Synthesis of glutamate and aspartate in rat brain synaptosomes

ATP and glutamine are the sources of endogenous ammonia in rat brain synaptosomes. The amount of endogenous ammonia formed from exogenous ATP is not sufficient to assure the maximum rate of aspartate and glutamate accumulation in the synaptosomes utilizing pyruvate + malate. Addition of exogenous NH4+ or depolarization of synaptosome plasma membranes with high K+ concentration led to a twofold increase in the rate of accumulation of these amino acids. This indicates that both exogenous and endogenous NH4+ is involved in the synthesis of aspartate and glutamate in nerve terminals. Accumulation of glutamate was stimulated by aminooxyacetate and inhibited by haloperidol which indicates that NH4+ is bound in the reaction catalysed by glutamate dehydrogenase. Endogenous oxaloacetate derived from pyruvate metabolism was the substrate for synthesis of aspartate. Additive inhibition of aspartate accumulation by fluorocitrate and (-) hydroxyacetate shows that, in addition to the tricarboxylic acid cycle, the reaction catalysed by ATP-citrate lyase serves in the synaptosomes as another source of oxaloacetate.     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

Pathways of glutamine and glutamate metabolism in resting and proliferating rat thymocytes: comparison between free and peptide-bound glutamine

Pathways of glutamine metabolism in resting and proliferating rat thymocytes were evaluated by in vitro incubations of freshly prepared or 60-h cultured cells for 1-2 h with [U14C]glutamine. Complete recovery of glutamine carbons utilized in products allowed quantification of the pathways of glutamine metabolism under the experimental conditions. Partial oxidation of glutamine via 2-oxoglutarate in a truncated citric acid cycle to CO2 and oxaloacetate, which then was converted to aspartate, accounted for 76 and 69%, respectively, of the glutamine metabolized beyond the stage of glutamate by resting and proliferating thymocytes. Complete oxidation to CO2 in the citric acid cycle via 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase accounted for 25 and 7%, respectively. In proliferating cells a substantial amount of glutamine carbons was also recovered in pyruvate, alanine, and especially lactate. The main route of glutamine and glutamate entrance into the citric acid cycle via 2-oxoglutarate in both cells is transamination by aspartate aminotransferase rather than oxidative deamination by glutamate dehydrogenase. In the presence of glucose as second substrate, glutamine utilization and aspartate formation markedly decreased, but complete oxidation of glutamine carbons to CO2 increased to 37 and 23%, respectively, in resting and proliferating cells. The dipeptide, glycyl-L-glutamine, which is more stable than free glutamine, can substitute for glutamine in thymocyte cultures at higher concentrations.     

The pathway of glutamate metabolism in rat brain mitochondria

1. The pathway of glutamate metabolism in non-synaptic rat brain mitochondria was investigated by measuring glutamate, aspartate and ammonia concentrations and oxygen uptakes in mitochondria metabolizing glutamate or glutamine under various conditions. 2. Brain mitochondria metabolizing 10mm-glutamate in the absence of malate produce aspartate at 15nmol/min per mg of protein, but no detectable ammonia. If amino-oxyacetate is added, the aspartate production is decreased by 80% and ammonia production is now observed at a rate of 6.3nmol/min per mg of protein. 3. Brain mitochondria metabolizing glutamate at various concentrations (0-10mm) in the presence of 2.5mm-malate produce aspartate at rates that are almost stoicheiometric with glutamate disappearance, with no detectable ammonia production. In the presence of amino-oxyacetate, although the rate of aspartate production is decreased by 75%, ammonia production is only just detectable (0.3nmol/min per mg of protein). 4. Brain mitochondria metabolizing 10mm-glutamine and 2.5mm-malate in States 3 and 4 were studied by using glutamine as a source of intramitochondrial glutamate without the involvement of mitochondrial translocases. The ammonia production due to the oxidative deamination of glutamate produced from the glutamine was estimated as 1nmol/min per mg of protein in State 3 and 3nmol/min per mg of protein in State 4. 5. Brain mitochondria metabolizing 10mm-glutamine in the presence of 1mm-amino-oxyacetate under State-3 conditions in the presence or absence of 2.5mm-malate showed no detectable aspartate production. In both cases, however, over the first 5min, ammonia production from the oxidative deamination of glutamate was 21-27nmol/min per mg of protein, but then decreased to approx. 1-1.5nmol/min per mg. 6. It is concluded that the oxidative deamination of glutamate by glutamate dehydrogenase is not a major route of metabolism of glutamate from either exogenous or endogenous (glutamine) sources in rat brain mitochondria.     

