• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.710-716
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• 2016

Gamma-aminobutyric acid (GABA) is a non-protein amino acid, which is an important inhibitor of neurotransmission in the human brain. GABA is also used as the precursor of biopolymer Nylon-4 production. In this study, the carbon flux from the tricarboxylic acid cycle was directed to the GABA shunt pathway for the production of GABA from glucose. The GABA shunt enzymes succinate-semialdehyde dehydrogenase (GabD) and GABA aminotransferase (GabT) were co-localized along with the GABA transporter (GadC) by using a synthetic scaffold complex. The co-localized enzyme scaffold complex produced 0.71 g/l of GABA from 10 g/l of glucose. Inactivation of competing metabolic pathways in mutant E. coli strains XBM1 and XBM6 increased GABA production 13% to reach 0.80 g/l GABA by the enzymes co-localized and expressed in the mutant strains. The recombinant E. coli system developed in this study demonstrated the possibility of the pathway of the GABA shunt as a novel GABA production pathway.

• Plant Biotechnology Reports
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• 제5권3호
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• pp.225-234
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• 2011

Arabidopsis mutants with T-DNA insertion in seven calmodulin genes (CAM) were used to determine the specific role of CAM in the tolerance of plants to oxidative stress induced by paraquat and hydrogen peroxide ($H_2O_2$) treatments. Arabidopsis calmodulin mutants (cam) were screened for seedling growth, seed germination, induced oxidative damage, and levels of ${\gamma}$-aminobutyric acid (GABA) shunt metabolites. Only the cam5-4 and cam6-1 mutants exhibited an increased sensitivity to paraquat and $H_2O_2$ during seed germination and seedling growth. In response to treatments with $3{\mu}M$ paraquat and 1 mM $H_2O_2$, only the cam5-4, cam6-1 mutants showed significant changes in malonaldehyde (MDA) levels in root and shoot tissues, with highly increased levels of MDA. In terms of the GABA shunt metabolites, GABA was significantly elevated in root and shoot tissues in response to the paraquat treatments in comparison to alanine and glutamate, while the levels of all shunt metabolites increased in root tissue but not in the shoot tissue following the $H_2O_2$ treatments. GABA, alanine and glutamate levels were significantly increased in root and shoot of the cam1, cam4, cam5-4, and cam6-1 mutants in response to paraquat (0.5, 1 and $3{\mu}M$), while they were increased only in the root tissue of the cam1, cam4, cam5-4, and cam6-1 mutants in response to $H_2O_2$ (200 and $500{\mu}M$, 1 mM). These data show that the cam5-4 and cam6-1 mutants were sensitive to the induced oxidative stress treatments in terms of seed germination, seedling growth, and oxidative damage. The accumulation of GABA shunt metabolites as a consequence of the induced oxidative stress treatments (paraquat and $H_2O_2$ treatments) suggests that the GABA shunt pathway and the accumulation of GABA metabolites may contribute in antioxidant machinery associated with reactive oxygen species and in the acquisition of tolerance in response to induced oxidative stress in Arabidopsis seedlings.

• BMB Reports
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• 제31권3호
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• pp.233-239
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• 1998

A GABA synthesizing enzyme, glutamate decarboxylase, has been purified from bovine brain by several chromatographic procedures. The preparation appeared homogeneous on SDS-PAGE. The enzyme is a homodimeric protein with a molecular mass of 120 kDa. The activation of glutamate decarboxylase by ginesenosides from Panax ginseng C.A. Meyer has been studied. Preincubation of the enzyme with total ginsenoside, $Rb_2$ and Rc ginsenosides, increased glutamate decarboxylase activities in a dose-dependent manner. There was a reproducible decrease in $K_m$, in addition to a increase in $V_{max}$, in response to increasing concentrations of the Rc ginsenoside fraction. Upon addition of the ginsenoside to the enzyme, a decrease in flurorescence intensity was discernible, together with an increase in emission anisotropy. Judging from the anisotropy values, the ginsenoside is rapidly trapped by the protein matrix. Total ginsenoside was administered to rats and the rat brains were removed for the measurement of the changes of GABA shunt regulating enzyme activities. Among the GABA shunt regulating enzymes, only the glutamate decarboxylase activities were increased after ginsenoside treatment. Therefore, it is suggested that the ginsenosides may elevate the GABA level in brain by activation of glutamate decarboxylase and the enzymatic activation might be due to the conformational change induced by binding of ginsenoside to the enzyme.

