Use of high-performance liquid chromatography for assay of glutamic acid decarboxylase: Its limitation in use for post-mortem brain
15 Dec 1991-Journal of Chromatography B: Biomedical Sciences and Applications (Elsevier)-Vol. 571, pp 235-240
TL;DR: In this paper, a simple and rapid liquid chromatographic (HPLC) technique was used to measure GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period.
About: This article is published in Journal of Chromatography B: Biomedical Sciences and Applications.The article was published on 1991-12-15. It has received 6 citations till now. The article focuses on the topics: Glutamate decarboxylase.
Citations
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TL;DR: Comparison of kinetic data, pH, and temperature optima as well as of the mode of interaction with pyridoxal phosphate as a cofactor revealed several significant differences between the two isoenzymes reflecting their somewhat different physiological and molecular features.
Abstract: Two isoforms of glutamate decarboxylase (GAD65kDa and GAD67kDa) from human brain, which had previously been overexpressed in Escherichia coli as fusion proteins containing a glutathione-S-transferase domain, were purified by affinity chromatography on glutathione Sepharose 4B. Both isoforms were also expressed in Saccharomyces cerevisiae. After modification of a HPLC based assay, the enzymes were characterized with respect to their biochemical properties. Comparison of kinetic data, pH, and temperature optima as well as of the mode of interaction with pyridoxal phosphate as a cofactor revealed several significant differences between the two isoenzymes reflecting their somewhat different physiological and molecular features. Investigation of the influence of 4‘-O-methylpyridoxine (ginkgotoxin) (1), a neurotoxin occurring in Ginkgo biloba L., on the different isoenzymes, indicates that the phosphorylated form of the toxin, 4‘-O-methylpyridoxine-5‘-phosphate (2), decreases GAD65kDa activity, although in unph...
32 citations
TL;DR: Old rats exposed to chronic mild stress were characterized by an increased risk to develop anhedonia and there was significant decrease in GAD enzyme activity and increased tau protein hyperphosphorylation in old Rats exposed to CMS compared to control.
Abstract: Effects of chronic stress are not completely understood. They may underlie depression and dementia. This study assessed the association between chronic stress, glutamate levels, tau- protein phosphorylation, and nitric-oxide in old rats exposed to chronic mild stress (CMS). Old (> 15 months) male Wistar rats were exposed to CMS. Comparison groups included old and young control rats, young CMS-exposed, and old CMS-exposed rats treated with the neuronal nitric-oxide synthase (nNOS) enzyme inhibitor, 7-nitroindazole (20 mg/kg/day i.p.). Hippocampal glutamate levels and glutamate decarboxylase (GAD) activity were determined and tau protein phosphorylation was assessed. Age was a significant ( p = 0.025) source of variation in glutamate level [811.71 ± 218.1, 665.9 ± 124.9 µmol/g tissue protein (M ± SD) in young and old control rats, respectively]. Old rats exposed to CMS were characterized by an increased risk to develop anhedonia. There was significant ( p = 0.035) decrease in GAD enzyme activity (− 60.06%) and increased tau protein hyperphosphorylation in old rats exposed to CMS compared to control. Administration of 7-nitroindazole to CMS-exposed old rats significantly ( p = 0.002) increased GAD activity, decreased glutamate levels (7.19 ± 3.19 vs. 763.9 ± 91 µmol/g tissue protein; p = 0.0005), and decreased phosphorylation of tau proteins compared to CMS exposed rats.
16 citations
TL;DR: Results suggest that NAP-22 could participate in the transport of GAD65 and GAD67 to the presynaptic termini and their retention on the synaptic vesicles as an anchoring protein.
12 citations
TL;DR: The identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick) are reported and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.
6 citations
31 Oct 2006
2 citations
References
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TL;DR: Reductions in cerebral GAD require to be interpreted with caution in view of the sensitivity of this enzyme to premortem status but that reductions in cerebral CAT may be a more reliable index of pathological change in senile (Alzheimer-type) dementia.
715 citations
TL;DR: Experiments with hydroxylamine and α-hydrazinophenylacetic acid showed that the rate of loss of enzymic activity during preincubation of the brain homogenate with these agents was less than in absence of inhibitor, a finding consistent with the interpretation that the major mode of inhibition by carbonyltrapping agents is by combination with the holoenzyme.
328 citations
TL;DR: The results indicated that the enzymes GAD and CAT are remarkably stable in human brain during routine post-mortem handling, although a substantial loss of both catecholamines probably occurs in the first few hours after death.