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.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells

1. Pyruvate strongly inhibited aspartate production by mitochondria isolated from Ehrlich ascites-tumour cells, and rat kidney and liver respiring in the presence of glutamine or glutamate; the production of (14)CO(2) from l-[U-(14)C]glutamine was not inhibited though that from l-[U-(14)C]glutamate was inhibited by more than 50%. 2. Inhibition of aspartate production during glutamine oxidation by intact Ehrlich ascites-tumour cells in the presence of glucose was not accompanied by inhibition of CO(2) production. 3. The addition of amino-oxyacetate, which almost completely suppressed aspartate production, did not inhibit the respiration of the mitochondria in the presence of glutamine, though the respiration in the presence of glutamate was inhibited. 4. Glutamate stimulated the respiration of kidney mitochondria in the presence of glutamine, but the production of aspartate was the same as that in the presence of glutamate alone. 5. The results suggest that the oxidation of glutamate produced by the activity of mitochondrial glutaminase can proceed almost completely through the glutamate dehydrogenase pathway if the transamination pathway is inhibited. This indicates that the oxidation of glutamate is not limited by a high [NADPH]/[NADP(+)] ratio. 6. It is suggested that under physiological conditions the transamination pathway is a less favourable route for the oxidation of glutamate (produced by hydrolysis of glutamine) in Ehrlich ascites-tumour cells, and perhaps also kidney, than the glutamate dehydrogenase pathway, as the production of acetyl-CoA strongly inhibits the first mechanism. The predominance of the transamination pathway in the oxidation of glutamate by isolated mitochondria can be explained by a restricted permeability of the inner mitochondrial membrane to glutamate and by a more favourable location of glutamate-oxaloacetate transaminase compared with that of glutamate dehydrogenase.     

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.     

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase ( l -glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase ( l -glutamate: NAD oxidoreductase EC 1.4.1.2).
The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase ( l -glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.
In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.     

Amino acid sequence of the trypsin-generated C3d fragment from human complement factor C3.

Energy metabolism in proliferating cultured rat thymocytes was compared with that of freshly prepared non-proliferating resting cells. Cultured rat thymocytes enter a proliferative cycle after stimulation by concanavalin A and Lymphocult T (interleukin-2), with maximal rates of DNA synthesis at 60 h. Compared with incubated resting thymocytes, glucose metabolism by incubated proliferating thymocytes was 53-fold increased; 90% of the amount of glucose utilized was converted into lactate, whereas resting cells metabolized only 56% to lactate. However, the latter oxidized 27% of glucose to CO2, as opposed to 1.1% by the proliferating cells. Activities of hexokinase, 6-phosphofructokinase, pyruvate kinase and aldolase in proliferating thymocytes were increased 12-, 17-, 30- and 24-fold respectively, whereas the rate of pyruvate oxidation was enhanced only 3-fold. The relatively low capacity of pyruvate degradation in proliferating thymocytes might be the reason for almost complete conversion of glucose into lactate by these cells. Glutamine utilization by rat thymocytes was 8-fold increased during proliferation. The major end products of glutamine metabolism are glutamate, aspartate, CO2 and ammonia. A complete recovery of glutamine carbon and nitrogen in the products was obtained. The amount of glutamate formed by phosphate-dependent glutaminase which entered the citric acid cycle was enhanced 5-fold in the proliferating cells: 76% was converted into 2-oxoglutarate by aspartate aminotransferase, present in high activity, and the remaining 24% by glutamate dehydrogenase. With resting cells the same percentages were obtained (75 and 25). Maximal activities of glutaminase, glutamate dehydrogenase and aspartate aminotransferase were increased 3-, 12- and 6-fold respectively in proliferating cells; 32% of the glutamate metabolized in the citric acid cycle was recovered in CO2 and 61% in aspartate. In resting cells this proportion was 41% and 59% and in mitogen-stimulated cells 39% and 65% respectively. Addition of glucose (4 mM) or malate (2 mM) strongly decreased the rates of glutamine utilization and glutamate conversion into 2-oxoglutarate by proliferating thymocytes and also affected the pathways of further glutamate metabolism. Addition of 2 mM-pyruvate did not alter the rate of glutamine utilization by proliferating thymocytes, but decreased the rate of metabolism beyond the stage of glutamate significantly. Formation of acetyl-CoA in the presence of pyruvate might explain the relatively enhanced oxidation of glutamate to CO2 (56%) by proliferating thymocytes.     

