Glutamate Metabolizing Enzymes in Prefrontal Cortex of Alzheimer’s Disease Patients

Amounts of glutamate metabolizing enzymes such as glutamate dehydrogenase (GDH), glutamine synthetase (GS), GS-like protein (GSLP), and phosphate-activated glutaminase (PAG) were compared in prefrontal cortex of control subjects and patients with Alzheimer disease (AD). The target proteins were quantified by ECL-Western immunoblotting in extracts from brain tissue prepared by two different techniques separating enzymes preferentially associated with cytoplasm (GDH I and II isoenzymes, GS, and partially GSLP) and membrane (GDH III, PAG, and partially GSLP) fractions. Amounts of all listed enzymes were found significantly increased in the patient group compared with controls. Some links between the measured values were observed in the control, but not in the AD patient group. The results may suggest for the pathological interruption of regulatory relations between distinct enzymes of glutamate metabolism in brain of AD patients.     

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells,and in mouse cerebral cortex and cerebellum in vivo

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in culture neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.Abbreviations used Asp-T aspartate aminotransferase (EC 2.6.1.1) - GABA-T GABA aminotransferase (EC 2.6.1.19) - GAD glutamate decarboxylase (EC 4.1.1.15) - gamma-GT gamma-glutamyl transferase (gamma-glutamyl transpeptidase) (EC. 2.3.2.2) - Glu glutamate - GDH glutamate dehydrogenase (EC 1.4.1.3) - GS glutamine synthetase (EC 6.3.1.2) - MAG maleate activated glutaminase - Orn-T ornithine aminotransferase (EC 2.6.1.13) - PAG phosphate activated glutaminase (EC 3.5.1.1)     

Enzymes of ammonia detoxication after portacaval shunt in the rat. II. Enzymes of glutamate metabolism.

Besides the synthesis of urea, ammonia detoxication at high concentrations can also be effected through enzyme reactions involved in glutamic acid metabolism. These mechanisms are also operative in extrahepatic tissues. Hyperammonemia is also found in the animal model of the portacaval shunt (PCS) rat. This model was chosen to study the activities of glutamate dehydrogenase, glutamine synthetase and glutaminase I in liver, brain and kidney 10, 20 and 30 days after PCS. In brain and kidney ammonia is detoxified mainly by the glutamate dehydrogenase and glutamine synthetase reactions whereas in the liver these enzyme reactions play a minor role.     

Ammonium and amino acid metabolism in excised leaves of wheat (Triticum aestivum) senescing in the dark

Several parameters of amino acid metabolism were studied in detached primary leaves of wheat (Triticum aestivum L. cv. Castell) during a 14 day incubation period in the dark. Protein loss was accompanied by a 5-fold increase in the total amount of free amino acids during the first 4 days of the incubation period with asparagine being the most important. Beyond this stage a pronounced intracellular accumulation of ammonium occured. A gradual decrease in the levels of free amino acids and ammonium at the later stages of senescence could in part be accounted for by leakage from the leaves. Additionally, some nitrogen was lost due to ammonia volatilization. The rapid decay of the glutamine synthetase (GS; EC 6.3.1.2)-glutamate synthase (Fd-GOGAT; EC 1.4.7.1) system and the fast decline of glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) activity appear to be predominant features of senescence in the dark. Decreasing Fd-GOGAT activity was slightly compensated by a small and temporary increase in the activity of NADH-GOGAT (EC 1.4.1.14). Glutamateoxalocetate transaminase (GOT: EC 2.6.1.1) activity, although declining continuously, proved to be much more persistent. Changes in glutamate dehydrogenase (GDH; EC 1.4.1.3) activity closely resembled the profile of ammonium evolution in the leaves and NADP-isocitrate dehydrogenase (IDH; EC 1.1.1.42) activity revealed a temporary maximum during the period of rapid increase in GDH activity. Increased activity of GDH could also be induced by exogenous ammonium. Ammonium accumulation could, at least partly, be caused by increased asparaginase (EC 3.5.1.1) activity which accompanied the rapid conversion of asparagine to aspartic acid. Asparagine aminotransferase (EC 2.6.1.14) activity declined sharply from the beginning of the senescence period. Although the activity profile of glutaminase (EC 3.5.1.2) was similar to that of asparaginase, glutamine was of little importance quantitatively and an analogous relationship between glutamine and glutamic acid could not be detected.     

Cerebral Ammonia Metabolism in Hyperammonemic Rats

The short-term metabolic fate of blood-borne [13N]ammonia was determined in the brains of chronically (8- or 14-week portacaval-shunted rats) or acutely (urease-treated) hyperammonemic rats. Using a "freeze-blowing" technique it was shown that the overwhelming route for metabolism of blood-borne [13N]ammonia in normal, chronically hyperammonemic and acutely hyperammonemic rat brain was incorporation into glutamine (amide). However, the rate of turnover of [13N]ammonia to L-[amide-13N]glutamine was slower in the hyperammonemic rat brain than in the normal rat brain. The activities of several enzymes involved in cerebral ammonia and glutamate metabolism were also measured in the brains of 14-week portacaval-shunted rats. The rat brain appears to have little capacity to adapt to chronic hyperammonemia because there were no differences in activity compared with those of weight-matched controls for the following brain enzymes involved in glutamate/ammonia metabolism: glutamine synthetase, glutamate dehydrogenase, aspartate aminotransferase, glutamine transaminase, glutaminase, and glutamate decarboxylase. The present findings are discussed in the context of the known deleterious effects on the CNS of high ammonia levels in a variety of diseases.     

