Aspartate aminotransferase isotope exchange reactions: implications for glutamate/glutamine shuttle hypothesis

George A. Kimmich, James A. Roussie, and Joan Randles

Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptor---oxaloacetate (OA) or alpha -ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between 3H-labeled Glu or 3H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of 3H2O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [3H]KG to Glu by glutamic dehydrogenase (GDH) or of [3H]OA to malate by malic dehydrogenase (MDH) "protects" the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.

glutamate/oxaloacetate transaminase; transaminase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MORE THAN 60% of all central nervous system (CNS) synapses are glutamatergic (22), indicative of the predominant role that glutamate (Glu) must play in the numerous neurotransmitter-mediated events required for brain function. Nevertheless, Glu becomes a serious neurotoxin if extracellular levels similar to those achieved during neuronal activity are sustained for even short time intervals (5, 11, 20, 23, 25, 28, 33, 41). Ordinarily, neurotoxicity is avoided because of the activity of sodium-coupled transport systems in cells surrounding glutamatergic synapses. Glu uptake by these systems restores the low extracellular level (~1 µM) characteristic of quiescent neurons.

Both neurons and glial cells have capability for Glu reuptake (1, 7, 18, 27, 29). However, when antisense DNA is used in "knockdown" experiments to block expression of specific transporter isoforms, the most severe pathophysiology occurs when either of the two primarily astrocytic isoforms (GLT1 or GLAST) is eliminated (28). Gene "knockout" experiments confirm this conclusion. GLT1 knockouts exhibit lethal spontaneous seizure activity (34), and mice depleted of GLAST survive but show reduced motor coordination (38). These experiments imply that Glu reuptake by astrocytes is particularly important for avoiding neurotoxicity. However, a problem is created for glutamatergic neurons even if astrocytes take up only a portion of the synaptic Glu. Neurons are believed to be incapable of catalyzing net Glu synthesis from the two- and three-carbon precursors provided by long-chain fatty acid and glucose metabolism, respectively, because they do not express pyruvate carboxylase, the anaplerotic enzyme required for net formation of TCA cycle intermediates (17, 31, 43). Instead, metabolic intermediates with four or five carbon atoms are thought to be necessary for returning carbon from astrocytes to neurons to allow continuing resynthesis of the neuronal Glu required for sustaining neurotransmission. Carbon must be returned that is at least equal in amount to that represented by astrocytic Glu capture.

Of the various intermediates receiving attention, glutamine (Gln) is often viewed as one of the most likely candidates for carbon return from astrocyte to neuron. Astrocytes have Gln synthetase that can produce Gln from Glu in a single step (26). Moreover, neurons have capability for sodium-coupled Gln uptake and can resynthesize Glu in a single step catalyzed by phosphate-activated glutaminase (15). There are no known neuronal Gln receptors, so extracellular return of carbon as Gln would not induce inappropriate neural activity. The proposed neuron-astrocyte-neuron carbon cycle is commonly called the Glu/Gln shuttle hypothesis (3, 30, 35, 37).

Questions regarding the shuttle hypothesis have been raised on the basis that astrocytes may metabolize some of the Glu they "capture" for energy production via the TCA cycle. To the extent that this occurs, neurons that release Glu will face a carbon deficit unless four- or five-carbon intermediates in some form(s) other than Gln are also returned. Most of the proposed limitations to the shuttle concept are based on observations showing that astrocytes take up isotopically labeled Glu rapidly and produce a wide variety of labeled TCA cycle and other metabolic intermediates (8, 9, 14, 32, 33, 39, 40, 42). These labeled intermediates are considered alternative candidates to Gln for shuttling carbon back to glutamatergic neurons. The higher the rate of formation of a particular intermediate and the larger its pool size, the more credence it is awarded as an alternative carbon shuttle participant.

The work presented here shows that aspartate aminotransferase (AAT; EC 2.6.1.1) catalyzes a rapid exchange of isotope between Glu and its cognate keto acid alpha -ketoglutarate (KG), in the absence of oxaloacetate (OA) or other keto acid acceptors for the amino group. The enzyme also catalyzes aspartate (Asp)/OA isotope exchange. The exchange reactions allow rapid incorporation of isotope from Glu into KG or from Asp into OA. In the whole cell context, label exchanged into either keto acid pool will then be distributed to other TCA cycle intermediates even though no net consumption of Glu or Asp occurs in the process. Astrocytes are known to express AAT in high activity (10, 19), so it is likely that similar aminotransferase-catalyzed isotope exchange events occur in intact cells. To the extent they do, rapid labeling of TCA cycle intermediates from labeled Glu exaggerates the calculated rate of net KG production and thus the need for alternatives to Gln for replenishing the neuronal Glu pool.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Glu/KG exchange. Enzyme-catalyzed partial reactions were characterized by determining whether AAT can transfer isotope between tritiated amino acid and its cognate keto acid in the absence of the substrate keto acid required for the full net reaction. Glu/KG exchange was studied at room temperature (20-25°C) by incubating 0.5 U AAT/ml with 50 µM [2,3-3H]Glu [0.5-1 × 105 disintegrations per minute (dpm)/ml] and 10-200 µM of unlabeled KG in a medium containing (in mM) 100 KCl, 10 NaCl, and 10 Tris · PO4 buffer (pH 7.0). Control experiments in which AAT was omitted were run in parallel. The total incubation volume was 0.1-1.0 ml depending on the number of samples to be taken. One unit of AAT is defined as the amount required to convert 1 µmol/min of KG to Glu at pH 7.5 and 37°C with Glu and OA present at saturating concentrations. Incubation with AAT was allowed for intervals from 5 to 120 min. Aliquots (100 µl) were taken from the incubation mixture at the times indicated, and enzyme activity was terminated by adding each sample to 100 µl of either 0.1 M NaOH or 1 mM aminooxyacetate (AOA), an AAT inhibitor (16). Enzyme-catalyzed transfer of the amino group from [2,3-3H]Glu to unlabeled KG produces KG labeled with tritium at C-3 and unlabeled Glu. Each 3H-labeled Glu molecule undergoing Glu/KG exchange liberates tritium at C-2 to the incubation medium as 3H2O. The amount of C-3 tritium present at C-3 of the keto acid was measured by adding NaOH to force tautomerization of keto acid to the enol form
Each keto-enol transition for a given molecule releases half of the C-3 tritium as 3H2O. Label loss after net conversion of [2,3-3H]Glu to [3-3H]KG proves that all keto acid C-3 3H is lost as 3H2O when NaOH is added. The amount of labeled keto acid was determined by the amount of "volatile" isotope produced by NaOH treatment and lost during subsequent sample evaporation.