• Biomolecules & Therapeutics
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• 제1권1호
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• pp.93-97
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• 1993

We have previously reported that an NADPH-dependent succinic semialdehyde reductase was purified homogeneously from bovine brain by several chromatographic procedures, and was found to be a monomeric protein with a molecular mass of 28 kDa (Cho et al., Eur. J. Biochem. 1993). Since succinic semialdehyde is an important intermediate in the ${\gamma}$-aminobutyrate(GABA) shunt and GABA level is associated with various forms of human neurological disorders, we have investigated the effects of anticonvulsant drugs on the succinic semialdehrde reductase. Among the drugs tested, sodium valproate and diphenylhydantoin inhibited the enzyme activity, while some other drugs, barbiturate and chlorpromazine, had no inhibitory effects on the enzyme activity. The purified enzyme was also injected as an immunogen into Balb/c mice to obtain monoclonal antibodies (mob) and several mobs to the protein were produced from the fusion experiments.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.269-269
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• 1994

GABA shunt에 관여하는 두 가지 효소인, GABA transminase와 Succinic semialdehyde reductase에 대한 단일 클론 항체를 생산하고 이들 항체의 특성을 살펴보았다. 소의 뇌에서 순수 정제된 효소를 동물에 주사한 후 immunodot-blot 분석법에 의하여 일차적으로 항체를 분비하는 hybridoma를 골라낸 후 생산된 단일 클론 항체가 특이적으로 이들 효소와 반응하는 가를 알아보기 위하여 Western blot 분석을 실시하였다. 뇌조직에서 추출한 총단백질을 SDS 전기영동법에 의하여 분리한 후 이들 항체를 처리한 결과, GABA-T에 대한 항체는 특이적으로 분자량이 50kDa에 해당하는 단백질 밴드만을 인지하였고 SSA reductase에 대한 항체의 경우 분자량이 34kDa 크기의 단백질 밴드와 반응하였다. 이들 분자량은 순수 정제된 소뇌의 효소 단백질의 크기에 해당하는 것을 확인하였다. 이들 소뇌의 효소에 대한 항체를 추적물체로 사용하여 다른 포유동물자 조류의 효소와 비교하는 cross-reactivity 연구를 수행하였다. 소, 돼지, 토끼, 쥐 (rat), 개, 고양이 그리고 닭의 뇌를 제거한 후 총단백질을 추출하고 Western blot을 해본 결과 GABA transaminase의 경우 조류를 제외하고 다른 포유동물에서는 같은 분자량의 단일 단백질 밴드를 확인하였고 SSA reductase의 경우 조류를 포함하는 모든 동물에서 같은 분자량의 밴드를 확인하였다 이상의 결과로 미루어 보아 포유류 뇌에 있는 이들 효소들은 변역학적으로 아주 유사하다고 사료된다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.71-71
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• 1995

The succinic semialdehyde dehydrogenase which is one of the key enzyme of GABA shunt in CNS has been purified from bovine brain homogeneously for the first time. The molecular mass of the native enzyme was estimated to be approximately 110,000 on gel filtration, The subunit molecular mass was determined by SDS-PAGE to be 54,000. These results indicate that the enzyme is a dimeric protein made up to identical subunits. Chemical modification studies of the enzyme suggest that the critical lysyl, connected with catalytic activity of the enzyme, The binding of IAF-SSDH(enzyme tagged with fluoreceine) to GABA transaminase which catalyzes the degradation of GABA was monitored by steady emission anisotropy. The changes of fluorescence anisotropy by interactions between two enzymes suggest that the formation of enzyme cluster must be invoved in the regulation of GABA concentration in brain tissues. The inhibitory effects of some antiepileptic and anticonvulsant drugs on the enzyme were also examined.

