• Applied Biological Chemistry
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• 제40권6호
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• pp.478-483
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• 1997

E. coli K-12 균주에서 ${\gamma}-Glutamylcysteine$ synthetase를 정제하고 효소적 방법에 의한 글루타치온 합성에 관련된 특성을 검사하였다. 정제한 효소의 활성은 L-glutamate의 농도가 60 mM 까지 증가와 더불어 증가하였으나, 60 mM L-cysteine 에서는 50% 그리고 45 mM glycine 에서는 40%의 효소활성이 감소되었다. 효소의 활성은 반응산물 중의 하나인 ADP 뿐만 아니라 환원형 글루타치온에 의해서 감소되었다. 그러므로 환원형 글루타치온 뿐만 아니라 glutathione synthetase의 기질인 glycine은 ${\gamma}-glutamylcysteine$ synthetase 활성을 저해하므로 글루타치온 생산을 위해서는 ${\gamma}-glutamylcysteine$ synthetase 반응과 glutathione synthetase의 두 분리된 반응으로 이루어진 생반응계를 고안하는 것이 바람직하다. 또한 글루타치온 합성반응으로 부터 생성되는 ADP는 ${\gamma}-glutamylcysteine$ synthetase의 활성을 감소시키므로 글루타치온 합성을 위해서 ATP 재생계의 도입이 필요하다.

• 미생물학회지
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• 제29권5호
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• pp.278-283
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• 1991

Two amino acid residues ($Ala^{494}$ and $Ser^{495}$ of E. coli .gamma.-glutamylcysteine synthetase have been investigated whether they are the site of feedback inhibition by site specific mutagenesis. Single substitution of $serine^{495}$ (S495F), and double substitutions of alanine$^{494}$ and $serine^{495}$ (A494G-S495F) resulted in the inactivation of the .gamma.-glutamylcysteine synthetase activity. Substitution of $alanine^{494}$ with $glycine^{494}$ resulted in a higher level of feedback inhibition. These results suggest that $serine^{495}$ in .gamma.-glutamylcysteine synthetase is required for its catalytic acitvity and $alanine^{494}$ is presumably related to the feeback inhibition site.

• 대한화학회지
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• 제38권1호
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• pp.69-73
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• 1994

백서에 paraquat(PQ)투여로 간, 신장 및 폐 glutathione량이 감소되는데, 간과 신장은 유사한 감소양상을 나타냈고, 폐에서 감소율이 가장 높았으며, 혈액에서는 유의한 변화를 나타내지 않았다. PQ투여로 γ-glutamylcysteine synthetase와 γ-glutamyl transpeptidase 활성이 감소되었다. 따라서 간과 신장의 glutathione 감소는 γ-glutamylcysteine synthetase 활성이 감소되어 glutathione 합성저하에 의해 나타난 결과로 생각되고, 폐에서는 간과 신장의 γ-glutamylcysteine synthetase 활성감소로 glutathione합성이 감소되고, 폐 γ-glutamyl transpeptidase활성감소로 인해 혈액으로부터 폐세포로 glutathione 이동이 감소될 것으로 추정되므로 폐 γ-glutamyl transpeptidase 활성감소는 glutathione량 감소의 한 원인이 될 것으로 생각되며, 혈액에서는 γ-glutamylcysteine synthetase와 γ-glutamyl transpeptidase활성감소로 glutathione의 혈액내로 유입과 타장기로 유출이 모두 저하되어 glutathione량의 변화가 없는 것으로 생각된다. PQ에 의한 장기내 glutathione량 감소는 유리기 소거기능이 저하되어 PQ에 의해서 생성된 유리기 제거가 미흡할 것으로 생각되므로 PQ독성의 한 요인이 될 것으로 추측된다.

• BMB Reports
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• 제31권3호
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• pp.254-257
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• 1998

The gshI gene from the Escherichia coli K-12 strain codes for ${\gamma}-glutamylcysteine$ synthetase which mediates the rate-limiting step of glutathione biosynthesis. The isolated gshI gene from E. coli K-12 has an unusual translation initiation codon, UUG. The 494th amino acid is Ala rather than Gly which was found in a mutant strain E. coli B. In order to improve the translational rate of the gshI gene of E. coli K-12, the initiation codon, UUG, was changed to the usual AUG codon by the site-specific mutagenesis. This change has resulted in a 53% increase of ${\gamma}-glutamylcysteine$ synthetase activity. The enzyme activity was also improved by replacing $Ala^{494}$ with Val (A494V) or Leu (A494L). The replacement of $Ser^{495}$ with Thr (S495T) also resulted in a 62% increase of the enzyme activity. Therefore, the specific activity of ${\gamma}-glutamylcysteine$ synthetase was increased with the increasing chain length of the aliphathic amino acid at the site of the 494th amino acid (Ala<$Val{\leq}Leu$).

• 한국초지학회:학술대회논문집
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• 한국초지조사료학회 2005년도 학술심포지엄, 제43회 학술발표회
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• pp.158-159
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• 2005

• 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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• 제39권6호
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• pp.654-660
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• 1995

The present study was attempted to investigate the mechanism of oxidative cellular injuries which occur in diabetic rats by determining changes of antioxidant enzymes activity in the lung of alloxan-induced diabetic rats, the contents of glutathione in the lung, liver, blood samples, and ${\gamma}$-glutamylcysteine synthetase activities in the liver. Superoxide dismutase activities (SOD), including Cu, Zn-SOD and Mn-SOD, decreased in the lung of diabetic rats compared with those of normal control rats. However, activities of catalase and glutathione peroxidase(GPX) activities were not affected in the lung of diabetic rats. In diabetic rats, glutathione contents in the lung, liver, and blood samples, as well as the activities of ${\gamma}$-glutamylcysteine synthetase in the livers which is known to be the key enzyme of glutatione biosynthesis, decreased significantly. From these experimental results, it is thought that the decrease in SOD activities in the lung, glutathione contents and ${\gamma}$-glutamylcysteine synthetase activities in some tissues in alloxan-induced diabetic rats may be the crucial cause of vullnerability to oxidative cellular injuries.

• 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.

• 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 Plant Biotechnology
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• 제7권3호
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• pp.195-202
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• 2005

The effects of constituent amino acids of glutathione (GSH), glutamate (Glu), cysteine (Cys) and glycine (Gly), on GSH synthesis in lettuce seedlings were examined in this study. The GSH concentration of the seedlings was increased to 5.1-fold and 1.6-fold the concentration of the control in the first leaves and roots, respectively, by simultaneous application of these constituent amino acids (Glu+Cys+Gly) at 100 mg/l to the culture solution for two days. In the first leaves and roots of these seedlings, the concentration of GSH was 180.4 and 14.6 nmole/gFW, and non-essential amino acids including Glu, Cys and Gly occupied 93.2% and 84.0% of the total free amino acids, respectively. Application of Cys greatly increased the concentration of GSH in the roots, and application of 50 mg/l Cys increased it to 26.1-fold the concentration in the control. The activity of GSH synthetase was higher in the leaves than in the roots, whereas the activity of ${\gamma}$-glutamylcysteine synthetase was higher in the roots than in the leaves.