After enzymatic activity was terminated, 100-µl samples were evaporated to dryness in a fume hood. The dry residue was dissolved in 200 µl of water, and tritium remaining in an aliquot was determined by liquid scintillation spectrometry (Beckman LS-200; 50% counting efficiency). Tritiated water in each sample at the time enzyme activity was stopped was calculated as the difference (in dpm) in evaporated samples compared with samples incubated without enzyme.

Although commercial preparations of tritiated Glu are produced as [2,3-3H]Glu, C-2 tritium is usually a low percentage of total label because of losses during storage by exchange with H+ in the solvent. The manufacturer characterized the preparation used here as having 8% of the total label at C-2. Evaporation of the initial tritiated stock resulted in loss of ~8% of the total dpm, suggesting that all of the original C-2 label may indeed have been lost, leaving tritium exclusively at C-3. To evaluate this possibility, the fraction of C-2 tritium was determined by cycling [2,3-3H]Glu through KG several times by the combined action of AAT and glutamic dehydrogenase (GDH), as shown by the following reaction sequence
50 &mgr;M [2,3<IT>-</IT><SUP>3</SUP>H]Glu<IT>+</IT>200<IT> &mgr;</IT>M OA <LIM><OP><IT>&cjs0807;→</IT></OP><UL>AAT</UL></LIM> [3<IT>-</IT><SUP>3</SUP>H]KG<IT>+</IT>Asp

[3-<SUP>3</SUP>H]KG<IT>+</IT>300<IT> &mgr;</IT>M NADH

<IT>+</IT>1 mM NH<SUP><IT>+</IT></SUP><SUB>4</SUB> <LIM><OP><IT>&cjs0807;→</IT></OP><UL>GDH</UL></LIM> [3<IT>-</IT><SUP>3</SUP>H]Glu<IT>+</IT>NAD<SUP><IT>+</IT></SUP>
The amount of Glu taken to KG and reconverted to Glu is dependent on the amount of OA available and was determined by spectrophotometric monitoring of the amount of NADH oxidized. The extent of cycling is dependent on the OA-to-Glu ratio provided that the NADH concentration exceeds that of OA. For the conditions indicated above, the amount of NADH oxidized shows that 3.2 times the initial amount of Glu is taken through a Gluright-arrowKGright-arrowGlu cycle before OA is expended as the system progresses toward equilibrium. KG remains at a low level throughout the incubation because the GDH equilibrium constant (Keq = 6.9 × 105 at pH 7) highly favors Glu formation (4). The only label lost after evaporation of samples taken from these experiments is released either from C-2 of the tritiated Glu sample or from the small amount of KG present after the GDH reaction is completed. GDH converts 93% of KG back to Glu under these conditions, as determined by spectroscopic measurements of NADH oxidation with 50 µM KG standards. Data shown in RESULTS indicate that little or no C-2 tritium is present in the labeled Glu and that volatile tritium originates solely from C-3 of KG.

The fraction of total label present in KG at isotopic equilibrium is equal to the mole fraction of KG. This value was used to calculate the theoretical limit to expected NaOH-induced label loss shown in Figs. 2, 3, 5, 6, and 7.
Maximal label loss<IT>=</IT>Fraction of <SUP>3</SUP>H in the KG pool

at equilibrium<IT>=</IT><FR><NU>[KG]</NU><DE>[Glu]<IT>+</IT>[KG]</DE></FR>
For samples stopped with AOA, the amount of 3H2O provides a measure of label released directly by the enzyme during Glu/KG exchange. Under these conditions, Glu/KG exchange results in loss of C-2 Glu tritium but C-3 tritium is retained in KG. In samples stopped with NaOH, the high pH of the treated sample forces KG to the enol form. Any KG labeled as a result of Glu/KG exchange loses its C-3 tritium as 3H2O during keto-enol tautomerization. The difference in the amount of label lost during evaporation of samples stopped with NaOH compared with that lost in samples stopped with AOA provides a measure of total tritium conveyed to the KG pool as a result of Glu/KG exchange. In some cases, KG in the sample was converted to Glu by adding 300 µM NADH, 1 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, and 0.1 U/ml of GDH before the addition of NaOH. Tritium is not released from C-3 of Glu by NaOH. All experiments were performed a minimum of three times with similar results in each trial. Data shown in Fig. 2 are means ± SE; all other data are mean values of duplicate samples taken for each set of conditions. The range for each set of duplicates is typically ~3% (9 of 10 sample pairs), or smaller than the symbol size used in the figures.

In some experiments Glu was converted to KG on a net basis. In these cases, AAT was incubated with (in µM) 50 Glu, 230 OA, and 300 NADH. After different time intervals, 1 U/ml of malic dehydrogenase (MDH) was added. The amount of OA remaining at the time of MDH addition was determined from the decrease in optical density at 340 nm due to NADH oxidation as OA is converted to malate (Mal). This approach provides a means of determining the fraction of [3H]Glu converted to labeled KG as a function of time and allows a comparison to the fraction of label that can then be released from KG by adding NaOH.

