The Glutamate Uptake System in Presynaptic Vesicles: Further Characterization of Structural Requirements for Inhibitors and Substrates

Noncyclic fluorine-substituted and cyclic analogs of glutamic acid were tested for their ability to inhibit glutamate uptake in isolated bovine presynaptic vesicles, in order to assess the specific structural requirements of the glutamate translocation system in the vesicle membrane. Cyclic analogs that permitted close interaction between the positive and negative charges of the glutamate molecule were effective inhibitors; maximum inhibitory potency was observed with L-trans-1-aminocyclopentane-1,3-dicarboxylic acid (l-t-ACPD), while d-t-ACPD was less active. Analogs with a larger or smaller ring (as in trans-1-aminocyclohexane-1,3-dicarboxylic acid or trans-1-aminocyclobutane-1,3-dicarboxylic acid) were also inhibitory, but somewhat less so. trans-ACPD was also taken up by the vesicles with a time course and ATP dependence similar to uptake of glutamate, and this uptake was inhibited by glutamate. The K _m value for t-ACPD uptake was similar to its K _i for inhibition of glutamate uptake, while its rate of uptake was lower than that of glutamate. Fluorine-substituted noncyclic analogs with substitutions at the 4-carbon were less effective than glutamic acid itself, although 4,4-difluoroglutamic acid was equal in activity to the unsubstituted compound. Inhibition by these derivatives appeared to be competitive in nature, and they probably were also transported by the vesicle uptake system. Special issue article in honor of Dr. Frode Fonnum.     

Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum

The phototrophic green sulphur bacterium Chlorobium vibrioforme f. thiosulfatophilum assimilated ammonia via glutamine synthetase and glutamate synthase when grown with ammonia up to 30 mM, but above this level glutamate dehydrogenase was the key enzyme. Glutamine synthetase purified 42-fold was found to be adenylylated. The -glutamyltransferase activity of the enzyme was markedly inhibited by alanine, glycine, serine and lysine, and these amino acids in various combinations showed cumulative inhibition. Adenine nucleotides also inhibited enzyme activity, especially ATP. Glutamate synthase purified 222-fold had a maximum absorption at 440 nm which was reduced by sodium dithionite, and the enzyme was inhibited by atebrin indicating the presence of a flavin component. The enzyme had specific requirements for NADH, -ketoglutarate and l-glutamine, the K _m values for these were 13.5, 270 and 769 M respectively. Glutamate synthase was sensitive to feedback inhibition by amino acids, adenine nucleotides and other metabolites and the combined effects of these inhibitors was cumulative.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamic dehydrogenase     

β-Dl-Methylene-aspartate,an inhibitor of aspartate aminotransferase,potently inhibitsl-glutamate uptake into astrocytes

[³H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [³H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability.     

Absence of a role of gamma-glutamyl transpeptidase in the transport of amino acids by rat renal brushborder membrane vesicles

Summary The role of the enzyme, gamma-glutamyl transpeptidase on the uptake of amino acids by the brushborder membrane of the rat proximal tubule was examined by inhibiting it with AT-125 (l-[S, 5S]--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). AT-125 inhibited 98% of the activity of gamma-glutamyl transpeptidase when incubated for 20 min at 37°C with rat brushborder membrane vesicles. AT-125 given to ratsin vivo inhibited 90% of the activity of gamma-glutamyl transpeptidase in subsequently isolated brushborder membrane vesicles from these animals. AT-125 inhibition of gamma-glutamyl transpeptidase bothin vivo andin vitro had no effect on the brushborder membrane uptake of cystine. Similarly, there was no effect of gamma-glutamyl transpeptidase inhibition by AT-125 on glutamine, proline, glycine, methionine, leucine or lysine uptake by brushborder membrane vesicles. Furthermore, the uptake of cystine by isolated rat renal cortical tubule fragments, in which the complete gamma-glutamyl cycle is present, was unaffected by AT-125 inhibition of gamma-glutamyl transpeptidase. Therefore, in the two model systems studied, gamma-glutamyl transpeptidase did not appear to play a role in the transport of amino acids by the renal brushborder membrane.     