THE COMPARTMENTATION OF GLUTAMATE AND ITS METABOLITES IN FRACTIONS OF NEURON CELL BODIES AND NEUROPIL; STUDIED BY INTRAVENTRICULAR INJECTION OF [U-14C]GLUTAMATE

Abstract—

• 1 The in vivo metabolism of glutamate in rat neuron cell bodies and neuropil was studied after intraventricular injection of (U-¹⁴C)glutamic acid followed by separation of the tissue into neuronal and neuropil fractions.

• 2 The losses of amino acid and of radioactivity during the fractionation were equivalent. Recoveries were: glutamate, 32; glutamine, 15; aspartate, 25; GABA, 41; alanine, 30 per cent. In the washed cell fractions glutamine was 45 per cent and alanine 132 per cent higher in the neuronal fraction, glutamate was 62, GABA 77 and aspartate 95 per cent of neuropil levels. This contrasted with results obtained previously for in vitro incorporation. Calculation from these results indicated that 28 per cent of the original cell suspension was neuronal, 72 per cent neuropil. In the final cell preparations, 29 per cent of the neuron cell bodies and 26 per cent of the neuropil were recovered.

• 3 Specific activity of glutamate in the neuronal fraction 15 min after injection was higher than in the original suspension, but had declined to 30 per cent of its initial value by 2 h. In the neuropil, specific activity of glutamate was below that of the cell suspension at 15 min, but at later times rose above it by up to 40 per cent.

• 4 Radioactivity was detected in aspartate and glutamine 15 min after injection and GABA by 60 min after injection. In the original cell suspension the specific activity of glutamine was higher than that of glutamate at all times (the Waelsch effect) but aspartate and GABA were lower than glutamate.

• 5 In the neuronal fraction the specific activity of glutamine was below that of glutamate at all times, indicating a precursor-product relationship. In the neuropil fraction, glutamine specific activity remained above glutamate for the first hour.

• 6 These results are discussed in relation to the interpretation of the Waelsch effect in terms of metabolic compartmentation.

     

Central respiratory effects of glutamine synthesis inhibition in dogs

Glutamic acid is an excitatory neurotransmitter that may have a significant role in the central chemical drive of ventilation. Therefore cardiorespiratory function was measured in pentobarbital sodium-anesthetized dogs before and after central inhibition of glutamate metabolism by means of methionine sulfoximine (MSO), a specific inhibitor of glutamine synthase (GS) catalyzing amidation of glutamate to glutamine. GS was inhibited centrally by perfusing the ventriculocisternal space with artificial cerebrospinal fluid (CSF) containing 92.5 mmol MSO per liter at a fixed pH, perfusion rate, and pressure. After GS inhibition, CSF transfer rate of [13N]glutamine synthesized from 13NH4+ amidation of glutamate was reduced five-fold, and minute ventilation increased from 2.90 +/- 0.41 (SE) l/min (0.164 +/- 0.020 l.min-1.kg body wt-1) to 4.46 +/- 0.52 l/min (0.254 +/- 0.029 l.min-1.kg body wt-1). This increase in ventilation with endogenous glutamate and the increase in ventilation previously observed during ventriculocisternal perfusion of exogenous glutamate are compared quantitatively via a model of central neurotransmitter glutamate chemoreception. The results support the hypothesis that the endogenous brain glutamate is important in the central chemical drive of ventilation.     

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)     

The effect of diethyl- -fluoroglutarate on phosphates and glutamate in slices of rabbit cerebral cortex

Abstract— The diethyl ester of α-fluoroglutarate (DEFG), an inhibitor of glutamate dehydrogenase, was prepared, and its effect on glutamate and phosphates in slices of rabbit cerebral cortex was examined. The primary effect of the drug on cortical slices incubating in a Krebs-Ringer glucose medium was to decrease the tissue levels of glutamate in association with decreased levels and turnover of high-energy phosphates. Assimilation of exogenous glutamate by the slices was partially blocked in the presence of the drug and severely depressed oxidative phosphorylation resulted when glutamate and DEFG were both present in the incubation mixture. The results suggested a significant relationship between the activity of cerebral glutamate dehydrogenase and oxidative phosphorylation. During incubation in a Krebs-Ringer glucose medium the endogenous pool of free amino acids in the cortical slice partitioned with the medium. Little or no glutamate, aspartate or GABA was present in the medium after incubation, but glycine, alanine, threonine, serine and glutamine did partition to varying degrees, with over one-half of the glutamine present in the incubation medium. With the exception of ‘leakage’ of aspartate, the partitioning patterns were relatively unaffected by the presence of added glutamate or DEFG.     