Asp/OA exchange. Isotope exchange associated with this partial reaction was evaluated by procedures fundamentally similar to those described in Glu/KG exchange. In this case, AAT was incubated at room temperature with 50 µM [2,3-3H]Asp (0.5-1.0 × 105 dpm/ml) and 10-200 µM of unlabeled OA. KG and Glu were absent. Again, reactions were stopped with NaOH to determine the amount of label released during the exchange process and by enolization of the [3H]OA product. The amount of tritium at C-2 of the tritiated Asp was evaluated by net conversion of Asp to Mal by incubating (in µM) 50 [2,3-3H]Asp, 250 KG, and 300 NADH with 0.5 U/ml AAT and 1 U/ml MDH. Parallel experiments in which NADH oxidation was monitored in a spectrophotometer allowed determination of the total amount of Asp converted to OA under these conditions.

Materials. [2,3-3H]Glu and [2,3-3H]Asp were purchased from New England Nuclear. AAT, GDH, and MDH were purchased from Sigma-Aldrich (St. Louis, MO) and stored at 4°C until used. All other chemical reagents were purchased from Sigma-Aldrich at the highest purity available.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Aminotransferases catalyze transfer of an amino group from an amino acid donor to a keto acid acceptor via a pyridoxal cofactor covalently bound to the enzyme (21, 24). A pyridoximine intermediate formed during the reaction sequence from the original amino group remains bound to the enzyme. Formation of the intermediate results in conversion of amino acid to its cognate keto acid, the first product of the complete net reaction. The reaction mechanism is "ping-pong" in that the cognate keto acid is released from the enzyme before binding of the second substrate (also a keto acid) required for the full reaction sequence. After binding the substrate keto acid, the amino group is transferred from the enzyme-pyridoximine intermediate to produce a new amino acid as the second product of the reaction. This transfer results in concomitant restoration of the pyridoxal form of the free enzyme. Mechanisms of this type are designated as ping-pong because the enzyme shifts back and forth between two different catalytic forms during the full reaction sequence. The net reaction and a schematic representation of the ping-pong mechanism for the full sequence catalyzed by AAT are


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where Enz is enzyme. Ping-pong reaction mechanisms always involve "partial" reactions related to the formation and subsequent expenditure of an enzyme intermediate. If the free energy change for a partial reaction is moderate in magnitude, so that unidirectional forward and reverse rates are similar and sufficiently rapid, significant bidirectional fluxes will occur between one substrate and its product in the absence of the second substrate and product. No net interconversion of substrate and product occurs except that due to formation of the tiny amount of pyridoximine-enzyme intermediate necessary to support the steady-state partial reaction sequence. Two partial reactions can be predicted for the AAT reaction mechanism---one between Glu and KG and another between Asp and OA. Occurrence of either partial reaction results in loss of a hydrogen atom from C-2 of the amino acid to the aqueous medium, as shown schematically for the Glu/KG partial reaction (absence of OA and Asp)
and the Asp/OA partial reaction (absence of KG and Glu)
If partial reactions indeed occur for AAT, they may be detected by the formation of 3H2O from an amino acid substrate tritiated at C-2. As shown above, if the amino acid is tritiated at both C-2 and C-3, the keto acid product retains the C-3 tritium. Because amino acid C-2 tritium is usually limited in amount because of spontaneous losses during storage, the degree to which either partial reaction has occurred is better determined by the amount of tritium in the keto acid pool as described in METHODS.

The degree to which AAT catalyzes a Glu/KG partial reaction was evaluated by incubating the enzyme with 50 µM [2,3-3H]Glu and either 50 or 200 µM of unlabeled KG. As shown in Fig. 1, the amount of tritium retained after sample evaporation decreases as a function of time and in a manner that is dependent on the amount of KG present. Much more label is released when samples are treated with NaOH instead of AOA, showing that most of the volatile tritium was released by enolization of the KG pool rather than by the enzyme during the exchange process.


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Fig. 1.   Aspartate aminotransferase (AAT)-catalyzed isotope exchange between 3H-labeled glutamate (Glu) and unlabeled alpha -ketoglutarate (KG). Loss of tritium as a function of time after incubation of 0.5 U/ml AAT at room temperature (23°C) with 50 µM [2,3-3H]Glu and 50 or 200 µM unlabeled KG is shown. Enzyme activity was stopped at the indicated time by adding 1 M NaOH in 3 trials and with aminooxyacetate (AOA), an aminotransferase inhibitor, in the remaining trial as indicated. AOA was added at the start of 1 experiment to inhibit AAT, and NaOH was added at the indicated times as a means of detecting any base-induced loss of tritium from [3H]Glu not due to enzyme-catalyzed isotope exchange into KG. The data for each time course are representative of 3 experiments with similar results in each case. Each data point shown here and in Figs. 3-7 is the mean value for duplicate samples taken from the same experiment. Range for the duplicates typically is <3%, or smaller than the symbol size used. dpm, Disintegrations per minute.

Figure 2 shows the amount of tritium remaining after a 60-min incubation interval when 50 µM [3H]Glu was incubated with six different concentrations of unlabeled KG. Total tritium loss is markedly dependent on the size of the KG pool, as suggested by the results shown in Fig. 1. The maximal tritium loss expected for each KG concentration is also shown in Fig. 2. These values were calculated from the amount of label expected in the KG pool if isotopic equilibrium had been achieved between Glu and KG. After a 60-min incubation interval, the tritium loss observed for each KG concentration was ~80% of the calculated maximal value.


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Fig. 2.   Base-induced tritium loss as a function of concentration of unlabeled KG after a 60-min incubation to allow Glu/KG isotope exchange. The incubation mixture included 50 µM [2,3-3H]Glu and 0.5 U/ml AAT. Enzyme activity was stopped by addition of 1 M NaOH before evaporation of samples. Each data point is the mean ± SE for 3 separate experiments. The dashed line shows the theoretical limit of the expected tritium loss, assuming that NaOH liberates all isotopic label in the KG pool and that isotopic equilibrium was achieved between Glu and KG.