Uptake of amino acids by actidione-treated yeast cells

Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10⁻⁴ m for glycine, 2.5×10⁻⁴ m forl-cysteine, 6×10⁻⁵ and 4×10⁻⁴ m forl-lysine, 3×10⁻⁵ and 6×10⁻⁴ m forl-methionine, 7–18×10⁻⁵ and 1.6×10⁻³ m forl-aspartic acid and 6×10⁻⁵ and 2×10⁻³ m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan (and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine, arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Studies on the mode of action of the antibacterial agent diethyltin dichloride onEscherichia coli

The mode of action of diethyltin dichloride as a representative of the antibacterial dialkyl-substituted tin compounds was studied forEscherichia coli. The oxygen uptake by resting cells ofEicoli with glucose, lactate, pyruvate or glutamate as substrates is strongly inhibited by diethyltin dichloride at a concentration of 0.08mm which is still below the minimum inhibitory concentration for growth on a rich medium. Inhibition of oxygen uptake with glucose or lactate as substrates is accompanied by an increase in the amount of pyruvic acid accumulating. A slight rise in the concentration of -ketoglutaric acid results from the inhibition of succinate and malate oxidation by diethyltin dichloride, while inhibition of -keto acid production is obtained with glutamate as a substrate. Anaerobically, with glucose as a substrate, ethanol formation as well as the production of carbon dioxide and hydrogen are inhibited by diethyltin dichloride. In many respects the effects of diethyltin dichloride on metabolism resemble those of arsenite.The inhibition of pyruvate metabolism probably results in a lack of acetyl-CoA, and this is suggested to be a possible cause of growth inhibition.     

Location of amino acid and carbohydrate transport sites in the surface membrane of normal and transformed mammalian cells

Summary Amino acid and carbohydrate transport in normal and malignant transformed hamster cells was studied after binding of the protein Concanavalin A (Con. A) to the surface membrane. Experimental conditions were used so that a similar number of Con. A molecules were bound to both types of cells. The transport of amino acids was inhibited after Con. A binding in the transformed cells but not in normal cells. This was found with the metabolizable amino acidsl-leucine,l-arginine,l-glutamic acid, andl-glutamine, and with the non-metabolizable amino acids cycloleucine and -aminoisobutyric acid. Transport ofd-glucose andd-galactose was more inhibited by Con. A in transformed than in normal cells, and in both types of cellsd-glucose was inhibited more thand-galactose. The inhibition by Con. A on transport was specific, since there was no effect on the transport ofl-fucose in either normal or transformed cells. Con. A also did not effect the entry of 3-0-methyl-d-glucose.These observations can be used to locate amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A. The results indicate that Con. A sites are associated in normal cells with transport sites ford-glucose and to a lesser extentd-galactose, and in transformed cells with transport sites for amino acids and to a greater extent than in normal cells withd-glucose andd-galactose. Malignant transformation of normal cells therefore results in a change in the location of amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A.     

Studies on aspergilli. VI. The utilization of amino acids in mixtures by some ascosporic members of the aspergillus nidulans group

Summary The utilization of certain amino acids when supplied in three different combinations, (a) glycine, serine, valine, histidine (b) methionine, valine, alanine, arginine (c) leucine, tryptophane, glutamic acid, aspartic acid, byA. nidulans (Eidam)Wint;A. rugulosus Thom andRaper;A. variecolor (Berk. andBr.)Thom andRaper;A. quadrilineatus Thom andRaper andA. violaceus Fennel andRaper was studied through circular partition chromatography. It was found that these Aspergilli which are so closely related morphologically exhibited different rate of assimilation of amino acids. Amino acids in mixtures were utilized better than when supplied singly. Although different species had their own preference to certain amino acids yet there was a simultaneous utilization of both good and poor amino acids.     

Hypotaurine transport in brain slices

Hypotaurine uptake was compared to taurine and GABA uptakes in brain slices under identical experimental conditions. The slices effectively concentrated both hypotaurine and GABA from the medium, whereas taurine was taken up more slowly. The uptakes of these three structurally related amino acids were all saturable, consisting of one low-and one high-affinity transport component. The kinetic parameters of hypotaurine uptake were of the same order of magnitude as those of GABA uptake. All uptake systems were sensitive to temperature, metabolic poisons, and sodium omission. Hypotaurine uptake was inhibited by GABA,l-2,4-diaminobutyric acid (l-DABA), cysteic acid, and -alanine, but not by taurine. Taurine uptake was strongly reduced by hypotaurine, -alanine, andl-DABA, as well as by GABA, whereas GABA uptake was affected only by cystamine,l-DABA, and nipecotic acid.The uptake processes of hypotaurine, taurine, and GABA were thus fairly similar and showed properties characteristic for neurotransmitter uptake. Hypotaurine uptake resembled more GABA than taurine uptake. The present inhibition studies suggest that there may exist only one common two-component transport system for these three amino acids.     

