Brain Glutamate Dehydrogenase Research Articles

Effect of pyridoxine and insulin administration on brain glutamate dehydrogenase activity and blood glucose control in streptozotocin-induced diabetic rats

Blood glucose level and kinetic parameters of glutamate dehydrogenase (GDH) were measured in the cerebellum, brain stem and cerebral cortex of control, insulin treated, pyridoxine treated, pyridoxine and insulin treated and untreated streptozotocin-diabetic rats. The combined administration of insulin and pyridoxine was found to be better in controlling the hyperglycaemia. Insulin with pyridoxine treatment brought back the increased maximal velocity of GDH during diabetes to control state. Also, there was an increase in Michaelis-Menten constant. These results suggest that pyridoxine and insulin together serve a better control for diabetes.

Activation of two types of brain glutamate dehydrogenase isoproteins by gabapentin

The stimulatory effects of gabapentin on the activities of two types of glutamate dehydrogenase (GDH) isoproteins homogeneously purified from bovine brain have been studied at various conditions. When the effects of different gabapentin concentrations on GDH activities were studied in the direction of reductive amination of 2-oxoglutarate with NADPH as a coenzyme, a marked activation was observed for both isoproteins, whereas both isoproteins showed activation to a lesser extent with NADH as a coenzyme. Stimulatory effects of gabapentin on GDH activities in the direction of the oxidative deamination of glutamate were also observed, but to a much lesser extent than reductive amination. There were big differences between the two GDH isoproteins in their sensitivity to the action of gabapentin. The largest activation was observed with GDH II when NADPH was used as a coenzyme. Half-maximal stimulation was reached at around 1.5 mM. Gabapentin relieved the inhibition of GDH isoproteins by GTP and this resulted in an increase in the apparent activation by gabapentin in the presence of GTP. 2-Oxoglutarate was found to give rise to high substrate inhibition and gabapentin reduced the substrate inhibition in the presence of 0.2 mM NADH. Since there are neurodegenerative disorders in which GDH activity is decreased, the therapeutic modulation of the activity of this enzyme may be clinically useful.

Brain glutamate dehydrogenase changes in streptozotocin diabetic rats as a function of age.

Kinetic parameters of brain glutamate dehydrogenase (GDH) were compared in the brain stem, cerebellum and cerebral cortex of three weeks and one year old streptozotocin (STZ) induced four day diabetic rats with respective controls. A single intrafemoral dose of STZ (60 mg/Kg body weight) was administered to induce diabetes in both age groups. After four days the blood glucose levels showed a significant increase in the diabetic animals of both age groups compared with the respective controls. The increase in blood glucose was significant in one year old compared to the three weeks old diabetic rats. The Vmax of the enzyme was decreased in all the brain regions studied, of the three weeks old diabetic rats without any significant change in the Km. In the adult the Vmax of GDH was increased in cerebellum and brain stem but was unchanged in the cerebral cortex. The Km was unchanged in cerebellum and cerebral cortex but was increased in the brain stem. These results suggest there may be an important regulatory role of the glutamate pathway in brain neural network disturbances and neuronal degeneration in diabetes as a function of age.

Expression and localization of brain Glutamate dehydrogenase with its monoclonal antibody

Glutamate dehydrogenase (GDH) is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study, we investigated the distribution of the GDH‐immunoreactive cells in the rat brain using monoclonal antibodies against bovine brain GDH isoprotein. GDH‐immunoreactive cell were distributed in the basal ganglia, thalamus and the nuclei belong to substantia innominata, and its connecting area, subthalamic nucleus, zona incerta, and substantia nigra. We could see GDH‐immunoreactive cells in the hippocampus, septal nuclei associated with the limbic system, the anterior thalamic nuclei connecting between the hypothalamus and limbic system, and its associated structures, amygdaloid nuclear complex, the dorsal raphe and median raphe nuclei and the reticular formation of the midbrain. The GDH‐immunoreactive cells were shown in the pyramidal neurons of the cerebral cortex, the Purkinje cells of the cerebella cortex, their associated structures, ventral thalamic n...

