\pm}0.006$, L1210VcR은 $1.16{\pm}0.060$, L1210Cis가 $2.19{\pm}0.282$으로 모세포에 비하여 내성세포에서 통계적으로 유의한 증가를 관찰하였다. Buthionine sulfoxamine(BSO)를 1 ${\mu}M$농도로 첨가하여 12시간 배양한 세포들에서의 glutathione농도는 L1210이 88%, L1210AdR가 85%, L1210VcR이 89%, 그리고 L1210Cis는 79%의 감소를 보였다. GCS의 활성도(nM/mg protein/min)는 L1210이 104인데 비하여 L1210AdR가 128, L1210VcR는 227, 및 L1210Cis는 212로 증가하였다. GGT의 활성도(nM/mg protein/min)는 L1210이 $2.15{\pm}0.531$이었고, L1210AdR은 $2.80{\pm}0.498$, L1210VcR은 $2.42{\pm}0.389$, 그리고 L1210Cis는 $2.98{\pm}0.623$으로 내성인 세포들에서 증가하였으며 L1210AdR과 L1210Cis에서 유의하였다. GST활성도(nM/mg protein/min)는 L1210이 $16.70{\pm}4.798$이었고, L1210AdR은 $14.51{\pm}3.402$, L1210VcR은 $19.52{\pm}4.255$, L1210Cis $17.77{\pm}4.495$로 L1210VcR과 L1210Cis가 약간의 증가를 보였으며, L1210AdR은 오히려 감소를 보였다. DNA의 slot blot에서 GCS, GGT, GST 유전자의 모세포와 내성세포간에 별다른 차이를 보이지 않았다. Northern hybridization에서 GCS는 약 4.5kb 크기의 band, GST-${\pi}$는 약 1.05kb 크기의 band를 보였으며 내성세포 모두에서 발현 증가가 관찰되었다. GGT의 경우 크기가 다른 6개의 band가 보였으며 특히 11.5 kb크기의 band에서 L1210AdR과 L1 210VcR의 발현이 증가하였으며, L1210VcR에서는 L1210과 다른 내성세포에서 보이는 1.95kb크기의 band가 보이지 않고 2.2kb 크기의 다른 band가 관찰되었다. 이상에서 L1210AdR과 L1210VcR의 내성에는 mdr1 유전자가 관여하고, L1210Cis의 내성에는 특히 glutathione이 중요하다. GCS, GGT 및 GST등의 활성도 및 유전자의 발현도 내성세포들에서 증가하였으며 이중 GCS는 내성세포내의 glutathione 합성에 가장 중요한 조절인자라 할 수 있다.

• BMB Reports
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• 제36권3호
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• pp.326-331
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• 2003

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSB) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, $\gamma$-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multi copy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride ($10\;{\mu}M$) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal8 amino acid-coding region were fused into the promoteriess $\beta$-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of $\beta$-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of $\gamma$-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.

• 식품보건융합연구
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• 제10권2호
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• pp.7-11
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• 2024

Glutathione (GSH) is a vital compound composed of glutamic acid, cysteine, and glycine, crucial for cellular functions including oxidative stress defense and detoxification. It has widespread applications in pharmaceuticals, cosmetics, and food industries due to its antioxidant properties and immune system support. Two primary methods for GSH synthesis are enzymatic and microbial fermentation. Enzymatic synthesis is efficient but costly, while microbial fermentation, particularly using yeast strains like Candida albicans, offers a cost-effective alternative. This study focuses on genetically modifying C. albicans mutants, specifically targeting glutathione reductase (GLR1) and gamma-glutamylcysteine synthetase (GCS1) genes, integral to GSH synthesis. By optimizing these mutants, the research aims to develop a model for efficient GSH production, potentially expanding its applications in the food industry.