Pyridoxal 5′-phosphate levels in brain after treatments which impair cerebral glucose metabolism

Pyridoxal 5-phosphate (PLP) concentrations were measured in brains of rats to determine whether a deficiency of this coenzyme was a common feature in hepatic coma, ethanol intoxication, and in animals treated withl-dopa or with 5-hydroxytryptophan (5-HTP) alone or with inhibitors of MAO or ofl-aromatic amino acid decarboxylase. These treatments have been shown previously to be associated with reduced conversion of glucose to amino acids in brain. Cerebral PLP concentrations were reduced after some of these treatments, notably injection of ethanol, orl-dopa alone or with -phenylisopropylhydrazine, an inhibitor of MAO, or of 5-HTP together withN-[-(chlorophenoxy)ethyl]cyclopropylamine hydrochloride, Lilly 51641, another MAO inhibitor. However, in other circumstances where inhibition of conversion of glucose to amino acids has been shown {treatment with 5-HTP, or with Lilly 51641 or with [N-(d,l-seryl)-N-2,3,4-trihydroxybenzyl]hydrazine, an inhibitor ofl-aromatic amino acid decarboxylase, together withl-dopa or with 5-HTP}, PLP levels in brain were unchanged, or were increased (in hepatectomized rats).     

Trichodesmium thiebautii; structure of a nitrogen-fixing marine blue-green alga (Cyanophyta)

Summary Trichodesmium thiebautii was collected, as floating bundles composed of uniseriate filaments aligned in parallel, from the Kuroshio waters off Shikoku Island, Japan. The ultrastructure of this alga had basically the same general features as the related speciesT. erythraeum first described byvan Baalen andBrown (1969). InT. thiebautii long electron dense fibers and concentrically lamellated bodies were observed which were either not reported previously, or did not occur inT. erythraeum. The peripheral wall layers were generally typical ofOscillatoria-type blue-green algae, but with a distinctive finely striated outer layer. Thylakoids per cell volume were very sparse compared to most other blue-green algae. Phycobilisomes, apparently hemidiscoidal in shape, typically occurred on the stromal side of the thylakoid surface. Large gas vesicle areas occupied the main volume of the cell, including cells which seemed to be actively growing. The gas vesicle areas were distributed throughout the cell, not only in the cell periphery as inT. erythraeum. Considerable complexity was suggested by the apparent cell compartmentation, particularly because the gas vesicle areas were delimited by one to several thylakoids. Only rarely were the gas vesicle areas traversed by thylakoids. Electron dense fibers (ca. 25 nm diameter) were always observed between the gas vesicles and were usually oriented parallel with them, but they were not rigid appearing as were the gas vesicles. The gas vesicles had a smaller diameter (ca. 45 nm) than most blue-greens. Concentrically lamellated bodies (ca. 1.0 m diameter) were observed in cells of some of the bundles. Each concentric layer was ca. 1.3 nm wide. These concentrically lamellated bodies may be characteristic of older cells. Cylindrical bodies were considerably smaller (ca. 120 nm diameter) and less complex than those reported forT. erythraeum.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

The nitrogen requirements ofGluconobacter,Acetobacter andFrateuria

The nitrogen requirements of 96Gluconobacter, 55Acetobacter and 7Frateuria strains were examined. Only someFrateuria strains were able to grow on 0.5% yeast extract broth or 0.5% peptone broth. In the presence ofd-glucose ord-mannitol as a carbon source, ammonium was used as the sole source of nitrogen by all three genera. With ethanol, only a fewAcetobacter strains grew on ammonium as a sole nitrogen source. Singlel-amino acids cannot serve as a sole source of carbon and nitrogen for growth ofGluconobacter, Acetobacter orFrateuria. The singlel-amino acids which were used by most strains as a sole nitrogen source for growth are: asparagine, aspartic acid, glutamine, glutamic acid, proline and alanine. SomeAcetobacter andGluconobacter strains deaminated alanine, asparagine, glutamic acid, threonine, serine and proline. NoFrateuria strain was able to develop on cysteine, glycine, threonine or tryptophan as a sole source of nitrogen for growth. An inhibitory effect of valine may explain the absence of growth on this amino acid. No amino acid is “essential” forGluconobacter, Acetobacter orFrateuria.     

