Biosynthetic origin of [R-(Z)]-4-amino-3-chloro-2-pentenedioic acid in Streptomyces viridogenes

The biosynthesis of the chlorinated amino acid [R-(Z)]-4-amino-3-chloro-2-pentenedioic acid (ACPA) was investigated. Feeding studies with Streptomyces viridogenes were conducted in resting cells. Substantial incorporation from [(15)N]- and [(13)C]-enriched glutamate and proline indicated that the biosynthetic origin of ACPA is one of these amino acids. Experiments with deuterated glutamate and proline imply that chlorination does not occur via a radical mechanism, but rather suggest that a FADH(2)-dependent halogenase is involved.     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.     

Phospholipase A2 Modulates Different Subtypes of Excitatory Amino Acid Receptors: Autoradiographic Evidence

Abstract: Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A₂ (PLA₂) on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA₂ pretreatment induced a significant increase in α-[³H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([³H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [³H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [³H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [³H]glutamate binding site were also increased by PLA₂ pretreatment. [³H]Kainate binding and NMDA-sensitive [³H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA₂ effect was reversed by EGTA, the PLA₂ inhibitor p-bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA₂. Arachidonic acid failed to mimic the PLA₂ effect on metabotropic binding. These results indicate that PLA₂ can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.     

Amino acid synthesis during hyperosmotic stress in penaeus aztecus postlarvae

• 1.1. Glycine, proline, and taurine are the quantitatively most important amino acid osmolytes in Penaeus aztecus postlarvae.
• 2.2. Taurine dominates the amino acid pool in low salinity, while proline dominates the amino acid pool at higher salinities.
• 3.3. Although not major contributors to the pool, glutamate and alanine are constitutively synthesized from [¹⁴C]glucose and [¹⁴C]glutamate under constant salinity and under hyperosmotic stress treatments.
• 4.4. Proline synthesis from [¹⁴C]-precursors is apparent under constant high (but not low) salinity and is significantly induced by hyperosmotic stress.
• 5.5. No appreciable glycine synthesis was observed from [¹⁴C]glucose or [¹⁴C]glutamate under any experimental conditions.

     

Effects of X-irradiation induced loss of cerebellar granule cells on the synaptosomal levels and the high affinity uptake of amino acids.

Abstract— Crude synaptosomal (P₂) preparations were obtained from the cerebella of rats in which the granule cell population had been selectively reduced by X-irradiation treatment and from the cerebella of control animals. In the P₂ fraction from control cerebella, the level of glutamate was greater than any other of the 5 amino acids measured and was 2-fold higher than taurine, which was present at the next highest level. The content of taurine was slightly higher than that found for aspartate and was 3-fold greater than that observed for GABA. Alanine and glycine were present in the lowest amounts. The levels of glutamate and aspartate were significantly (P < 0.05) lower by 25 and 15%, respectively, in the P₂ fraction isolated from the X-irradiated cerebella in comparison to control values. The content of taurine, GABA, glycine, and alanine were not changed by the X-irradiation treatment. The uptake of 1.0 μm -l -[³H]glutamate and l -[³H]aspartate was reduced approx 20% by X-irradiation treatment, whereas the uptake of 1.0 μm -[³H]GABA and [³H]taurine was unchanged. A more detailed kinetic analysis of l -[³H]glutamate uptake revealed there was a 20% decrease in the V_max value with X-irradiation treatment and no change in the apparent K_m value. In a second study, the uptake of l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was measured using P₂ fractions obtained from the cerebella of rats in which the population of granule, stellate and basket cells had been reduced by X-irradiation treatment. The uptake of 1.0μm -l -[³H]glutamate, l -[³H]aspartate and [³H]GABA was significantly (P < 0.05) reduced to 57, 68, and 59%, respectively, of control values. A more detailed kinetic analysis of [³H]GABA uptake revealed no significant change in the apparent K_m and a 35% decrease in the V_max value. The data are discussed in terms of glutamate being the excitatory neurotransmitter released from granule cells and GABA being the inhibitory neurotransmitter released from basket cells.     

Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids,arachidonic acid,and GABA

In rat brain slices the synthesis of [³H]phosphoinositides and the production of [³H]inositol monophosphate (IP₁) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [³H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A₂. Arachidonic acid caused a rapid and dose-dependent inhibition of [³H]phosphoinositide synthesis and of NE-stimulated [³H]IP₁ production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A₂ with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A₂ were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A₂. Further studies were carried out in slices that were prelabelled with [³H]inositol in an attempt to separate modulatory effects on [³H]phosphoinositide synthesis and agonist-stimulated [³H]IP₁ production. Several excitatory amino acid agonists inhibited NE-stimulated [³H]IP₁ production. This inhibitory inter-action could be due to impaired synthesis of [³H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [³H]IP₁ production, indicating that continuous regeneration of [³H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid AP₄ dl-2-amino-4-phosphonobutyric acid - BPB bromphenacyl bromide - BSA bovine serum albumin - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - DFMO -difluoromethylornithine - DIDS diisothiocyanotostilbene-2,2-disulfonic acid - EGTA ethyleneglycol-bis-N - N, N N-tetraacetic acid - GABA -aminobutyric acid - GDEE glutamate diethyl ether - -GG -glutamylglycine - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - NDGA nordihydroguaiaretic acid - NE norepinephrine - NMDA N-methyl-d-aspartate     

Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K⁺ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R-phenylisopropyladenosine. All these effects were reversed by the selective adenosine A₁ receptor antagonist 8-cyclo-pentyltheophylline. In the same synaptosomal preparation, K⁺ (30 mM) strongly stimulated the release of the preloaded [³H]adenosine in a partially Ca²⁺-dependent and tetrodotoxin (TTX)-sensitive manner. Moreover, in the same experimental conditions, both l -glutamate and l -aspartate enhanced the release of [³H]adenosine derivatives ([³H]ADD). The gluta-mate-evoked release was dose dependent and appeared to be Ca²⁺ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d -glutamate and d -aspartate were able to stimulate [³H]ADD release. In contrast, the specific glutamate agonists N-methyl-d -aspartate, kainate, and quisqualate failed to stimulate [³H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg²⁺ and l -glutamate-evoked release was not inhibited by adding the specific antagonists 2-amino-5-phosphonovaleric acid or 6–7-dinitroquinoxaline-2, 3-dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ-anunobutyric acid stimulated [³H]ADD release in a dose-related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.     

Influence of amino acids and organic antimetabolites on growth and biosynthesis of the chemoautotroph Thiobacillus neapolitanus strain C

Summary The growth of Thiobacillus neapolitanus strain C in liquid cultures was depressed by phenylalanine, p-fluorophenylalanine, cysteine, methionine, nor-leucine, azetidine-2-carboxylic acid, and chloramphenicol, but was little affected by glutamic acid, glycine, proline, azathymine, or oligomycin.Growing cultures assimilated ¹⁴C-labelled glycine, glutamic acid, phenylalanine, and tyrosine into protein. Tyrosine and phenylalamine were incorporated unchanged, but glutamate was used also for synthesis of arginine and proline. Glycine-¹⁴C contributed also to adenine and guanine synthesis. The extremely large amounts of phenylalanine incorporated into protein could indicate its toxicity to depend on its producing abnormal protein synthesis. Azetidine-2-carboxylic acid appeared to lower the amount of proline in the protein.Assimilation of glutamate and glycine by non-growing organisms was almost entirely dependent on energy from thiosulphate oxidation, thus suggesting a cause of obligate chemoautotrophy. Chloramphenicol specifically inhibited this thiosulphate-dependent incorporation of glutamate, glycine or CO₂ into protein at concentrations which did not affect total CO₂-fixation. Provided that energy is available from thiosulphate-oxidation this Thiobacillus is thus able to (a) activate exogenous amino acids; (b) incorporate them and CO₂ into protein by a chloramphenicol sensitive mechanism; (c) synthesise proline and arginine from glutamate; or adenine and guanine from glycine. Its biosynthesis thus depends on mechanisms like those of heterotrophs but requires to be driven by a chemolithotrophic energy supply.     

Glutamate metabolism in a human intestinal epithelial cell layer model

Plasma glutamate concentrations are constant despite dynamic changes in diets. Most likely, virtually all the dietary glutamate is metabolized in the gut. The present study investigated permeability and metabolism of dietary glutamate in a Caco-2 intestinal epithelial cell layer model by tracing the fate of [U-¹³C] or [¹⁵N]glutamate added to the apical medium. For comparison, several other labelled essential and non-essential amino acids were tested as well. Almost all the labelled glutamate in the apical medium (98% and 96% at 24 h of the culture, respectively) was incorporated in the cell layer, while it barely appeared at the basolateral side, indicating an almost complete utilization of glutamate. Indeed, the ¹³C was incorporated into alanine, proline, ornithine, and glutamine, and the ¹⁵N was incorporated into alanine, glutamine, ornithine, proline, branched chain amino acids and also found as ammonia indicative of oxidation. In contrast, substantial apical-to-basolateral transport of amino acids (8–85% of uptake) other than glutamate and aspartate was evident in studies using amino acid tracers labelled with ¹³C, ¹⁵N or D. These results suggest that the intestinal epithelial cell monolayer utilizes dietary glutamate which adds to maintaining glutamate homeostasis in the body.