Regulation of two soluble forms of brain glutamate dehydrogenase isoproteins by protein kinases

We isolated two soluble forms of glutamate dehydrogenase isoproteins, GDH I and GDH II, from bovine brain. The regulation of GDH I and GDH II by phosphorylation and dephosphorylation has been examined in various conditions. There were dose‐ and time‐ dependent activation of the GDH isoproteins when phosphorylated by cAMP‐dependent protein kinase. The phosphorylated GDH had 1.1 mol of covalently bound phosphate/mol of subunit and a 2‐fold increased specific activity. The phosphorylated amino acid was identified as serine. When treated with alkaline phosphatase, the activities of the phosphorylated GDH isoproteins were reduced in dose and time dependent manner and returned to those of unphosphorylated enzymes. There were no significant differences between GDH I and GDH II in their sensitivities to the action of phosphorylation and dephosphorylation, demonstrating that the microenvironmental structures of the phosphorylation site in GDH isoproteins are similar to each other. These results suggest that the in...

Essential Active‐Site Lysine of Brain Glutamate Dehydrogenase Isoproteins

Two soluble forms of bovine brain glutamate dehydrogenase (GDH) isoproteins were inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff's base formation with amino groups of the enzyme. Sodium borohydride reduction of the pyridoxal 5'-phosphate-inactivated GDH isoproteins produced a stable pyridoxyl enzyme derivative that could not be reactivated by dialysis. The pyridoxyl enzyme was studied through fluorescence spectroscopy. No substrates or coenzymes separately gave complete protection against pyridoxal 5'-phosphate. A combination of 10 mM 2-oxoglutarate with 2 mM NADH, however, gave complete protection against the inactivation. Tryptic peptides of the isoproteins, modified with and without protection, resulted in a selective modification of one lysine. In both GDH isoproteins, the sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other GDH species.

Identification of a peptide of the guanosine triphosphate binding site within brain glutamate dehydrogenase isoproteins using 8-azidoguanosine triphosphate.

Photoaffinity labeling with [gamma-32P]8N3GTP (8-azidoguanosine triphosphate) was used to identify the guanine binding peptides of the GTT binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. 8N3GTP, without photolysis, mimicked the inhibitory properties of GTP on GDH I and GDH II activities. Saturation of photoinsertion of GDH isoproteins revealed an apparent Kd of 8 microM (GDH I) and 24 microM (GDH II) for [gamma-32P]8N3GTP. Ion exchange and reversed-phase high-performance liquid chromatography (HPLC) were used to isolate photolabel-containing peptides generated with trypsin. This identified a portion of the guanine binding domain within the GTP binding site is the region containing the sequence I-S-G-A-S-E-X-D-I-V-H-S-A-L-A-Y-T-M E-R (GDH I) and I-S-G-A-S-E-X-D-I-V-H-S-G-L-A-Y-T-M-E-R (GDH II). The symbol X indicates a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designated as a photolabeled lysine since the sequences including the lysine residue in question have a complete identity with those of the other GDH species known. Also, trypsin was unable to cleave the photolabeled peptide at this site. Photolabeling of these peptides was prevented by the presence of GTP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as GTP. These results demonstrate selectivity of the photoprobe for the GTP binding site and suggest that the peptide identified using the photoprobe is located in the GTP binding domain of the brain GDH isoproteins.

Modification of brain glutamate dehydrogenase isoproteins with pyridoxal 5′-phosphate

Two soluble forms of brain glutamate dehydrogenase isoproteins were inactivated by pyridoxal 5'-phosphate. Restoration of catalytic activity can be accomplished by dialysis and addition of an excess of cysteine or lysine. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff base formation with amino groups of the enzyme. Inactivation became irreversible after reduction with NaBH4 and the NaBH4-reduced enzyme showed a characteristic absorption peak at 325 nm. Using spectral titration at 325 nm, the stoichiometry was 2 mol/mol of GDH subunit without protection and 1 mol/mol with protection, indicating the complete masking of one mol of lysine. The results with analogs of pyridoxal 5'-phosphate show that the aldehyde group, but not the phosphate group, is required for efficient inactivation.