In experiments similar to those summarized in Figs. 1 and 2, NADH and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> were included in the incubation mixture. After 60 min of incubation to permit isotope exchange to the KG pool, GDH was added to some of the samples and incubation continued for another 40 min to allow conversion of any labeled KG to Glu. Samples taken when only AAT is present lose 70% of the total label after NaOH treatment and evaporation. After GDH treatment, nearly all of the tritium is "protected" by conversion of KG to Glu and was retained after NaOH and evaporation as shown in Fig. 3. When both AAT and GDH were present at the start of the experiment, only 7% of the total label was lost after the 100-min incubation interval, the same amount as when GDH was added after 60 min.


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Fig. 3.   Isotope exchange between 50 µM [3H]Glu and 200 µM unlabeled KG followed by net conversion of [3H]KG to [3H]Glu by 1 U/ml glutamic dehydrogenase (GDH). GDH (open circle ) protected virtually 100% of the tritium otherwise lost after NaOH treatment and evaporation when only AAT was present (). When AAT and GDH were both present from the start of the experiment (black-triangle), 7% of the total tritium was still lost, indicating that a small pool of KG may not have been converted to Glu and was still available for supporting isotope exchange.

Figure 4 shows results obtained when tritiated Glu was converted to KG on a net basis by incubating AAT with 50 µM Glu and 230 µM OA. The amount of OA remaining after various incubation intervals, determined from the optical density change after MDH was added to each sample, showed that 41.5 µM OA was utilized after an 80-min incubation, indicating that an equal amount of Glu (83%) was converted to KG during that time. The percentage of label lost after NaOH treatment after various time intervals was determined in parallel experiments under identical conditions but with [2,3-3H]Glu present. The percentage of label lost during evaporation is approximately equal to the percentage of Glu converted to KG in each sample, as shown.


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Fig. 4.   Time dependence and % net conversion of [3H]Glu to [3H]KG during incubation of 0.5 U/ml AAT with 50 µM [2,3-3H]Glu and 250 µM oxaloacetate (OA; left axis). The % of tritium remaining in nonvolatile form after NaOH treatment is shown on right axis. The amount of Glu converted to KG is equal to the amount of OA consumed in the net AAT reaction, i.e., the difference between initial OA and residual OA at the time the sample was taken. Residual OA was determined from the change in optical density after 1 U/ml malic dehydrogenase (MDH) was added to the reaction mixture. The reaction mixture included 300 µM NADH.

In another approach, Glu was converted to KG and then cycled back to Glu by incubating 50 µM [2,3-3H]Glu with AAT and GDH in the presence of 200 µM OA, 300 µM NADH, and 1 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP> as described in METHODS. The decrease in NADH showed that 3.2 times the initial amount of Glu was cycled from Glu to KG and then back to Glu before OA became limiting. Despite the high degree of Glu turnover with both enzymes present, only 5% of the total tritium was lost after treatment of the final incubation mixture with NaOH and subsequent evaporation (Fig. 5). This loss probably is C-3 tritium released from the small amount of KG not cycled back to Glu rather than from C-2 of the [3H]Glu substrate (see DISCUSSION). When only AAT was present to allow net KG formation, ~70% of the total tritium was lost.


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Fig. 5.   Net conversion of tritiated Glu to tritiated KG followed by GDH-catalyzed reconversion of tritiated KG back to Glu. Tritium remaining in nonvolatile form after evaporation of NaOH-treated samples is shown as a function of time during incubation of 0.5 U/ml AAT with 50 µM [2,3-3H]Glu, 200 µM OA, 300 µM NADH, and 1 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. When only AAT was present during the 2-h incubation interval, 65% of the isotope was converted to 3H2O after NaOH treatment and lost during subsequent evaporation. When GDH was added after 60% of the isotope had been converted to a potentially volatile form, but before NaOH was added to stop enzymatic activity, the label was progressively "protected" from later release by NaOH. When AAT and GDH were both present at the outset, only 4% of the label was released by NaOH treatment. GOT, glutamate/oxaloacetate transaminase.

Experiments similar to those described above show that AAT also catalyzes isotope exchange between Asp and OA. When AAT was incubated with 50 µM [2,3-3H]Asp and variable amounts of unlabeled OA, the amount of NaOH-induced label loss was a function of the Asp-to-OA ratio, as shown in Fig. 6. If MDH was added after allowing 60 min of isotope exchange with 200 µM OA, much of the label otherwise released by NaOH was protected, because of conversion of [3H]OA to [3H]Mal. However, 24% of the total label was still lost during evaporation, as shown. In some cases, AAT was incubated with 50 µM [3H]Asp, 250 µM KG, and MDH so that [3H]OA produced from Asp by AAT was converted immediately to Mal. Under these circumstances, ~25% of the total label also is lost after treatment with NaOH and evaporation, as shown in Fig. 7. Net conversion of Asp to OA and the Asp/OA exchange reaction are blocked by AOA.


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Fig. 6.   AAT-catalyzed isotope exchange between 50 µM [3H]Asp and unlabeled OA as a function of OA concentration. In one case, MDH was added before NaOH treatment but after 75% of the Asp tritium was shifted to OA where it is subject to base-induced release. Two-thirds of the potentially releasable label was protected from release during subsequent NaOH treatment. Lack of protection for the other one-third indicates that ~24% of the total tritium is at C-2 of the [2,3-3H]Asp. The dotted line shows the theoretical limit for 3H2O formation expected if isotopic equilibrium had been achieved before NaOH treatment, after correction for the loss of C-2 tritium. The reaction medium contained 300 µM NADH.