Abstract: Measurements have been made of dopamine, noradrenaline, glutamate decarboxylase (GAD) and choline acetylase (CAT) in a variety of regions from human post-mortem brain tissue, and the results analysed as a function of ante-mortem and post-mortem factors which may influence such measurements. The agonal status of the patient emerged as a major influence on the post-mortem measurement of GAD activity, without significantly affecting the other biochemical parameters. Of the other ante-mortem factors investigated, there appeared to be no differences between male and female, no circadian fluctuations, and few significant age-related changes, in any of the biochemical parameters measured in different brain regions. Moreover, the results indicated that the enzymes GAD and CAT are remarkably stable in human brain during routine post-mortem handling. Similar findings were made for dopamine and noradrenaline, although a substantial loss of both catecholamines probably occurs in the first few hours after death.
207 citations
TL;DR: The above findings suggest minimally the presence of aldehyde, sulfhydryl and positively charged groups at or near the active site of the holoenzyme.
Abstract: —l-Glutamate decarboxylase purified from mouse brain was found to be highly sensitive to the sulfhydryl reagents, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) and p-chloromerburibenzoate (PCMB), which were competitive inhibitors (Ki for DTNB is 1·1 · 10−8m). Iodoacetamide and iodoacetic acid were less effective inhibitors than DTNB and PCMB. The mercapto acids, 3-mercaptopropionic, 2-mercaptopropionic, and 2-mercaptoacetic acids were potent competitive inhibitors with Ki values of 1·8, 53 and 300 μm, respectively. 2-Mercaptoethanol was less effective. Aminooxyacetic acid was the most potent carbonyl-trapping reagent tested inhibiting the enzyme activity completely at 1·6 μm, followed by hydroxylamine, hydrazine, semicarbazide, and d-penicillamine. Carboxylic acids with a net negative charge were strong competitive inhibitors e.g. d-glutamate (Ki 0·9 mm), α-ketoglutarate (Ki, l·2mm), fumarate (Ki,1·8 mm), dl-β-hydroxyglutamate (Ki, 2·8 mm), l-aspartate (ki, 3·1 mm) and glutarate (Ki, 3·5 mm). 2-Aminophosphonobutyric and 2-aminophosphonopropionic acids, phosphonic analogs of glutamate and aspartate, respectively, had no effect at l0mm. γ-Aminobutyric acid, l-glutamine, l-γ-methylene-glutamine, and α,γ-diaminoglutaric acid, amino acids with no net negative charge at neutral pH, had no effect at 5 mm. Glutaric and α-ketoglutaric acids were the most potent inhibitors among the various dicarboxylic and α-keto-dicarboxylic acids tested (Ki, 3·5 and 1·2 mm, respectively). Compounds with one carbon less, succinic and oxalacetic acids, or with one carbon more, adipic and α-ketoadipic acids, were less inhibitory. The monovalent cations, Li+, Na+, NH4+, and Cs+ had no effect on l-glutamate decarboxylase activity in concentrations up to 10mm. Divalent cations, on the other hand, were very potent inhibitors. Among eleven divalent cations tested, Zn2+ was the most potent inhibitor, inhibiting to the extent of 50 per cent at 10μm. The decreasing order of inhibitory potency was: Zn2+ > Cd2+, Hg2+, Cu2+ > Ni2+ > Mn2+ Co2+ > Ba2+ > Ca2+ > Mg2+ > Sr+2, The anions, I−, Br−, Cl− and F− were only weak inhibitors. The Ki value for Cl− was 17mm. The above findings suggest minimally the presence of aldehyde, sulfhydryl and positively charged groups at or near the active site of the holoenzyme. Intermediates of glycolysis had little effect on l-glutamate decarboxylase activity, but intermediates of the tricarboxylic acid cycle, e.g. α-ketoglutarate (Ki= 1·2 mm) and fumarate (Ki= 1·8 mm) were relatively potent inhibitors. The nucleotides, ATP, ADP, AMP, cyclic AMP, GTP, GDP, GMP, and cyclic GMP were weak inhibitors. l-Norepinephrine (Ki= 1·3 mm) and serotonin were potent inhibitors, while acetylcholine, dopamine and histamine were less effective. Ethanol and dioxane inhibited the enzyme activity to the extent of 20-50 per cent at 10 per cent (v/v), while slight activation was observed at low concentrations (0·1-1 per cent) of both solvents. The possible role of Zn2+ and some metabolites in the regulation of steady-state levels of γ-aminobutyric acid also was discussed.
185 citations
TL;DR: Studies of the phosphate activation of glutaminase activity, hitherto studied in the tissues of the rat, have been extended to include other species and revealed that a higher concentration of phosphate was necessary to achieve an appreciable ac- celeration of desamidation than was the case in extracts of brain or liver.
97 citations