Metabolic pathways for cytotoxic end product formation from glutamate- and aspartate-containing peptides by Porphyromonas gingivalis

Metabolic pathways involved in the formation of cytotoxic end products by Porphyromonas gingivalis were studied. The washed cells of P. gingivalis ATCC 33277 utilized peptides but not single amino acids. Since glutamate and aspartate moieties in the peptides were consumed most intensively, a dipeptide of glutamate or aspartate was then tested as a metabolic substrate of P. gingivalis. P. gingivalis cells metabolized glutamylglutamate to butyrate, propionate, acetate, and ammonia, and they metabolized aspartylaspartate to butyrate, succinate, acetate, and ammonia. Based on the detection of metabolic enzymes in the cell extracts and stoichiometric calculations (carbon recovery and oxidation/reduction ratio) during dipeptide degradation, the following metabolic pathways were proposed. Incorporated glutamylglutamate and aspartylaspartate are hydrolyzed to glutamate and aspartate, respectively, by dipeptidase. Glutamate is deaminated and oxidized to succinyl-coenzyme A (CoA) by glutamate dehydrogenase and 2-oxoglutarate oxidoreductase. Aspartate is deaminated into fumarate by aspartate ammonia-lyase and then reduced to succinyl-CoA by fumarate reductase and acyl-CoA:acetate CoA-transferase or oxidized to acetyl-CoA by a sequential reaction of fumarase, malate dehydrogenase, oxaloacetate decarboxylase, and pyruvate oxidoreductase. The succinyl-CoA is reduced to butyryl-CoA by a series of enzymes, including succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and butyryl-CoA oxidoreductase. A part of succinyl-CoA could be converted to propionyl-CoA through the reactions initiated by methylmalonyl-CoA mutase. The butyryl- and propionyl-CoAs thus formed could then be converted into acetyl-CoA by acyl-CoA:acetate CoA-transferase with the formation of corresponding cytotoxic end products, butyrate and propionate. The formed acetyl-CoA could then be metabolized further to acetate.     

AMINO ACID METABOLISM AND AMMONIA FORMATION IN BRAIN SLICES

The formation of ammonia and changes in the contents of free amino acids have been investigated in slices of guinea pig cerebral cortex incubated under the following conditions: (1) aerobically in glucose-free saline; (2) aerobically in glucose-free saline containing 10 mM-bromofuroic acid, an inhibitor of glutamate dehydrogenase (EC 1.4.1.2); (3) aerobically in saline containing 11-1 mM-glucose and (4) anaerobically in glucose-free saline. Ammonia was formed at a steady rate aerobically in glucose-free medium. The formation of ammonia was largely suppressed in the absence of oxygen or in the presence of glucose whereas the inhibitor of glutamate dehydrogenase produced about 50 per cent inhibition. Other inhibitors of glutamate dehydrogenase exerted a similar effect. Ammonia formation was also inhibited by some inhibitors of aminotransferases but not by others. Inhibition was generally more pronounced during the second and third hour of incubation. With the exception of glutamine which decreased slightly, the contents of all amino acids increased markedly during the anaerobic incubation. During aerobic incubation in a glucose-free medium, there was an almost complete disappearance of glutamic acid and GABA. Glutamine also decreased, but to a relatively smaller extent. The content of all other amino acids increased during aerobic incubation in glucose-free medium, although to a lesser extent than under anaerobic conditions. The greater increase of amino acids appearing anaerobically in comparison to the increase or decrease occurring under aerobic conditions corresponded closely to the greater amount of ammonia formed aerobically over that formed anaerobically. This finding is interpreted as indicating a similar degree of proteolysis under anaerobic and aerobic conditions; aerobically, the amino acids are partly metabolized with the concomitant liberation of ammonia. In glucose-supplemented medium, the content of glutamine was markedly increased. The content of glutamate and aspartate remained unchanged, whereas that of some other amino acids increased but to a lesser extent than in the absence of glucose. Proteolysis in the presence of glucose was estimated at about 65 per cent of that in its absence. In the presence of bromofuroate the rate of disappearance of glutamate was unchanged, but there was a larger increase in the content of aspartate and a smaller decrease of GABA and glutamine. Other changes did not differ significantly from those observed in the absence of bromofuroate. We conclude that the metabolism of amino acids in general and of glutamic acid in particular differs according to whether they are already present within the brain slice or are added to the incubation medium. Only the endogenous amino acids appear to be able to serve as precursors of ammonia and as substrates for energy production.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINOACIDS. GLUTAMATE RECEPTOR OF THE SHRIMP MUSCLE