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Fig. 7.   Net conversion of tritiated Asp to tritiated OA followed by MDH-catalyzed conversion of tritiated OA to malate. Tritium remaining in nonvolatile form after evaporation of NaOH-treated samples is shown as a function of time during incubation of 0.5 U/ml AAT with (in µM) 50 [2,3-3H]Asp, 250 KG, and 300 NADH (). MDH added after 60 min protected over half of the tritium otherwise released by NaOH, but 24% remained releasable (open circle ). When AAT and MDH were present from the start (black-triangle), 22% of the label was lost after NaOH and evaporation. The top dashed line is the average of 9 samples taken over 90 min but with AOA present from the start of incubation (triangle ). The bottom dashed line is the loss expected from the amount of OA produced from Asp, determined by spectroscopic measurement, after correction for the calculated tritium loss from C-2. The difference between this value and the observed label loss relates to MDH "pulling" the AAT reaction toward completion more fully than when only transaminase is present.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented here show that AAT catalyzes two partial reactions in addition to the two-substrate, two-product net reaction usually considered. Each partial reaction leads to isotope exchange between an amino acid substrate and its cognate keto acid, under conditions where net interconversion of the two reactants cannot occur. Release of label in volatile form (3H2O) after NaOH-induced enolization of keto acid is a rapid and sensitive method for detecting each partial reaction. When the full net reaction is allowed, the percentage of [3H]Glu converted to KG proves to be the same as the percentage of total tritium released after evaporation of NaOH-treated samples (Fig. 4), showing that base releases tritium from KG quantitatively. Production of volatile tritium requires AAT, is time dependent, and is inhibited by AOA, an aminotransferase inhibitor (Fig. 1). Nearly all of the tritium released in Fig. 1 is dependent on NaOH addition, showing that little releasable tritium came from C-2 of Glu. This was confirmed by experiments in which tritiated KG was converted to [3H]Glu with GDH. In one case, labeled KG was produced by exchange with tritiated Glu (Fig. 3). In the other, [3H]Glu was cycled three or four times on a net basis to KG and then back to Glu (Fig. 5). Only 5-7% of the total label was lost in each case. Both experiments show that strong base does not induce tritium release from amino acid. The cycling experiment in particular indicates that no more than 5% of tritium in the original [2,3-3H]Glu could have been at C-2.

It is important to recognize that, in both experiments, a small pool of KG remains after GDH action that may explain the small amount of label lost after NaOH treatment. The Keq for AAT is ~1.0, and the reported Keq for Glu formation from KG by GDH is 6.9 × 105 at pH 7.0 (4). With these values and the initial concentrations of Glu, OA, NADH, and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> present in the experiment shown in Fig. 5, it can be calculated that <1% of the initial 50 µM Glu would be present as KG if both reactions achieve equilibrium. Spectrophotometric measurement of NADH oxidized during the 60-min incubation interval shows that the reaction actually reaches 93% of the calculated equilibrium value. The 5-7% label loss in the experiments shown in Figs. 3 and 5 therefore can be explained by base-induced loss of C-3 tritium from the small pool of labeled KG remaining in the incubation mixture because of the lack of full equilibrium for GDH. We conclude that virtually all the volatile tritium produced from Glu because of Glu/KG exchange results from base-induced enolization of [3H]KG, with consequent release of C-3 tritium.

As shown in Figs. 2 and 6, isotope exchange is dependent on relative pool sizes of the amino/keto acid pair. Figures 2, 3, and 5 include a comparison of the observed fraction of tritium released during sample evaporation and the maximum fraction predicted to be in KG. As shown, the approach to equilibrium is dependent on the time allowed for the exchange process and on the KG-to-Glu ratio. For the AAT concentration used (0.5 U/ml), approximate isotopic equilibrium is established in 60 min at room temperature when Glu and KG are present at 50 and 10 µM, respectively. As the concentration of KG is raised, more total tritium is transferred to the keto pool but longer incubation times are required to approach equilibrium. At 500 µM KG, isotope in the KG pool after 1 h is ~80% of the calculated equilibrium value.

Although label in volatile form is routinely detected after NaOH treatment to induce release of C-3 tritium, it is also possible that some label originates from spontaneous enolization of keto acid before addition of base. Tautomeric tritium loss is probably modest in magnitude because of the very low percentage of enol KG expected at pH 7.0. Nevertheless, if rate constants are high for the keto-enol transition, some C-3 label could be lost. If appreciable tautomerization occurs at pH 7.0, then label will remain in the incubation medium as 3H2O after conversion of labeled KG to Glu and be lost during sample evaporation. As described above, the 5-7% tritium loss after sample treatment with GDH probably originates from NaOH-induced enolization of the small amount of KG (~3.5 µM) remaining after enzyme treatment. Base-induced tritium release from this small KG pool fully accounts for the observed tritium loss and implies that tautomeric label release due to spontaneous tautomerization at pH 7 is negligible.

Figures 6 and 7 show that AAT also catalyzes an Asp/OA exchange. Exchange between 50 µM [3H]Asp and 200 µM unlabeled OA results in release of ~75% of the total label after 60 min.

However, if MDH is then added to convert labeled OA to MAL, only two-thirds of the volatile label present before MDH is protected (Fig. 6), in contrast to the case with Glu, where GDH added after [3H]Glu/KG exchange protects nearly all tritium from NaOH-induced loss. The 24% label loss after MDH treatment could be due to spontaneous tautomerization of OA occurring more readily than for KG. Insight into this possibility is provided in Fig. 7. In this case, net conversion of [3H]Asp to OA by incubation of AAT with 250 µM unlabeled KG resulted in loss of 59% of the total label. Spectroscopic data showed that 60% of the 50 µM Asp was converted to OA if only AAT was present, in good agreement with the fraction of label lost. When MDH is then added, it "pulls" the Asp right-arrow MAL conversion 99% to completion. After MDH is added at 60 min, the base-induced tritium loss is still 25%. The same loss (24%) is observed if both AAT and MDH are present from the start of the experiment, despite the fact that the OA pool is much smaller in this case during the entire incubation interval. The data are consistent with a substantial percentage of total label in C-2 of [3H]Asp (unlike the case for [3H]Glu), with no spontaneous tautomerization of either KG or OA.