Effect of Cortico-Striate Pathway Lesion on the Activities of Enzymes Involved in Synthesis and Metabolism of Amino Acid Neurotransmitters in the Striatum

The activities of several enzymes involved in the metabolism of aspartate and glutamate were measured in striatal (nucleus caudatus and putamen) homogenates 2-3, 6-7, and 35-40 days following frontoparietal and frontal cortical ablation. The activity of glutamine synthetase (GS) was substantially increased (46-48%) on the operated side 6-7 days following the lesion whereas smaller changes were observed at 2-3 and 35-40 days after lesion. In contrast, decreased levels of glutaminase and malate dehydrogenase (MDH) were observed by 6-7 days while no significant change was found at either 2-3 or 35-40 after the lesion. The activities of glutamate dehydrogenase (GDH) and glutamate decarboxylase (GAD) were elevated after 35-40 days whereas no changes in the levels of either GDH or aspartate aminotransferase (ASAT) were found at 2-3 or 6-7 days after the fronto-parietal decortication. When only the frontal cortex was removed quantitatively similar changes were observed in striatal GS and glutaminase activity. The content of glutamate and glutamine in the denervated striatum followed qualitatively the changes in glutaminase and GS. The results indicate that the degeneration of cortico-striatal terminals causes a profound glial reaction in the striatum, and both glutaminase and MDH are present in relatively high concentrations in the corticostriatal terminals.     

The localisation of enzymes of nitrogen assimilation in maize leaves and their activities during greening

The activities of nitrate reductase (EC1.6.6.1), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC6.3.1.2), glutamate synthase (EC1.4.7.1) and NAD(P)H-dependent glutamate dehydrogenase (EC 1.4.1.3) were investigated in mesophyll and bundle sheath cells of maize leaves (Zea mays L.). Whereas nitrate and nitrite reductase appear to be restricted to the mesophyll and GDH to the bundle sheath, glutamine synthetase and glutamate synthase are active in both tissues.During the greening process, the activities of nitrate and nitrite reductase increased markedly, but glutamine synthetase, glutamate synthase and glutamate dehydrogenase changed little.Abbreviations BDH British Drug Houses - EDTA Ethylene diamine tetra-acetic acid - GDH Glutamate dehydrogenase - NADH Nicotinamide-adenine dinucleotide reduced form - NADPH Nicotnamide-adenine dinucleotide phosphate reduced form - PMSF Phenylmethyl sulphonyl fluoride     

A mathematical model for the growth of a single cell of E. coli on a glucose/glutamine/ammonium medium

Extension of a single cell model of E. coli B/r to make predictions of culture response to variations in glutamine/glucose/ammonium ion concentrations is described. A biphasic glutamine transport system, a nitrogen metabolism scheme that includes glutamate dehydrogenase (GDH), glutamine synthetase/glutamate synthase (GS/GOGAT), the glutaminase routes, and a transaminase mechanism for glutamine carbon usage are added to the prototype model. The predictions of the extended model with regard to nutrient concentrations and cell size compare well with the experimental data and the prototype model predictions, demonstrating the capability of the integrated kinetic model to illustrate important enzymological interactions in a biological system. The discrepancies between the experimental data and the model predictions on growth yield suggest that a more detailed regulatory system of the TCA cycle is required for a more accurate energy budget.     

Glutamine from Glial Cells Is Essential for the Maintenance of the Nerve Terminal Pool of Glutamate: Immunogold Evidence from Hippocampal Slice Cultures

Abstract: The immunogold labeling for glutamate and glutamine was studied at the electron microscopic level in hippocampal slice cultures following inhibition of l -glutamine synthetase [ l -glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2]. In control cultures, glutamate-like immunoreactivity was highest in terminals, intermediate in pyramidal cell bodies, and low in glial cells. Glutamine-like immunoreactivity was high in glial cells, intermediate in pyramidal cell bodies, and low in terminals. After inhibition of glutamine synthetase with l -methionine sulfoximine, glutamate-like immunoreactivity was reduced by 52% in terminals and increased nearly fourfold in glia. Glutamine-like immunoreactivity was reduced by 66% in glia following l -methionine sulfoximine, but changed little in other compartments. In cultures that were treated with both l -methionine sulfoximine and glutamine (1.0 m M ), glutamate-like immunoreactivity was maintained at control levels in terminals, whereas in glia glutamate-like immunoreactivity was increased and glutamine-like immunoreactivity was decreased to a similar extent as in cultures treated with l -methionine sulfoximine alone. We conclude that (a) glutamate accumulates in glia when the flux through glutamine synthetase is blocked, emphasizing the importance of this pathway for the handling of glutamate; and (b) glutamine is necessary for the maintenance of a normal level of glutamate in terminals, and neither reuptake nor de novo synthesis through pathways other than the glutaminase reaction is sufficient.     