• 한국버섯학회지
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• 제15권1호
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• pp.38-44
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• 2017

최근 식용과 약용버섯으로 각광을 받고 있는 꽃송이버섯(Sparassis latifolia 혹은 S. crispa)은 베타-글루칸이 풍부하여 다양한 생리활성을 나타낸다고 알려져 있다. 꽃송이버섯에 다량 포함된 베타-글루칸과 더불어 뇌기능을 증진시킬 수 있는 영양성분이 보강된다면 약용버섯의 기능성이 더욱 증대되어 농가소득 증대와 국민 건강증진에 큰 기여를 할 것으로 판단된다. 본 연구에서는 꽃송이버섯 균사체 배양 시 아미노산을 첨가하였을 시 균사 생장이 촉진됨과 함께 GABA 함량이 증대되었고, ${\beta}-glucan$ 함량에는 유의적인 효과가 없는 것을 확인하였다. 균사의 생장에는 glutamic acid와 ornithine 모두 효과가 있었으나, GABA 함량에 대해서는 glutamic acid의 첨가가 훨씬 효과적임을 확인하였다. 이는 꽃송이버섯 균사체의 GABA-shunt에 glutamic acid가 영향을 미친 것으로 사료된다. 생성된 GABA의 소멸과 다른 화합물의 신규 생성에 대해서는 아직 결과를 도출하지 못하였으므로, 이 부분에 대한 추가 연구가 필요하리라 사료된다.

• 한국미생물·생명공학회지
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• 제47권1호
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• pp.139-147
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• 2019

Glutamate decarboxylase catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), contributing to pH homeostasis through proton consumption. The reaction is the first step toward the GABA shunt. To date, the enzymes involved in the glutamate metabolism of Photobacterium damselae subsp. piscicida have not been elucidated. In this study, an open reading frame of P. damselae subsp. piscicida, showing homology to the glutamate decarboxylase or putative pyridoxal-dependent aspartate 1-decarboxylase genes, was isolated and cloned into an expression vector to produce the recombinant enzyme. Preliminary gas chromatography-mass spectrometry characterization of the purified recombinant enzyme revealed that it catalyzed not only the decarboxylation of glutamate but also the transamination of GABA. This enzyme of P. damselae subsp. piscicida could be bifunctional, combining decarboxylase and transaminase activities in a single polypeptide chain.

• BMB Reports
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• 제32권3호
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• pp.254-258
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• 1999

The NADPH-dependent succinic semialdehyde reductase is one of the key enzymes in the brain GABA shunt, and it catalyzes the formation of the neuromodulator $\gamma$-hydroxybutyrate from succinic semi aldehyde. This enzyme was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of $1.1{\times}10^3\;M^{-1}min^{-1}$ at pH 7.0, $25^{\circ}C$, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. Complete inactivation of succinic semialdehyde reductase required the modification of five histidyl residues per molecule of enzyme. However, only one residue was calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity. The inactivation of the enzyme by DEP was prevented by preincubation of the enzyme with the coenzyme NADPH but not with the substrate succinic semialdehyde. These results suggest that an essential histidyl residue involved in the catalytic activity is located at or near the coenzyme binding site of the brain succinic semialdehyde reductase.

• Archives of Pharmacal Research
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• 제22권2호
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• pp.219-224
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• 1999

In our search for the anticonvulsant consitutent of Gastrodia elata repeated column chromatographies guided by activity assay led to isolation of an active compound, which was identified as gastrodin on the basis of spectral data. Brain succinic semialdehyde dehydrogenase (SSADH) was inactivated by preincubation with gastrodin in a time-dependent manner and the reaction was monitored by absorption and fluorescene spectroscopic methods. The inactivation followed pseudo-first-order kinetics with the second-rate order constant of $1.2{\times}10^{3} M^{-1} min^{-1}$. The time course of the reaction was significantly affected by the coenzyme NAD^{+}$, which affected complete protection against the loss of the catalytic activity, whereas substrate succinic semialdehyde failed to prevent the inactivation of the enzyme. It is postulated that the gastrodin is able to elevate the neurotransmitter GABA levels in central nervous system by inhibitory action on one of the GABA degradative enzymes, SSADH.