     

Inhibition of yeast Δ-pyrroline-5-carboxylate dehydrogenase by common amino acids and the regulation of proline catabolism

A yeast glutamate auxotroph (glt_{1 − 1}), blocked in the tricarboxylic acid cycle at aconitase, is shown to possess catabolic pathways to glutamate from proline, arginine and glutamine, and grows on any of these amino acids in a minimal medium. This mutant does not, however, grow on these amino acids in a medium containing the full complement of common amino acids minus glutamate. The mechanism of this growth failure involves partial inhibition of the catabolic routes to glutamate by more than half the common amino acids. In the case of proline catabolism, this inhibition is localized principally at the enzyme Δ¹-pyrroline-5-carboxylate: NAD(P)⁺ oxidoreductase by in vitro studies. Similar results with this enzyme prepared both from yeast and from beef kidney mitochondria suggest that the inhibition observed may be the basis of a regulatory mechanism of general significance.     

Interactions Between the Glutamate and Glycine Recognition Sites of the N-Methyl-d-Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies

Abstract: The N-methyl-d -aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P₂ membranes we have investigated the effect of glutamate recognition site ligands on [³H]glycine (agonist) and (±)4-trans-2-car-boxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]l -689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l -[³H]glutamate (agonist), dl -3-(2-carboxypiperazin-4-yl)-[³H]propyl-1 -phosphonate ([³H]-CPP; “C-7” antagonist), and cis-4-phosphonomethyl-2-[³H]piperidine carboxylate ([³H]CGS-19755; “C-5” antagonist) binding to the glutamate site. “C-7” glutamate site antagonists partially inhibited [³H]l -689,560 binding but had no effect on [³H]glycine binding, whereas “C-5” antagonists partially inhibited the binding of both radioligands. Glycine, d -serine, and d -cycloserine partially inhibited [³H]CGS-19755 binding but had little effect on l -[³H]-glutamate or [³H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (l -687,414), and 1-amino-1-carboxycyclobutane all enhanced [³H]CPP binding but had no effect on [³H]CGS-19755 binding, and (+)-HA-966 and l -687,414 inhibited l -[³H]glutamate binding. The association and dissociation rates of [³H]l -689,560 binding were decreased by CPP and d -2-amino-5-phosphonopentanoic acid (“C-5”). Saturation analysis of [³H]l -689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [³H]l -689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [³H]l -689,560 is discussed.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

Abstract: CO₂ fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with ¹⁴CO₂ and the incorporation of ¹⁴C into medium or cell extract products was determined. After chromatographic separation of ¹⁴C-labelled products, the fractions of ¹⁴C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the ¹⁴C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO₂ fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [¹⁴C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net ¹⁴CO₂ fixation, but did alter the distribution of ¹⁴C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or ¹⁴C incorporation). Low rates of conversion of α-[¹⁴C]ketoglutarate to [¹⁴C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Compartmentation of citric acid cycle metabolism in brain: effect of aminooxyacetic acid, ouabain and Ca2+ on the labelling of glutamate, glutamine, aspartate and gaba by [1-14C]acetate, [U-14C]glutamate and [U-14C]asparate

—(1) The effects of aminooxyacetic acid, ouabain and Ca²⁺ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium-[1-¹⁴C]acetate, l-[U-¹⁴C]glutamate and l-[U-¹⁴C]aspartate as tracer metabolites. (2) Aminooxyacetic acid (10^-3 m) inhibited the labelling of aspartate from [¹⁴C]acetate and [¹⁴C]glutamate, as well as the incorporation of label from [¹⁴C]aspartate into glutamate and glutamine. It also inhibited the labelling of GABA from all three radioactive precursors, as would be anticipated if there was inhibition of several transaminases as well as glutamate decarboxylase. The RSA of glutamine labelled from [1-¹⁴C]acetate was increased. This finding indicated that the glutamate pool which is utilized for glutamine formation is associated with glutamate dehydrogenase, and this enzyme appears to be related to the ‘synthetic tricarboxylic acid cycle’. AOAA exerted its major inhibitory effects on the citric acid‘energy cycle’with which transaminases are associated. (3) Ouabain (10^-5 m) inhibited the labelling of glutamine to a much greater extent than the labelling of glutamate from [1-¹⁴C]acetate. It also caused leakage of amino acids from the tissue into the medium. Its effect on the glutamate–glutamine system was interpreted to be a selective inhibition of the 'synthetic’citric acid cycle. (4) The omission of Ca²⁺ from the incubation medium was associated with formation of glutamine with RSA less than 1·0 when labelled from [U-¹⁴C]glutamate, [U-¹⁴C]aspartate and lower than normal when labelled from [1-¹⁴C]acetate.