Increase In Glutamate Decarboxylase Activity Research Articles

Watercore Pear Fruit Respiration Changed and Accumulated γ-Aminobutyric Acid (GABA) in Response to Inner Hypoxia Stress

Watercore is a physiological disorder which often occurs on the pear fruit and the excessive accumulation of sorbitol in fruit intercellular space is considered to be an important cause of watercore. Our previous studies found that the metabolic disorder of sugars may lead to hypoxia stress and disturb respiration, resulting in aggravated fruit rot and the formation of bitter substances. However, the further changes of respiration and the fruit response mechanism are not well understood. A comprehensive transcriptome analysis of ‘Akibae’ pear watercore fruit was performed in this study. The transcriptome results revealed the hypoxia stress significantly induced the expression of several key enzymes in the TCA cycle and may lead to the accumulation of succinic acid in watercore fruit. The glycolytic pathway was also significantly enhanced in watercore fruit. Moreover, the γ-aminobutyric acid (GABA) synthesis related genes, glutamate decarboxylase (GAD) genes and polyamine oxidase (PAO) genes, which associated with the GABA shunt and the polyamine degradation pathway were significantly upregulated. In addition, the PpGAD1 transcript level increased significantly along with the increase of GAD activity and GABA content in the watercore fruit. Above all, these findings suggested that the hypoxic response was marked by a significant increase of the hypoxia-inducible metabolites succinic acid and GABA and that PpGAD1 may play a key role in response to watercore by controlling the GABA synthesis.

Gamma-Aminobutyric Acid (GABA) Modulates Nitrate Concentrations and Metabolism in the Leaves of Pakchoi (Brassica campestris ssp. chinensis Makino) Treated with a Nitrogen-Rich Solution

Pakchoi plants were grown in 32 mM NO3− nutrient solution with or without 2.5 mM γ-aminobutyric acid (GABA) to investigate metabolite changes, gene and protein expression levels, and the activities of key enzymes related to nitrate metabolism in the leaves over a period of 0–12 days. High-nitrogen treatment enhanced plant growth and the NO3−, NO2−, NH4+, Gln, and Glu contents in the leaves; promoted the gene and protein expression of nitrate reductase (NR) and glutamate decarboxylase (GAD); and increased the activities of NR, nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and GAD. The endogenous GABA concentration in the leaves was enhanced in parallel with the increase in GAD activity. The GABA-treated leaves displayed the greatest increases in the gene and protein expression levels of NR and GAD and in the activities of NR, NiR, GS, GOGAT, and GAD. In addition, accelerated rates of nitrate reduction and assimilation were detected, and these changes occurred concurrently with the observed increases in gene or protein expression and enzyme activity. As a result, the concentrations of NH4+, Gln, Glu, and endogenous GABA were significantly elevated, and the NO3− and NO2− contents were significantly decreased, in GABA-treated leaves compared with plants exposed to nitrogen-rich conditions. Our results reveal a potential positive that GABA may act as a nitrogen source to improve the plant growth and the most prominent effect of decreasing nitrate contents by accelerating NO3− reduction and assimilation. Exogenous GABA plays an important role in reducing the NO3− content of leaves, and thereby improves the ability to harvest leafy vegetables containing higher levels of endogenous GABA.

Glutamate decarboxylase activity and gamma-aminobutyric acid content in Synechocystis sp. PCC 6803 under osmotic stress and different carbon sources

Glutamate decarboxylase (GAD), a pyridoxal 5′-phosphate-dependent enzyme, catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA) through irreversible decarboxylation reaction. In the present study, GAD activity and GABA content in Synechocystis sp. PCC 6803 were investigated in response to osmotic stress and various carbon sources. The highest GAD activity was detected in late log phase cells subjected to 24 h of osmotic stress by 50 mM NaCl and 100 mM sorbitol with a 2- and 5.5-fold increase in the enzyme activity, respectively. GABA content was found to be enhanced by osmotic stress induced by both sorbitol and NaCl. GAD activity was increased by fourfold when late log phase cells were grown mixotrophically for 24 h in medium containing 0.1 % glucose as carbon source, whereas a nearly fivefold increase in GABA content was observed under the same condition. The increase in GAD activity by osmotic stress induced by NaCl and sorbitol up to 50 and 100 mM, respectively, and glucose was also corroborated by the increase in the levels of gad transcripts. Our results suggest that GAD activity, GABA content, and transcription of the gene-encoding GAD in Synechocystis cells are affected by alternative carbon sources as well as osmotic stress.