Brain mitochondrial citrate synthase and glutamate dehydrogenase: Differential inhibition by fatty acyl coenzyme a derivatives

Organic acidemia is found in several metabolic encephalopathies (e.g., hepatic and valproate encephalopathies, Reye's syndrome, and hereditary organic acidemias). Although fatty acids are known to be neurotoxic, the underlying mechanisms have not been fully elucidated. It has been hypothesized that one mechanism underlying fatty acid neurotoxicity is the selective inhibition of rate-limiting and/or regulated tricarboxylic acid (TCA) cycle and related enzymes by fatty acyl-coenzyme A (CoA) derivatives. To test the hypothesis, this study has examined the effects of several fatty acyl-CoAs on citrate synthase (CS) and glutamate dehydrogenase (GDH) in brain mitochondria. At levels higher than 100 microM, butyryl-CoA (BCoA; a short-chain acyl-CoA; IC50 approximately 640 microM), octanoyl-CoA (OCoA; a medium-chain acyl-CoA; IC50 approximately 380 microM), n-decanoyl-CoA (DCoA; a medium-chain acyl-CoA; IC50 approximately 436 microM), and palmitoyl-CoA (PCoA; a long-chain acyl-CoA; IC50 approximately 340 microM) inhibited brain mitochondrial CS activity in a concentration-related manner. However, these fatty acyl-CoAs were less effective inhibitors (IC50 values for OCoA, DCoA, and PCoA being approximately 1260, 420, and 720 microM, respectively) of brain mitochondrial GDH activity. Compared to the other three acyl-CoAs investigated, BCoA was a very poor inhibitor of GDH. These results demonstrate that fatty acyl-CoAs are inhibitors of brain mitochondrial CS and GDH activities only at pathological/toxicological levels. Thus, the fatty acyl-CoA inhibition of brain mitochondrial CS and GDH is unlikely to assume major pathophysiological and/or pathogenetic importance.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hyperammonemia on brain amino acids in young and adult ferrets

Effects of arginine deficiency and hyperammonemia on the brain concentrations of amino acids and urea cycle enzyme activities in young and adult ferrets were investigated. Only young ferrets developed hyperammonemia and encephalopathy immediately after consuming the arginine-free diet. Brain ornithine and citrulline concentrations in young ferrets fed arginine containing diet were significantly lower than those in adult ferrets. Compared to rats and other animals, young and adult ferrets had lower concentrations of brain glutamic acid and glutamine. Unlike in other species, brain glutamine was not elevated in young, hyperammonemic ferrets. Brain arginase and glutamate dehydrogenase activities were significantly increased in young ferrets fed arginine-free diet. Young ferrets provide a useful animal model for investigating the neurotoxicity of acute hyperammonemia.

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.

Molecular cloning, structure and expression analysis of a full-length mouse brain glutamate dehydrogenase cDNA

We isolated and analysed a full-length mouse brain glutamate dehydrogenase (GLUD) cDNA as a preliminary step to use the mouse model for the investigation of GLUD function in neurotransmission and neurodegeneration. GLUD coding sequences were found highly conserved among mouse, human and rat. Northern blots revealed two transcripts with different ratios in different mouse organs implying some mechanism of tissue-specific expression. In contrast to human, mouse GLUD gene family appears not to contain an intronless member.

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.

Inhibition of ox brain glutamate dehydrogenase by perphenazine

Factors affecting the inhibition of ox brain glutamate dehydrogenase (GDH) by the anti-psychotic drug perphenazine have been studied. Inhibition was found to be of mixed type with respect to 2-oxoglutarate and competitive towards NADH. However the data indicate that perphenazine binds to a site distinct from the catalytic site to which NADH binds. Perphenazine also enhanced the high-substrate inhibition by these two substrates. Inhibition by perphenazine was not affected by the allosteric effector GTP but it was enhanced by increasing pH, in the range of 6.3 to 7.6, and diminished by increasing ionic strength. Low concentrations of perphenazine relieved the inhibition of GDH by phosphatidylserine and cardiolipin. However, at higher concentrations phosphatidylserine did not interfere with the inhibition by perphenazine whereas cardiolipin relieved it. The possible significance of these interactions in terms of the behaviour of this antipsychotic drug in vivo are discussed.

