• Fisheries and Aquatic Sciences
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• v.4 no.1
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• pp.25-31
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• 2001

4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semi aldehyde. We isolated and sequenced' a fish cDNA fragment that encodes 4-aminobutyrate aminotransferase. A brain cDNA library from flounder (Paralichthys olivaceus) was constructed using the ZAP- III XR vector and screened for the fish 4-aminobutyrate aminotransferase gene using a probe derived from the conserved sequences of known mammalian 4-aminobutyrate aminotransferases. A partial cDNA for 4-aminobutyrate aminotransferase was cloned and found to be 700 bp in length corresponding to 66 amino acids. Nucleotide sequence of the clone was aligned with NCBI (National Center for Biotechnology Information) DNA sequence data base. The result showed high sequence identity with previously reported mammalian 4-aminobutyrate aminotransferases. The trans­criptional level of flounder 4-aminobutyrate aminotransferase was detected with the presence of mRNA at different flounder tissues by reverse transcription-polymerase chain reaction (RT-PCR). The expression of flounder 4-aminobutyrate aminotransferase was also tested and detected from the flounder tissues of the brain, liver, kidney and pancreas.

• Journal of Microbiology and Biotechnology
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• v.6 no.3
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• pp.162-166
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• 1996

The protein coding sequence of the 4-aminobutyrate aminotransferase was amplified by polymerase chain reaction (PCR) from a previously cloned cDNA of pig brain using a pair of primers based on the published sequence. The amplified DNA was introduced into a T7 expression vector. Recombinant 4-aminobutyrate aminotransferases were overexpressed in Escherichia coli. The inclusion bodies were formed when enzyme was overexpressed. The unfolded, overproduced proteins were purified by chromatography with hydroxyapatite and refolded by a sequential dialysis method. The renatured 4-aminobutyrate aminotransferase regained the catalytic activity. However, the purified mutant protein did not show the catalytic function of 4-aminobutyrate aminotransferase.

• BMB Reports
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• v.32 no.2
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• pp.181-188
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• 1999

4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semialdehyde. Recombinant 4-aminobutyrate aminotransferases were overexpressed as their catalytically active forms in E. coli by coproduction with thioredoxin and their solubilities were also dramatically increased. In order to study the structural and functional aspects of the C-terminal domain of brain 4-aminobutyrate aminotransferase, we have constructed a C-terminal mutant of pig brain 4-aminobutyrate aminotransferase and analyzed the functional and structural roles of C-terminal amino acids residues on the enzyme. The deletion of five amino-acid residues from C-terminus did not interfere with the kinetic parameters and functional properties of the enzyme. Also, the deletion did not affect the dimeric structure of the protein aligned along the subunit interface at neutral pH. However, the deletion of the C-terminal region of the protein changed the stability of its dimeric structure at acidic pH. The dissociation of the enzyme acidic, facilitated by the deletion of five amino acids from C-terminus, abolished the catalytic activity.

• BMB Reports
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• v.33 no.1
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• pp.43-48
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• 2000

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It converts the neurotransmitter 4-aminobutyric acid to succinic semialdehyde. In order to study the structural and functional aspects of catalytically active Cys residues of pig brain 4-aminobutyrate aminotransferase, we purified the active form in E. coli by coproduction of thioredoxin. The structural arrangement for functional requirements of a dimeric protein using a bifunctional sultbydryl reagent was then characterized, and the spatial proximity between the essential SH groups and a cofactor (pyridoxal-5'-phosphate) binding site was determined. The bifunctional sultbydryl reagent DMDS reacted with the enzyme at the ratio of one molecule per enzyme dimer. This resulted in an approximately 50% loss of enzymatic activity. The spatial proximity of the distance between the essential SH groups and the cofactor-binding site was determined by the energy transfer measurement technique. The result (approximate 20 ${\AA}$) suggested that cross-linking of two sulfhydryl groups with DMDS is not near a PLP binding site.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.224-229
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• 1993