Characterization of γ-Aminobutyric acid(GABA) produced by a lactic acid bacterium from button mushroom bed

<TEX>${\gamma}$</TEX>-Aminobutyric acid(GABA) is a four carbon non-protein amino acid that has several well-known physiological functions, such as a postsynaptic inhibitory neurotransmitter in the brain and induction of hypotensive and tranquilizer effects. A lactic acid bacterium was isolated from button mushroom bed, which is showing high GABA productivity by TLC or HPLC analysis. The strain was identified as Lactobacillus hilgardii by analysis of 16S rDNA gene sequence. When the maximum production of GABA by L. hilgardii was investigated with various concentration of monosodium glutamate, the yield of GABA reached to be 53.65 mM at 1% mono sodium glutamate (MSG) in flask cultivation. A Glutamate decarboxylase (GAD) enzyme, which was known to convert MSG to GABA, was purified from a cell-free extract of L. hilgardii and the molecular weights of purified GAD was estimated to 60,000 by SDS-PAGE. The optimum pH and temperature of GAD were at pH4.6 and at <TEX>$37^{\circ}C$</TEX>, respectively. The GAD activity was increased by the addition of sulfate ions such as ammonium sulfate, sodium sulfate and magnesium sulfate, indicating that the increase of hydrophobic interaction causes the increase of GAD activity.

Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005.

Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobacillus brevis IFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30 degrees C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effects was as follows: ammonium sulfate > sodium sulfate > magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with L-glutamic acid as a substrate and the K(m), kcat, and kcat/K(m) values were 9.3 mM, 6.5 S-1, and 7 x 10(2) M-1 S-1, respectively.

Effect of insulin on GABAergic development in the embryonic chick retina

We investigated the role of insulin in GABAergic differentiation in the embryonic chick retina at different embryonic ages using glutamate decarboxylase (GAD) and high-affinity GABA uptake as developmental markers. Both these GABAergic markers exhibit developmentally programmed increases in activity during retinogenesis that also occur in culture. Insulin stimulated GABA uptake in retina neurons at all embryonic ages in a dose-dependent manner and GAD activity by 30% in embryonic retina neurons after 11 days of development. The stimulation of GABA uptake by insulin was blocked by addition of ouabain suggesting a role for the Na +,K + ATPase. The same concentration of insulin caused a 76% stimulation of protein synthesis in these retinal cells, and previous work demonstrated that insulin also stimulates cholinergic differentiation in the chick retina (Hausman et al., Dev. Brain Res. 59, (1991) 31–37). Thus, there was no selective stimulation of GABAergic differentiation by insulin but likely a neurotrophic effect. The increase in GAD activity in neurons from post-11-day embryonic neurons contrasts with our previous findings at embryonic days 6–7 where there is little change in GAD activity after addition of insulin. It is possible that the failure of insulin to stimulate GAD activity during early retina development is due to the increased accumulation of GABA in the presence of insulin. GABA levels were increased more than two-fold by 100 ng/ml insulin.