It should be noted that AAT can also catalyze a direct exchange of C-3 tritium with solvent H+ under certain circumstances. Nuclear magnetic resonance (NMR) and proton magnetic resonance (PMR) studies show that C-3 deuterium/hydrogen exchange occurs when AAT is incubated in 2H2O with 100-200 mM Glu and 0.1-2 mM KG (2, 6). The exchange probably results from polarity of an imine intermediate formed by the enzyme that influences 3H lability at the adjacent C-3. Rearrangement of the initial imine to an enamine is thought to be facilitated by transfer of a C-3 hydrogen atom (as H+) to a basic group in close proximity on the enzyme. If the substrate proton extracted by the enzyme exchanges with a solvent deuteron, reversal of the imine-enamine transition allows deuterium incorporation to the pyridoxal-bound carbon skeleton that can either be converted back to Glu or appear as [2H]KG if the keto acid is released by the enzyme. With Glu as substrate, a small pool of KG in the medium is mandatory for allowing NMR detection of H/2H exchange, even though the required enzyme-bound intermediates could be expected to originate solely from Glu. KG probably prevents rapid dissociation of KG derived from the Glu substrate, allowing the enzyme to sustain a significant titer of bound [3H]KG in equilibrium with the 3H-enamine intermediate required for H/2H exchange. Exchange of C-3 tritium due to equilibria among imine-enamine enzyme intermediates is especially pronounced for alanine aminotransferase (2, 6).

It is important to recognize that the mechanism of aminotransferase-catalyzed H/2H exchange with solvent described above allows H+ (or tritium) release from C-3 of amino acid substrates as a direct result of enzyme activity (36). However, once tritium is released by this process, it would not be restored to the substrate by reversal of the AAT reaction or by action of another enzyme because of 3H dilution in the massive H+ pool represented by aqueous solvent. Our results clearly show that conversion of KG to Glu by GDH, or of OA to MAL by MDH, prevents formation of volatile label that otherwise is released by NaOH. The NaOH-induced label release must originate from the keto acid pool, rather than directly from 3H/solvent exchange at C-3 via an enzyme-bound intermediate.

As might be expected, the magnitude of C-3 H exchange with solvent is highly dependent on enzyme concentration. Substrate/solvent H/2H exchange observed by NMR occurs at enzyme concentrations 40- to 80-fold higher (20-40 U/ml) than those used here and with amino acid concentrations (100-200 mM) more than 100-fold higher (2, 6). The moderate enzyme and substrate concentrations used in our experiments prevent C-3 tritium release directly by the enzyme from making a significant contribution, relative to the rate of tritium exchange between the Glu and KG pools. Tritium release from an enzyme intermediate as the basis of 3H2O formation would be detected after stopping the enzyme activity with AOA. The tritium release as 3H2O described here does not occur if the AAT reaction is stopped with AOA; hence it is primarily (if not entirely) a result of release from the KG pool by strong base. Together, the data are consistent with an active isotope exchange between substrate and cognate keto acid, with little or no isotope release from enzyme-bound intermediate(s).

Glu/Gln shuttle. The Glu/KG partial reaction of AAT described here raises significant questions regarding some of the challenges raised against the Glu/Gln shuttle hypothesis. Most of those challenges call attention to the fact that astrocytes incubated with isotopically labeled Glu produce a wide variety of labeled TCA cycle and related metabolic intermediates. The rapidity of formation of these products has been interpreted as indicative of substantial net Glu metabolism by astrocytes rather than conversion to Gln and subsequent return of carbon in that form to glutamatergic neurons (9, 14, 32, 33, 39, 40). One study showed that astrocytes convert uniformly labeled-[13C]Glu to Glu and Gln labeled at carbons 1, 2, and 3 (33). This labeling pattern can be explained if part of the uniformly labeled KG product produced by AAT makes one turn of the TCA cycle and is then converted back to Glu either by GDH or AAT. The observation was interpreted as a partial restoration of Glu that had been converted to KG on a net basis, after one circuit through the cycle. Thus actual net Glu loss was less than that otherwise calculated from the amount of 14CO2 produced from [1-14C]Glu, which loses all its 14C label at KG dehydrogenase. The original interpretation of the data concluded that some net consumption of Glu occurs, even after correction for reconversion of KG to Glu.

Hassel and Brathe (12, 13) suggested that the apparent need for a carbon cycle is debatable because neurons can incorporate label from 14CO2 into Glu. They proposed that malic enzyme (ME) may allow conversion of pyruvate to Mal that is then converted to Glu via TCA cycle activity without a requirement for pyruvate carboxylase. However, enzymes that catalyze carboxylation events typically require biotin as cofactor, which is important to provide sufficient affinity for the CO2 substrate. ME is not a biotin enzyme. Our own observations indicate that ME produces pyruvate, not Mal. At physiological concentrations of Mal and pyruvate, >95% of the Mal present is converted to pyruvate with concomitant NADPH production, rather than formation of Mal and NADP. This is consistent with the conventional view that ME functions to produce NADPH to support reductive biosynthetic events.

AAT-catalyzed isotope exchange raises another explanation for the wide distribution of label among metabolites that is observed after incubating astrocytes with labeled Glu. The Glu/KG partial reaction can transfer labeled carbon from Glu to KG. All TCA cycle intermediates produced subsequently will be labeled, but this implies no net consumption of Glu. Moreover, isotope exchange is a two-way street. Once isotope is distributed among TCA cycle intermediates, formation of [1,2,3-13C]Glu from the original UL-[13C]Glu may imply nothing more than an ongoing exchange cycle that transfers isotope from differentially labeled KG back to Glu that now will gain the same labeling pattern as the KG involved in the exchange process. The initial partial reaction can allow rapid formation of labeled metabolic intermediates from labeled Glu with no net Glu oxidation. The second exchange event allows production of [1,2,3-13C]Glu from UL-[13C]Glu but would not imply net Glu synthesis. To the extent that any isotope exchange occurs, there is less net Glu oxidation than would otherwise be concluded from the distribution of isotope in metabolic intermediates. Labeling patterns alone do not provide insight into the magnitude of net Glu consumption or production.