Alanine and taurine transport by the gill epithelium of a marine bivalve: Effect of sodium on influx

Summary Marine mussels can accumulate amino acids from seawater into the epithelial cells of the gill against chemical gradients in excess of 5×10⁶ to 1. Uptake of both alanine and taurine into gill tissue isolated fromMytilus californianus was found to be dependent upon Na⁺ in the external solution. Uptake of these amino acids was described by Michaelis-Menten kinetics, and a reduction in external [Na⁺] (from 425 to 213mm) increased the apparent Michaelis constants (alanine, from 8 to 17 m; taurine, from 4 to 39 m) without a significant influence on theJ _max's of these processes. Fivemm harmaline, an inhibitor of Na-cotransport processes in many systems, reduced both alanine and taurine uptake by more than 95%; this inhibition appeared to be competitive in nature, with an apparentK _i of 43 m for the interaction with alanine uptake. Increasing the external [Na⁺] from 0 to 510mm produced a sigmoid activation of alanine and taurine uptake withK _Na's of approximately 325mm. The apparent Hill coefficients for this activation were 7.3 and 7.4 for alanine and taurine, respectively. These data are consistent with uptake mechanisms which require comparatively high concentrations of Na⁺ to activate transport, and which couple several Na⁺ ions to the transport of each amino acid. These characteristics, in conjunction with the previously demonstrated low passive permeability of the apical membrane to amino acids, result in systems capable of i) accumulating amino acids from seawater to help meet the nutritional needs of this animal, and ii) maintaining the high intracellular amino-acid concentrations associated with volume regulation in the gill.     

Analogue of aspartate and glutamate active at synapses are attractants forEscherichia coli

Summary 1. This research was carried out to compareEscherichia coli bacteria with animals in their response tol-aspartate andl-glutamate and their analogues.2. Various analogues of aspartate and glutamate known to be neurotransmitters at synapses were shown to be attractants forE. coli.3. The amino acid sequences of the animal receptors and the bacterial receptor, however, have no detectable relationship. Based on the amino acid sequence, evolutionarily the two systems appear not to be related.     

D-Amino acids of the amino acid pool and occurrence of racemase andD-amino acid oxidase activities inEscherichia coli B

Less than 20 % of the amino acid content of the amino acid pool ofEscherichia coli B exists in theD-form. Alanine, glutamic acid, and valine were shown by gas-chromatography to be partially in theD-form. OnlyD-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles butD-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid -D-amino acid - oxo acid which exists in the oxidation ofL-alanine does not exist with otherL-amino acids. It is likely that otherD-amino acids in the pool are formed in the presence ofD-amino acid oxidase orD-amino acid aminotransferase.     

Organization and expression inPseudomonas putida of the gene cluster involved in cephalosporin biosynthesis fromLysobacter lactamgenus YK90

TheLysobacter lactamgenus YK90pcbAB gene encoding -(l--aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is located immediately upstream of thepcbC gene in the same orientation in the gene cluster involved in cephalosporin biosynthesis. ThepcbAB gene encodes a large polypeptide composed of 3722 amino acid residues with a molecular mass of 411 593 Da. The predicted amino acid sequence has a high degree of similarity with those of known ACV synthetases from fungi and actinomycetes. Within thepcbAB amino acid sequence, three conserved and repeated domains of about 600 amino acids were identified. The domains also share a high degree of similarity with non-ribosomal peptide synthetases such as gramicidin synthatase 2 ofBacillus brevis. ThepcbAB gene was expressed under the control of thelac promoter inPseudomonas putida. Expression of the gene cluster involved in cephalosporin biosynthesis inP. putida led to the accumulation of -lactam antibiotics. Deletion analysis of an open-reading frame located between thecefE andcefD genes from the gene cluster revealed that it encoded deacetylcephalosporin C synthetase (cefF). From the results presented here and those of previous studies, the genes involved in cephalosporin biosynthesis inL. lactamgenus appear to be clustered in the orderpcb AB-pcbC- cefE-cefF-cefD-bla in the same orientation within a 17-kb region of DNA.     

Asymmetric hydrolysis of α-aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34

Summary Rhodococcus rhodochrous PA-34 isolated from soil as a propionitrile-utilizing microorganism, hydrolysed several -aminonitriles to optically active amino acids. The hydrolysis of -aminonitriles was found to be catalysed by a nitrilase. The characteristics of the purified enzyme revealed that this is a new nitrilase as it has a molecular mass of 45 kDa and acts as a monomer. The optimum pH and temperature for the activity of the purified enzyme were 7.5 and 35° C, respectively. Thiol-specific reagents caused inhibition whereas chelators did not significantly alter the activity of this enzyme. The amino acids produced were of L-form, except for alanine. In the case of leucine production from -aminoisocapronitrile, the enantiomeric ratio of L-leucine to D-leucine was about 60.     

Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons

Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM),d[³H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and ofd-[³H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously suppliedd-[³H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release ofd-[³H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased psuedospecific radioactivity of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of -ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed. This cytoplasmic origin of transmitter glutamate is in aggreement with the finding thatd-[³H] aspartate readily labels the transmitter pool even when synthesis of endogenous transmitter is impaired in the presence of AOAA or phenylsuccinate.Special issue dedicated to Dr Elling Kvamme