Acute metabolic effects of ammonia on the enzymes of glutamate metabolism in isolated astroglial cells

Enzymes of glutamate metabolism were studied in the astrocytes isolated from rats injected with a large dose of ammonium acetate and compared with those isolated from controls. The activities of glutamate dehydrogenase (GDH) and glutaminase decreased while those of glutamine synthetase (GS) and aspartate aminotransferase (AAT) increased both in convulsive and comatose states. The activity of alanine aminotransferase (A1AT) increased only in convulsive state. The results suggested that glutamate required for the formation of glutamine in astrocytes might have its origin in nerve endings and the depletion of citric acid cycle intermediates might occur in nerve endings at least in acute ammonia toxicity.     

Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver

Glutamine synthetase (EC 6.3.1.2) was localized within the matrix compartment of avian liver mitochondria. The submitochondrial localization of this enzyme was determined by the digitonin-Lubrol method of Schnaitman and Greenawalt (35). The matrix fraction contained over 74% of the glutamine synthetase activity and the major proportion of the matirx marker enzymes, malate dehydrogenase (71%), NADP-dependent isocitrate dehydrogenase (83%), and glutamate dehydrogenase (57%). The highest specific activities of these enzymes were also found in the matrix compartment. Oxidation of glutamine by avian liver mitochondria was substantially less than that of glutamate. Bromofuroate, an inhibitor of glutamate dehydrogenase, blocked oxidation of glutamate and of glutamine whereas aminoxyacetate, a transaminase inhibitor, had little or no effect with either substrate. These results indicate that glutamine metabolism is probably initiated by the conversion of glutamine to glutamate rather than to an alpha-keto acid. The localization of a glutaminase activity within avian liver mitochondria plus the absence of an active mitochondrial glutamine transaminase is consistent with the differential effects of the transaminase and glutamate dehydrogenase inhibitors. The high glutamine synthetase activity (40:1) suggests that mitochondrial catabolism of glutamine is minimal, freeing most of the glutamine synthesized for purine (uric acid) biosynthesis.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

Valproate Increases Glutaminase and Decreases Glutamine Synthetase Activities in Primary Cultures of Rat Brain Astrocytes

Abstract: It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l -[U-¹⁴C]glutamate, label incorporation into [¹⁴C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l -[U-¹⁴C]glutamate into [¹⁴C]aspartate also decreased with increasing concentrations of valproate. Following a 4-day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs-cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.     

Deletion of glutamate dehydrogenase 1 (Glud1) in the central nervous system affects glutamate handling without altering synaptic transmission

Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS‐specific GDH‐null mice (CnsGlud1^?/?); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns‐Glud1^?/? brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns‐Glud1^?/? mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns‐Glud1^?/? mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up‐regulation of astrocyte‐type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.     

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [¹³N]ammonia have shown that about 25?% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [¹³N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: l-Aspartate?+?GTP?+?H₂O?→?Fumarate?+?GDP?+?P_i?+?NH₃) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.     

Effect of the antiepileptic drug sodium valproate on glutamine and glutamate metabolism in isolated human kidney tubules

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.     

Pentylenetetrazole inhibits glutamate dehydrogenase and aspartate aminotransferase, and stimulates GABA aminotransferase in homogenates from rat cerebral cortex

The mechanism by which pentylenetetrazole provokes convulsions in animals has been investigated by measuring its influence in vitro on the activities of several enzymes of glutamate metabolism in rat brain homogenates. Pentylenetetrazole does not affect the specific activities of glutamine synthetase, glutaminase, or glutamate decarboxylase; it inhibits those of glutamate dehydrogenase and aspartate aminotransferase, and stimulates that of gamma-aminobutyric acid (GABA) aminotransferase. The overall consequence of the action of pentylenetetrazole on the activities of these enzymes should be an increase in the concentration of glutamate and a decrease in that of GABA. This modulation of glutamate and GABA metabolism by pentylenetetrazole could contribute to the triggering of convulsions.     

Acute metabolic effects of taurine on the enzymes metabolizing glutamate and gaba

100 mg of taurine per kg body weight had been administered intraperitoneally and 30 min after the administration the animals were sacrificed. Glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, glutaminase, glutamine synthetase, glutamate decarboxylase and GABA aminotransferase along with the content of glutamate and GABA in cerebral cortex, cerebellum and brain stem were studied and compared with the same obtained in the rats treated with normal saline in place of taurine. The results indicated a significant decrease in the activity of glutamate dehydrogenase in cerebral cortex and cerebellum and a significant increase in brain stem. Glutaminase and glutamine synthetase were found to increase significantly both in cerebral cortex and cerebellum. The activities of glutamate decarboxylase was found to increase in all the three regions along with a significant decrease in GABA aminotransferase while the content of glutamate showed a decrease in all the three brain regions, the content of GABA was observed to increase significantly. The above effects of taurine on the metabolism of glutamate and GABA are discussed in relation to the functional role of GABA and glutamate. The results indicate that taurine administration would result in a state of inhibition in brain.