Furthermore, astrocytes may catalyze net synthesis of Glu from KG via GDH activity under the same conditions in which simultaneous AAT-catalyzed isotope exchange allows formation of labeled metabolites. It is important to remember that equilibrium for the reaction catalyzed by GDH lies greatly in favor of Glu formation. At pH 7.4, the equilibrium ratio of Glu to KG established by GDH ranges from a few hundred- to a few thousandfold, depending on the NH<UP><SUB>4</SUB><SUP>+</SUP></UP> concentration and the NADH-to-NAD ratio sustained in the mitochondria. It is important to note that glucose was present in all of those studies in which [13C]Glu was used to study isotope distribution in metabolites. Astrocytes express pyruvate carboxylase (31, 43), the anaplerotic enzyme necessary for providing net synthesis of KG from glucose. These cells therefore have the capacity for net Glu production, even while exchange events cause isotope distribution in metabolites that would suggest net Glu metabolism. When glucose is present, the pool size of TCA cycle intermediates and other metabolites may convey more information regarding glucose metabolism than that of Glu. Accurate interpretation of metabolism from isotope distribution data is particularly complex and difficult if AAT-catalyzed partial reactions occur concomitantly.

The rate of AAT in vivo is believed to be seven to eight times faster than the rate-limiting step for TCA cycle activity. This pronounced rate difference allows cellular pools of AAT reactants and products to be near thermodynamic equilibrium. Therefore, it is tempting to assume that the net AAT reaction allows isotope introduced to the cellular Glu pool to achieve rapid isotopic equilibrium with the KG pool. However, the steady-state KG pool is ~20-fold larger than that for KG. Net AAT activity is limited by OA availability such that complete turnover of the entire steady-state OA pool delivers only a small amount of label from Glu to KG and then on to TCA cycle intermediates. In contrast, isotopic equilibrium due to the AAT partial reaction can label the KG pool ~20-fold faster. Indeed, label transfer by AAT from Glu to KG is nearly undetectable at physiological concentrations of OA compared with KG at its physiological concentration. We conclude that the Glu/KG partial reaction of AAT may be a much greater determinant of isotope incorporation from Glu to the TCA metabolite pool than the net AAT reaction with OA. Label incorporation to these metabolites must be interpreted with caution when assessing quantitative aspects of Glu metabolism. Otherwise, the role played by a Glu/Gln shuttle in the CNS carbon cycle may be underestimated.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-15365-27.


    FOOTNOTES

Address for reprint requests and other correspondence: G. A. Kimmich, Dept. of Biochemistry and Biophysics, School of Medicine and Dentistry, Univ. of Rochester, Rochester, NY 14642 (E-mail: george_kimmich{at}urmc.rochester.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 30, 2002;10.1152/ajpcell.00487.2001

Received 10 October 2001; accepted in final form 28 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amara, SG, and Kuhar MJ. Neurotransmitter transporters. Annu Rev Neurosci 16: 73-93, 1993[ISI][Medline].

2.   Babu, UM, and Johnston RB. Nuclear magnetic resonance studies of D2O-substrate exchange reactions catalyzed by glutamic pyruvic and glutamic oxaloacetic transaminases. Biochemistry 15: 5671-5678, 1976[ISI][Medline].

3.   Balazs, R, Machiyama Y, Hammond BJ, Julian T, and Richter D. The operation of the gamma-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro. Biochem J 116: 445-461, 1970[ISI][Medline].

4.   Bergmeyer, HU, Klotzsch H, Mollering H, Nelbock-Hochstetter M, and Beaucamp K. Methods of Enzymatic Analysis. New York: Academic, 1965, p. 967-1037.

5.   Choi, DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1: 623-634, 1988[ISI][Medline].

6.   Cooper, JL. Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange. J Biol Chem 251: 1088-1096, 1976[Abstract].

7.   Danbolt, NC. Glutamate uptake. Prog Neurobiol 65: 1-105, 2001[ISI][Medline].

8.   Dennis, SC, and Clark JB. The pathway of glutamate metabolism in rat brain mitochondria. Biochem J 168: 521-527, 1977[ISI][Medline].

9.   Farinelli, SE, and Niklas WJ. Glutamate metabolism in rat brain cortical astrocyte cultures. J Neurochem 58: 1905-1915, 1992[ISI][Medline].

10.   Fonnum, F. The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain. Biochem J 106: 401-412, 1968[ISI][Medline].

11.   Frandsen, A, and Schousboe A. Development of excitatory amino acid induced cytotoxicity in cultured neurons. Int J Dev Neurosci 8: 209-216, 1990[ISI][Medline].

12.   Hassel, B, and Brathe A. Cerebral metabolism of lactate in vivo: evidence for neuronal pyruvate carboxylation. J Cereb Blood Flow Metab 20: 327-336, 2000[ISI][Medline].

13.   Hassel, B, and Brathe A. Neuronal pyruvate carboxylation supports formation of transmitter glutamate. J Neurosci 20: 1342-1347, 2000[Abstract/Free Full Text].

14.   Hassel, B, Sonnewald U, and Fonnum F. Glial-neuronal interactions as studied by cerebral metabolism of [2-13C]-acetate and [1-13C]-glucose: an ex vivo 13C NMR spectroscopic study. J Neurochem 64: 2773-2782, 1995[ISI][Medline].

15.   Hogstad, S, Svenneby G, Torgner IA, Kvamme E, Hertz L, and Schousboe A. Glutaminase in neurons and astrocytes cultured from mouse brain; kinetic properties and effects of phosphate, glutamate, and ammonia. Neurochem Res 13: 383-388, 1988[ISI][Medline].