Comparison of human brain and liver glutamate dehydrogenase cDNAS

In order to investigate suggestions that more than one glutamate dehydrogenase (GDH) gene may be active in humans, seven human brain and seventeen human liver GDH cDNAs were isolated by probing with a 590 base cDNA from the coding region of human brain GDH. No sequence heterogeneity was revealed among any of the cDNAs by an oligonucleotide binding assay, nor did any cDNA appear to encode a hexapeptide contained in a published amino acid sequence of human liver GDH. Homologous regions of three liver and three brain cDNAs had identical sequences over more than 2 kb, including 3′ nontranslated regions. This suggests that identical GDH mRNAs are present in human brain and human liver. Although only one gene appears to be expressed, human genomic DNA blots show a pattern of hybridization consistent with the existence of more than one GDH gene.

Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate transmission

The principally mitochondrial enzyme glutamate dehydrogenase (GDH) exhibited low-intensity, uniform immunoreactivity in neurons and intense heterogeneous labeling of glial cells of rat brain. Simultaneous peroxidase labeling for GDH and immunoautoradiography for glial fibrillary acidic protein (GFAP) confirmed the astrocytic localization of the enzyme. Immunoreactivity in astrocytes, but not in neurons, required the presence of Triton X-100 as a solubilizing agent. Most of the intensely labeled glial processes were localized to regions previously reported as containing moderate to high densities of binding sites for the excitatory amino acids, L-glutamate or L-aspartate, and glutamatergic fibers. These included several forebrain regions, such as the superficial layers of the rostral neocortex, dorsal neostriatum, nucleus accumbens, septohippocampal nucleus, intralaminar thalamic nuclei, and external capsules. However, the central gray of the midbrain, the nuclei of the reticular formation, brain stem regions projecting to the cerebellum, and cranial nuclei of the trigeminal and vagal nerves also exhibited intense glial labeling for GDH, even though some of these regions are known to receive only weak glutamatergic projections. A second factor determining the distribution of GDH appeared to be neuronal activity, as assessed by correspondence with reported high densities of cytochrome oxidase. We conclude that GDH enriched in glial populations exists in a subcellular compartment distinct from that of neurons and may serve as one of the enzymes involved in glutamatergic transmission. Deficiencies of glial GDH and the consequent cytotoxic effects of high levels of excitatory amino acids may contribute to a number of neurodegenerative disorders.

Isolation of a human brain cDNA for glutamate dehydrogenase.

A cDNA has been isolated from a human brain expression library using anti-bovine glutamate dehydrogenase (GDH) antibodies. The cDNA has an open reading frame of 774 nucleotides, which codes for 258 amino acids. The 258-amino-acid sequence is 95% homologous to the carboxy terminus of human liver GDH. This high degree of homology indicates that the cDNA codes for brain GDH. Fourteen differences between the amino acid sequence deduced from this cDNA and the sequence reported for human liver GDH suggest that there may be two active human GDH genes. A cRNA probe synthesized from the cDNA detects a 3.7-kilobase (kb) mRNA from human brain. Rat liver and kidney each contain two GDH mRNAs, 3.5 and 2.8 kb, respectively. The 3.5-kb transcript is prominent in rat brain, whereas the 2.8-kb transcript is barely detectable, a result suggesting that GDH gene expression is differentially controlled in rat brain.

Glutamate and malate dehydrogenase activities in Joseph disease and olivopontocerebellar atrophy.

The activities of brain glutamate dehydrogenase and malate dehydrogenase were not statistically different in samples from patients with autosomal dominant olivopontocerebellar atrophy or Joseph disease compared with control subject samples. These two enzymes are thus not involved in the pathogenesis of these two separate dominantly inherited diseases.

Glutamate dehydrogenase in human brain: regional distribution and properties.

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.

Effect of hypothermia on brain glutamate dehydrogenase activity

Effect of hypothermia on brain glutamate dehydrogenase activity