• Journal of Applied Biological Chemistry
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• v.42 no.1
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• pp.19-24
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• 1999

Plants have been shown to contains $Ca^{2+}$/calmodulin-stimulated GAD and NAD kinase. To test how calmodulin and calmodulin methylation affect the activation of GAD and NAD kinase, GAD and NAD kinase were partially purified from tobacco plants. GAD was also partially purified from E. coli transformed with a plasmid carrying a cloned tobacco GAD gene. We find that GAD from the transformed E. coli showed 60-fold $Ca^{2+}$/calmodulin-dependent activation. However, GAD from tobacco plants was stimulated only about 3.8-fold by the addition of calmodulin in the presence of calcium, suggesting high background activity of the enzyme was possibly due to bound endogenous tobacco calmodulin. There were no significant differences in the tobacco GAD activator properties between calmodulins. A monoclonal antibody against petunia GAD interacted strongly with both GAD from tobacco plants and GAD from cloned gene. NAD kinase from tobacco plants showed a complete $Ca^{2+}$/calmodulin dependency for activity. Unmethylated calmodulins activated GAD in a manner similar to methylated calmodulin. However, the maximum level of NAD kinase activation obtained with unmethylated calmodulins is approximately 4-fold higher than methylated calmodutins. These data suggested that endogenous tobacco calmodulin may interact more tightly with GAD than NAD kinase and that calmodulin methylation affects the activator properties of calmodulins for tobacco NAD kinase but not for GAD.

• BMB Reports
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• v.38 no.5
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• pp.595-601
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• 2005

In this study, we have isolated a rice (Oryza sativa L.) glutamate decarboxylase (RicGAD) clone from a root cDNA library, using a partial Arabidopsis thaliana GAD gene as a probe. The rice root cDNA library was constructed with mRNA, which had been derived from the roots of rice seedlings subjected to phosphorus deprivation. Nucleotide sequence analysis indicated that the RicGAD clone was 1,712 bp long, and harbors a complete open reading frame of 505 amino acids. The 505 amino acid sequence deduced from this RicGAD clone exhibited 67.7% and 61.9% identity with OsGAD1 (AB056060) and OsGAD2 (AB056061) in the database, respectively. The 505 amino acid sequence also exhibited 62.9, 64.1, and 64.2% identity to Arabidopsis GAD (U9937), Nicotiana tabacum GAD (AF020425), and Petunia hybrida GAD (L16797), respectively. The RicGAD was found to possess a highly conserved tryptophan residue, but lacks the lysine cluster at the C-proximal position, as well as other stretches of positively charged residues. The GAD sequence was expressed heterologously using the high copy number plasmid, pVUCH. Our activation analysis revealed that the maximal activation of the RicGAD occurred in the presence of both $Ca^{2+}$ and calmodulin. The GAD-encoded 56~58 kDa protein was identified via Western blot analysis, using an anti-GAD monoclonal antibody. The results of our RT-PCR analyses revealed that RicGAD is expressed predominantly in rice roots obtained from rice seedlings grown under phosphorus deprivation conditions, and in non-germinated brown rice, which is known to have a limited phosphorus bioavailability. These results indicate that RicGAD is a $Ca^{2+}$/calmodulin-dependent enzyme, and that RicGAD is expressed primarily under phosphate deprivation conditions.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.696-703
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• 2015

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37℃ for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55℃ and the activity was dependent on pyridoxal 5'-phosphate. The K_m and V_max of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

• Anxiety and mood
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• v.13 no.1
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• pp.10-16
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• 2017

Objective : This study aimed to investigate differences in demographic, clinical characteristics, and quality of life between panic-disorder patients with generalized anxiety disorder (PD+GAD) and without generalized anxiety disorder (PD-GAD). Methods : We examined data from 218 patients diagnosed with PD+GAD (150 patients) and PD-GAD (68patients). The following instruments were applied: Stress coping strategies, Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), Panic Disorder Severity Scale (PDSS), Anxiety Sensitivity Index-Revised (ASI-R), Albany Panic and Phobia Questionnaire (APPQ), NEO-neuroticism(NEO-N), Short Form health survey-36 (SF-36). Results : Compared to the PD-GAD group, the PD+GAD group had higher scores in emotion-focused coping strategies and clinical severity, such as BDI, BAI, PDSS, ASI, APPQ, and neuroticism. The PD+ GAD group showed lower scores in most scales in SF-36 status than PD-GAD group. Conclusions : This study shows that PD+GAD patients are different from PD-GAD patients in coping strategies, clinical severity and quality of life. It emphasizes the need of personalized therapy in clinical approach among patients with PD+GAD.

• Preventive Nutrition and Food Science
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• v.9 no.4
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• pp.324-329
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• 2004

In order to investigate the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in lactic acid bacteria, we cloned a glutamate decarboxylase (GAD) gene from Lactobacillus plantarum using polymerase chain reaction (PCR). One PCR product DNA was obtained and inserted into a TA cloning vector with a T7 promoter. The recombinant plasmid was used to transform E. coli. The insertion of the product was con­firmed by EcoRI digestion of the plasmid purified from the transformed E. coli. Nucleotide sequence analysis showed that the insert is a full-length Lactobacillus plantarum GAD and that the sequence is $100\%$ and $72\%$ identical to the regions of Lactobacillus plantarum GAD and Lactococcus lactis GAD sequences deposited in GenBank, accession nos: NP786643 and NP267446, respectively. The amino acid sequence deduced from the cloned Lactobacillus plantarum GAD gene showed $100\%$ and $68\%$ identities to the GAD sequences deduced from the genes of the NP786643 and NP267446, respectively. To express the GAD protein in E. coli, an expression vector with the GAD gene (pkk/GAD) was constructed and used to transform the UT481 E. coli strain and the expression was confirmed by analyzing the enzyme activity. The Lactobacillus plantarum GAD gene obtained may facilitate the study of the molecular mechanisms regulating GABA metabolism in lactic acid bacteria.

• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.9-17
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• 2021

A γ-aminobutyric acid (GABA)-producing microorganism was isolated from galchi (hairtail fish, Trichiurus lepturus) jeotgal, a Korean salted and fermented seafood. The G144 isolate produced GABA excessively when incubated in MRS broth containing monosodium glutamate (MSG, 3%, w/v). G144 was identified as Lactobacillus brevis through 16S rRNA and recA gene sequencing. gadB and gadC encoding glutamate decarboxylase (GAD) and glutamate/GABA antiporter, respectively, were cloned and gadB was located downstream of gadC. The operon structure of gadCB was confirmed by reverse transcription (RT)-polymerase chain reaction. gadB was overexpressed in Escherichia coli and recombinant GAD was purified and its size was 54.4 kDa as evidenced by SDS-PAGE results. Maximum GAD activity was observed at pH 5.0 and 40℃ and the activity was dependent on pyridoxal 5'-phophate. The K_m and V_max of GAD were 8.6 mM and 0.01 mM/min, respectively.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.73-73
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• 1995

We investigated the effect of derivatized phospholipid, P-pyridoxyl dipalmiuylphosphatidylethanolamine (P-pyr-DPPE), on the catalytic activity of purified porcine brain glutamate decarboxylase(GAD) which catalyzes the synthesis of GABA known as major inhibitory neurotransmitter in CNS. When the P-pyr-DPPE was incorporated into dipalmitdylphosphatidylcholine(DPPC) or phosphatidylserine(PS) vesicles, these vesicles enhanced the catalytic activity of GAD. P-pyr-DPPE also interacted with apoglutamate decarboxylase(apoGAD) and produced the free pyridoxal-5-phosphate(PLP) which is the natural cofactor of GAD. This result indicated that apoGAD catalyzed the cleavage reaction of the P-pyridoxyl moiety of the derivatized phopholipid to generate free PLP, and then free PLP bound to the apoGAD resulting in restroration of the catalytic activity of the enzyme.

• YAKHAK HOEJI
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• v.42 no.3
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• pp.248-256
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• 1998

We had already reported that we purified N-${\beta}$-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), a novel antibacterial substance from the immunized adult Sarcoph aga peregrina (Flesh fly). We found that the antibacterial activity of synthetic 5-S-GAD is equal to that of authentic 5-S-GAD without a specificity of antibacterial activity against Gram positive and Gram negative. Significant synergism was detected between 5-S-GAD and streptomycin against streptomycin resistant strain E.coli K12 594. It has an antitumor activity against several tumor cell lines at a concentration of $100{\mu}M$. However, no cytotoxic activity against murine macrophage was detected at a concentration of $500{\mu}M$. Furthermore, haemolytic activity against sheep erythrocytes was not detected at the same concentration. We suggest that the S-conjugation of glutathion with dihydroxyphenylalanine might be important to increase antibacterial activity of dihydroxyphenylalanme.

• Korean Journal of Biological Psychiatry
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• v.12 no.1
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• pp.13-19
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• 2005

Objective:The purposes of this study were to investigate heart rate variability(HRV) in patients with generalized anxiety disorder(GAD) compared with major depressive disorder in Korea. Methods:Fifty-six GAD patients(20 male and 36 female) was classified into their comorbid psychiatric illness. Among them, Twenty-five patients(10 male and 15 female) who do not have any psychiatric comorbidity were compared with 30 major depressive disorder patients(12 male and 18 female). Clinical symptoms, HRV and MMPI were analysed between two group. Results:Comorbid psychiatric illnesses of GAD were ranked into no diagnosis(44.6%), MDD(32.1%), panic disorder(10.7%), social phobia(5.3%), PTSD(1.7%), OCD(1.7%), MDD+panic disorder(1.7%) and MDD+specific phobia(1.7%). GAD patients showed low functioning in HRV, but degree of decreasing HRV is not so severe compared with MDD patient. Balance of sympathetic and parasympathetic nerve tone is more severely impaired in GAD patients compared with MDD patient. The score of MMPI did not reveal any differences between two groups. Conclusions:The result showed that HRV can differenciate GAD and MDD patients. GAD patients could show decreased HRV functioning, less than MDD patients. But autonomic imbalance could be more severe in GAD than MDD patients.

• KSBB Journal
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• v.16 no.1
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• pp.36-40
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• 2001

In order to study the molecular mechanism of $\gamma$-aminobutyric acid (GABA) production in plants, we cloned and sequenced a partial glutamate decarboxylase (GAD) cDNA from the Arabidopsis thaliana cDNA library, using primers targeted at highly conserved sequences of the petunia GAD gene. The cDNA fragment was inserted into TA cloning vector with T7 promoter and the recombinant plasmid obtained was used to transform E. coli. The plasmid DNA purified from the transformed E. coli was digested with EcoRI and the presence of the insert was confirmed. Nucleotide sequence analysis showed that the fragment is a partial Arabidopsis thaliana GAD gene and that the sequence showed 98% and 78% identity to the region of the putative Arabidopsis thaliana GAD sequences deposited in GenBank, Accession nos: U46665 and U10034, respectively. The amino acid sequence deduced from the partial Arabidopsis thaliana GAD gene showed 99% and 91% identities to the GAD sequences deduced from the genes of the U46665 and U10034, respectively. The partial cDNA sequence determined may facilitate the study of the molecular mechanism of GABA metabolism in plants.