16.   John, RA, and Charteris A. The reaction of amino-oxyacetate with pyridoxal phosphate-dependent enzymes. Biochem J 171: 771-779, 1978[ISI][Medline].

17.   Kaufman, E, and Driscoll BF. Carbon dioxide fixation in neuronal and astroglial cells in culture. J Neurochem 58: 258-262, 1992[ISI][Medline].

18.   Lester, HA, Mager S, Quick MW, and Corey JL. Permeation properties of neurotransmitter transporters. Annu Rev Pharmacol Toxicol 34: 219-249, 1994[ISI][Medline].

19.   Magee, SC, and Phillips AT. Molecular properties of the multiple aspartate aminotransferases purified from rat brain. Biochemistry 10: 3397, 1971[ISI][Medline].

20.   Meldrum, B. Amino acids as dietary excitotoxins---a contribution to understanding neurodegenerative disease. Trends Pharmacol Sci 11: 379-387, 1990[ISI][Medline].

21.   Metzler, ED, Ikawa M, and Snell EE. A general mechanism for vitamin B6-catalyzed reactions. J Am Chem Soc 76: 648-652, 1954[ISI].

22.   Nicholls, DG. CNS Neuro-Transmitters and Neuromodulators: Glutamate. New York: CRC, 1995, p. 35-52.

23.   Olney, JW. Excitotoxic amino acids and neuropsychiatric disorders. Annu Rev Pharmacol Toxicol 30: 47-71, 1990[ISI][Medline].

24.   Oshima, T, and Tamiya N. Mechanism of transaminase action. Biochem J 78: 116-119, 1961[ISI].

25.   Patel, AJ, Weir MD, Hunt A, Tahourdin CSM, and Thomas DGT Distribution of glutamine synthetase and glial fibrillary acidic protein and correlation of glutamine synthetase with glutamate decarboxylase in different regions of the rat cerebral nervous system. Brain Res 331: 1-9, 1985[ISI][Medline].

26.   Rothman, JM, and Olney JW. Glutamate and the pathology of hypoxic/ischemic brain damage. Ann Neurol 19: 105-111, 1986[ISI][Medline].

27.   Rothman, S. Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J Neurosci 4: 1884-1891, 1984[Abstract].

28.   Rothstein, JD, Hoberg MDH, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, and Welty DF. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16: 675-686, 1996[ISI][Medline].

29.   Rothstein, JD, Martin L, Levey AI, Dykes-Hobert M, Jin L, Wu D, Nash N, and Kuncl RW. Localization of neuronal and glial glutamate transporters. Neuron 13: 713-725, 1994[ISI][Medline].

30.   Schousboe, A, Westergaard N, Waagepetersen HS, Larsson OM, Bakken IJ, and Sonnewald U. Trafficking between glia and neurons of TCA cycle intermediates and related metabolites. Glia 21: 99-105, 1997[ISI][Medline].

31.   Shank, RP, Bennett GS, Freytag SO, and Campbell GLM Pyruvate carboxylase: an astrocyte specific enzyme implicated in the replenishment of amino acid neurotransmitter pools. Brain Res 329: 364-367, 1985[ISI][Medline].

32.   Sonnewald, U, Westergaard N, Petersen SB, Unsgard G, and Schousboe A. Metabolism of [U-13C]-glutamate in astrocytes studied by 13C NMR spectroscopy: incorporation of more label into lactate than into glutamine demonstrates the importance of the tricarboxylic acid cycle. J Neurochem 61: 1179-1182, 1993[ISI][Medline].

33.   Sonnewald, U, Westergaard N, and Schousboe A. Glutamate transport and metabolism in astrocytes. Glia 21: 56-63, 1997[ISI][Medline].

34.   Tanaka, K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, Iwama H, Nishikawa T, Ichihara N, Hori S, Takimoto M, and Wada K. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276: 1699-1702, 1997[Abstract/Free Full Text].

35.   Van den Berg, CJ, and Garfinkel D. A simulation study of brain compartments: metabolism of glutamate and related substances in mouse brain. Biochem J 123: 211-218, 1971[ISI][Medline].

36.   Walter, U, Luthe H, Gerhart F, and Soling HD. Hydrogen exchange at the carbon of amino acids during transamination. Eur J Biochem 39: 395-403, 1975.

37.   Waniewski, RA, and Martin DL. Exogenous glutamate is metabolized to glutamine and exported by rat primary astrocyte cultures. J Neurochem 47: 304-313, 1986[ISI][Medline].

38.   Watase, K, Hashimoto K, Kano M, Yamada K, Watanabe M, Inoue Y, Okuyama S, Sakagawa T, Ogawa S, Kawashima N, Hori S, Takimoto M, Wada K, and Tanaka K. Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. Eur J Neurosci 10: 976-988, 1998[ISI][Medline].

39.   Westergaard, N, Sonnewald U, and Schousboe A. Release of alpha -ketoglutarate, malate and succinate from cultured astrocytes: possible role in amino acid neurotransmitter homeostasis. Neurosci Lett 176: 105-109, 1994[ISI][Medline].

40.   Westergaard, N, Sonnewald U, and Schousboe A. Metabolic trafficking between neurons and astrocytes: the glutamate glutamine cycle revisited. Dev Neurosci 17: 203-211, 1995[ISI][Medline].

41.   Whetsell, WO, and Shapira NA. Biology of disease---neuroexcitation, excitotoxicity and human neurological disease. Lab Invest 68: 372-387, 1993[ISI][Medline].

42.   Yu, AC, Schousboe A, and Hertz L. Metabolic fate of 14C-labeled glutamate in astrocytes in primary cultures. J Neurochem 39: 954-960, 1982[ISI][Medline].

43.   Yu, ACH, Drejer J, Hertz L, and Schousboe A. Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J Neurochem 41: 1484-1487, 1983[ISI][Medline].


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