Role of the cell recognition molecule, cognin, in GABAergic differentiation in chick retina

Previous work showed that GABAergic differentiation in developing chick retina depends on insulin and cell interactions. Here, we investigated whether it depended on cell signaling mediated by retina cognin, a 50 kDa cell recognition molecule. Cognin mediates cell adhesion in vitro and occurs on retinal neurons that become both GABAergic and cholinergic. We investigated two markers of GABAergic differentiation: glutamate decarboxylase (GAD) activity and high-affinity GABA uptake. Both increase during differentiation of retinal neurons in culture and can be easily measured. We blocked cognin-mediated cell signaling with cognin antibody and found a reduction of the developmental increase in GAD activity in cultures of retinal neurons from 7 and 11 day chick embryos. There was no reduction of high-affinity GABA uptake. This suggested that cognin-mediated signaling was necessary for the normal developmental increase in GAD but not for high-affinity GABA uptake. These results contrasted with our previous observations on cholinergic differentiation in cultured retinal neurons. We found that cognin antibody blocked the normal developmental increase in choline acetyltransferase (ChAT) only if the cells were exposed before embryonic day 7. Thus, while both GAD and ChAT activity appear to be controlled by cell signaling involving cognin, the periods of developmental sensitivity for the two differentiation markers are different. Antibodies to other adhesion molecules, Ng-CAM, and N-cadherin, did not similarly affect GAD activity. Antibodies to laminin at a 10-fold higher concentration inhibited GAD activity only in early embryonic retina. Tests for protein synthesis and “housekeeping” enzyme activity demonstrated that the cognin antibody effect was selective for neuronal differentiation pathways. Thus, GABAergic differentiation in developing retina is sensitive to cell signaling mediated in part by cognin.

Influence of serum glucose on glutamate decarboxylase activity in the ventromedial nucleus of rats

Changes in the activity of glutamate decarboxylase (GAD), the rate-limiting enzyme in gamma-aminobutyric acid synthesis, in response to insulin-induced hypoglycemia were measured in several brain areas involved in glucoregulation. A 20% increase in GAD activity in the ventromedial nucleus (VMN) of rats was measured 30 min after injections of insulin. The increase was significantly correlated with the decrease in serum glucose (r = -0.74) and increased food intake (r = 0.44). The increase in VMN GAD activity was linear between 5 and 30 min after insulin injection and coincided with the decline in serum glucose. Pretreatment with a gastric preload of a 50% glucose solution attenuated the decline in serum glucose, the increase in food intake, and the increase in GAD activity. Inhibition of protein synthesis by puromycin did not block the decrease in glucose, but neither the increase in GAD activity in the VMN nor the increase in food intake in response to insulin was observed. The increase in VMN GAD activity also was present 6 h after insulin injection if rats were denied access to food. No consistent changes in GAD activity were measured in any brain area other than the VMN. The results of these trials suggest that the increase in VMN GAD activity is associated with the decrease in serum glucose and may be involved in the increased feeding associated with insulin administration.

Chronic administration of amphetamine increases glutamic acid decarboxylase activity in the rat substantia nigra

The glutamate decarboxylase activity in the rat cerebellum, frontal cerebral cortex, hypothalamus, substantia nigra and nucleus caudatus, was measured after either acute or chronic administration of (+)-amphetamine (1.35 and 5.4 mg/kg, i.p.). It was found that following 45 days of treatment the highest dose of the drug induced a selective increase of glutamate decarboxylase activity in the substantia nigra. Also in addition to the known changes in body weight, behavior and food-intake, some of the rats (5 out of 80 rats) treated with the highest dose of (+)-amphetamine developed a self-multilating behavior. These results suggest that after the repeated administration of a high dose of (+)-amphetamine the activity of the striatonigral GABAergic pathway is increased and supports the idea that γ-aminobutyric acid output neurons might convey dopamine-related functions.

Striatal dopamine depletion, dopamine receptor stimulation, and GABA metabolism: implications for the therapy of Parkinson's disease.