Abstract— –Muscle of shrimps ( Artemisia longinaris ) were extracted with chloroform-methanol (2:1, v/v) and the proteolipids were separated by column chromatography on Sephadex LH-20. Three peaks of protein were eluted with chloroform and one with chloroform-methanol (4:1, v/v). Only the first peak eluted between 16 and 26 ml of chloroform showed binding for l -(¹⁴C]-glutamate. The type of saturation curve obtained suggests the existence of single type of binding site. The saturation is reached at one mole of l -glutamate per 320,000 g protein and the purification achieved about 3200-fold. The protein binding-glutamate does not bind GABA, aspartate or glutamine. The binding of l -[¹⁴C]-glutamate was inhibited by dl -α -methyl glutamic acid and l -glutamic acid diethyl ester. The binding properties of this hydrophobic protein fraction suggest that it may represent the isolated glutamate receptor of the shrimp muscle.     

Locations of amino acids in brain slices from the rat. Tetrodotoxin-sensitive release of amino acids

1. Amino acids, particularly glutamate, gamma-aminobutyrate, aspartate and glycine, were released from rat brain slices on incubation with protoveratrine (especially in a Ca(2+)-deficient medium) or with ouabain or in the absence of glucose. Release was partially or wholly suppressed by tetrodotoxin. 2. Tetrodotoxin did not affect the release of glutamine under various incubation conditions, nor did protoveratrine accelerate it. 3. Protoveratrine caused an increased rate of formation of glutamine in incubated brain slices. 4. Increased K(+) in the incubation medium caused release of gamma-aminobutyrate, the process being partly suppressed by tetrodotoxin. 5. Incubation of brain slices in a glucose-free medium led to increased production of aspartate and to diminished tissue contents of glutamates, glutamine and glycine. 6. Use of tetrodotoxin to suppress the release of amino acids from neurons in slices caused by the joint action of protoveratrine and ouabain (the latter being added to diminish reuptake of amino acids), it was shown that the major pools of glutamate, aspartate, glycine, serine and probably gamma-aminobutyrate are in the neurons. 7. The major pool of glutamine lies not in the neurons but in the glia. 8. The tricarboxylic cycle inhibitors, fluoroacetate and malonate, exerted different effects on amino acid contents in, and on amino acid release from, brain slices incubated in the presence of protoveratrine. Fluoroacetate (3mm) diminished the content of glutamine, increased that of glutamate and gamma-aminobutyrate and did not affect respiration. Malonate (2mm) diminished aspartate and gamma-aminobutyrate content, suppressed respiration and did not affect glutamine content. It is suggested that malonate acts mainly on the neurons, and that fluoroacetate acts mainly on the glia, at the concentrations quoted. 9. Glutamine was more effective than glutamate as a precursor of gamma-aminobutyrate. 10. It is suggested that glutamate released from neurons is partly taken up by glia and converted there into glutamine. This is returned to the neurons where it is hydrolysed and converted into glutamate and gamma-aminobutyrate.     

Glutamate Metabolism in Rat Cortical Astrocyte Cultures

Glutamate metabolism in rat cortical astrocyte cultures was studied to evaluate the relative rates of flux of glutamate carbon through oxidative pathways and through glutamine synthetase (GS). Rates of 14CO2 production from [1-14C]glutamate were determined, as was the metabolic fate of [14C(U)]glutamate in the presence and absence of the transaminase inhibitor aminooxyacetic acid and of methionine sulfoximine, an irreversible inhibitor of GS. The effects of subculturing and dibutyryl cyclic AMP treatment of astrocytes on these parameters were also examined. The vast majority of exogenously added glutamate was converted to glutamine and exported into the extracellular medium. Inhibition of GS led to a sustained and greatly elevated intracellular glutamate level, thereby demonstrating the predominance of this pathway in the astrocytic metabolism of glutamate. Nevertheless, there was some glutamate oxidation in the astrocyte culture, as evidenced by aspartate production and labeling of intracellular aspartate pools. Inhibition of aspartate aminotransferase caused a greater than 70% decrease in 14CO2 production from [1-14C]glutamate. Inhibition of GS caused an increase in aspartate production. It is concluded that transamination of glutamate rather than oxidative deamination catalyzed by glutamate dehydrogenase is the first step in the entry of glutamate carbon into the citric acid cycle in cultured astrocytes. This scheme of glutamate metabolism was not qualitatively altered by subculturing or by treatment of the cultures with dibutyryl cyclic AMP.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.