GABA transminase (4-aminobutyrate aminotransferase), which catalyzes the breakdown of the major inhibitory neurotransmitter, GABA, in mammalian brain, was inactivated by preincubation with the mycotoxin patulin. The time course of the reaction was significantly affected by the substrate .alpha.-ketoglutarate, which aforded complete protection against the loss of catalytic activity. The recovery from the inhibition of patulin by the addition of dithiothreitol (DTT) supports that patulin reacts with the sulfhydryl residue in the catalytic domain of the enzyme. The reconstitution of the reduced enzyme and apoenzyme with pyridoxal-5-P(PLP) was inhibited by another mycotoxin, penicilic acid. This mycotoxin may interact with lysyl residue of the enzyme. Therefore, it is postulated that the critical sulfhydryl and lysyl residues in the catalytic domain of the enzyme react with mycotoxin patulin and penicillic acid, respectively.

• Journal of Applied Biological Chemistry
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• v.56 no.2
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• pp.89-93
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• 2013

Gamma-aminobutyric acid (GABA) aminotransferase (gabT) and succinic semialdehyde dehydrogenase (gabD) genes from Pseudomonas fluorescens KCCM 12537 were cloned into a single pETDuet-1 vector and co-expressed in Escherichia coli BL21(DE3) simultaneously. The mixture of both enzymes, called GABase, is the key enzyme for the enzymatic analysis of GABA. The molecular mass of the GABA aminotransferase and succinic semialdehyde dehydrogenase were determined to be 52.8 and 46.7 kDa following computations performed with the pI/Mw program, respectively. The GABase activity between pH 6.0 and 9.0 for 24 h at $4^{\circ}C$ remained over 75%, but under pH 6.0 decreased rapidly. The GABase activity between 25 and $35^{\circ}C$ by the treatment at pH 8.6 for 30 min remained over 80%, but over $35^{\circ}C$ decreased rapidly. When the activity against GABA was defined as 100%, the purified GABase activity against 5-aminovaleric acid having a similar structure to GABA showed 47.7% and GABase activity against ${\beta}$-alanine, ${\varepsilon}$-amino-n-caproic acid, $_L$-ornithine, $_L$-lysine, and $_L$-aspartic acid showed between 0.3 to 2.3%. The GABA content was analyzed with this co-expressed GABase, compared with the other GABase which was available commercially. As a result, the content of GABA extracted from brown rice, dark brown rice, and black rice were $26.4{\pm}3.5$, $40.5{\pm}4.7$ and $94.7{\pm}9.3{\mu}g/g$, which were similar data of other GABase in the error ranges.

• Journal of Life Science
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• v.25 no.8
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• pp.861-869
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• 2015

One of the domesticated species; the dog has been selectively bred for various aims by human. The dog has many breeds, which are artificially selected for specific behaviors and morphologies. Dogs contribute their life to human as working dogs for guide, rescue, detection or etc. Working dogs requires good personality, such as gentleness, robustness and patience for performing their special duty. Many studies have concentrated on finding genetic marker for selecting the high-quality working dog. In this study, we confirmed quantitative expression patterns of eight genes (ABAT; 4-Aminobutyrate Aminotransferase, PLCB1; Phospholipase C, Beta 1, SLC10A4; Solute Carrier Family 10, Member 4, WNT1; Wingless-Type MMTV Integration Site Family, Member 1, BARX2; BarH-Like Homeobox 2, NEUROD6; Neuronal Differentiation 6, SEPT9; Septin 9 and TBR1; T-Box, Brain, 1) among brains tissues from four dog breeds (Beagle, Sapsaree, Shepherd and Jindo), because these genes were expressed and have functions in brain mostly. Specially, BARX2, SEPT9, SLC10A4, TBR1 and WNT1 genes were highly expressed in Beagle and Jindo, and Sapsaree and German Shepherd were vice versa. The biological significance of total genes was estimated by database for annotation, visualization and integrated discovery (DAVID) to determine a different gene ontology (GO) class. In these analyses, we suppose to these eight genes could provide influential information for brain development, and intelligence of organisms. Taken together, these results could provide clues to discover biomarker related to functional traits in brain, and beneficial for selecting superior working dogs.