Dopamine-denervated rat striata exhibit increased synaptosomal gamma-aminobutyric acid (GABA) synthetic capacity (glutamate decarboxylase activity) without an alteration in GABA transport capacity. Stimulating denervated striatal dopamine receptors with apomorphine selectively increases in vivo steady-state striatal GABA turnover. The striatal response in animals to isolated loss of dopamine innervation is an increase in glutamate decarboxylase activity, not the decrease frequently found in the brains of patients with Parkinson's disease. Neurochemical expression of denervated striatal dopamine receptor stimulation may involve increased synaptic activity of striatal GABA neurons. For these reasons, enhancement of basal ganglia GABA function may improve responsiveness to dopamimetic therapies in patients with Parkinson's disease.

THE GAMMA‐AMINOBUTYRIC ACID (GABA) SYSTEM IN BRAIN DURING ACUTE AND CHRONIC ETHANOL INTOXICATION

Abstract— The effects of acute and chronic ethanol intoxication on the GAGA system of rats have been investigated. Under the terminal conditions provoked. by ethanol (6–8 g/kg, i.p.) the brain GABA content sharply increased. There was a simultaneous decrease of 35–40% in the glutamate decarboxylase (GAD) activity of the cerebellum and cerebral hemispheres. In contrast, the transaminase, GABA‐T was either unchanged, or it increased: by 28% only in cerebellum and by 1.5–2.0–fold in liver and kidney. It is suggested that effects of acute ethanol intoxication at different doses (2–8 g/kg) on the brain GABA system is connected with the phases of the functional condition of the CNS and a disturbance of homeostatic function. Chronic ethanol consumption caused a decrease in brain GABA. an increase of GAD activity in cerebellum and cerebral hemispheres, and no change in GABA‐T activity. The activity of this last enzyme was increased 1.5–2.0‐fold in liver and kidneys of rats consuming a diet containing 10% ethanol daily. A 50‐fold purified preparation of GABA‐T obtained from pig brain was inhibited by butanol‐l and propanol‐1 (0.03–0.6m) with no effect of ethanol. It is suggested that the mechanisms involved in the ethanol effect on nervous cells are linked with the GABA system and the phases of the functional condition of the CNS.

Changes in glycolipid glycosyltransferases and glutamate decarboxylase and their relationship to differentiation in neuroblastoma cells

Glycolipid glycosyltransferase activities involved in the biosynthesis in vitro of neutral and acidic glycosphingolipids were measured in C-1300 tumors and cloned cells derived therefrom. An adrenergic clone (NIE-115) was grown in tissue culture in the presence of dibutyryl cyclic AMP and the levels of glycosyltransferases were measured before and after differentiation. Increased activities of galactosyltransferases and sialyl-transferases with a concomitant increase in glutamate decarboxylase activity (the enzyme that catalyzes the synthesis of an inhibitory neurotransmitter, γ-aminobutyric acid) were observed.

Biochemical plasticity of short-axon interneurons: Increased glutamate decarboxylase activity in the denervated area of rat dentate gyrus following entorhinal lesion

The entorhinal cortex of adult rats was destroyed unilaterally, thus denervating the outer two-thirds of the molecular layer of the ipsilateral dentate gyrus. The biochemical response of short-axon GABAnergic interneurons intrinsic to the dentate gyrus was subsequently determined by laminar analysis of glutamate decarboxylase, an enzyme associated with boutons of these neurons. Glutamate decarboxylase activity was unaffected by entorhinal lesion for at least 10 days. However, when measured 29–38 days after operation, the absolute and specific activities of glutamate decarboxylase were respectively 45 and 63% greater in the outer part of the molecular layer of the operated side than in the corresponding zone of the control side. The difference between the two sides in specific activity was still significant, though reduced in magnitude, 90 days or more after operation. All statistically significant changes in glutamate decarboxylase activity were confined exclusively to the denervated region. Therefore, inhibitory projections of intrinsic interneurons, like intact excitatory afferents in close proximity to the degeneration, respond to entorhinal lesion in a manner suggesting increased input to the granule cells in compensation for the loss of excitatory entorhinal input. We suggest that additional GABAnergic boutons may be formed by axon sprouting or, alternatively, that an increase in glutamate decarboxylase activity within existing connections may be transsynaptically induced.