Nitrogen Assimilation in Mycorrhizas : Ammonium Assimilation in the N-Starved Ectomycorrhizal Fungus Cenococcum graniforme

Ammonium assimilation was followed in N-starved mycelia from the ectomycorrhizal Ascomycete Cenococcum graniforme. The evaluation of free amino acid pool levels after the addition of 5 millimolar NH₄⁺ indicated that the absorbed ammonium was assimilated rapidly. Post-feeding nitrogen content of amino acids was very different from the initial values. After 8 hours of NH₄⁺ feeding, glutamine accounted for the largest percentage of free amino acid nitrogen (43%). The addition of 5 millimolar methionine sulfoximine (MSX) to NH₄⁺-fed mycelia caused an inhibition of glutamine accumulation with a corresponding increase in glutamate and alanine levels.

Using ¹⁵N as a tracer, it was found that the greatest initial labeling was into glutamine and glutamate followed by aspartate, alanine, and ornithine. On inhibiting glutamine synthetase using MSX, ¹⁵N enrichment of glutamate, alanine, aspartate, and ornithine continued although labeling of glutamine was quite low. Moreover, the incorporation of ¹⁵N label in insoluble nitrogenous compounds was lower in the presence of MSX. From the composition of free amino acid pools, the ¹⁵N labeling pattern and effects of MSX, NH₄⁺ assimilation in C. graniforme mycelia appears to proceed via glutamate dehydrogenase pathway. This study also demonstrates that glutamine synthesis is an important reaction of ammonia utilization.

     

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.     

Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae

Nitrogenase biosynthesis in Klebsiella pneumoniae including mutant strains, which produce nitrogenase in the presence of NH₄⁺ (Shanmugam, K.T., Chan, Irene, and Morandi, C. (1975) Biochim. Biophys. Acta 408, 101–111) is repressed by a mixture of L-amino acids. Biochemical analysis shows that glutamine synthetase activity in strains SK-24, SK-28, and SK-29 is also repressed by amino acids, with no detectable effect on glutamate dehydrogenase. Among the various amino acids, L-glutamine in combination with L-aspartate was found to repress nitrogenase biosynthesis completely. In the presence of high concentrations of glutamine (1 mg/ml) even NH₄⁺ repressed nitrogenase biosynthesis in the strains SK-27, SK-37, SK-55 and SK-56. Under these conditions, increased glutamate dehydrogenase activity was also detected. Physiological studies show that nitrogenase derepressed strains are unable to utilize NH₄⁺ as sole source of nitrogen for biosynthesis of glutamate, whereas back mutations leading to NH₄⁺ utilization results in sensitivity to repression by NH₄⁺. These findings suggest that amino acids play an important role as regulators of nitrogen fixation.     

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

Amino acids and lactate efflux from in vitro incubated soleus muscles of suckling rats.

The net in vitro release of lactate, pyruvate, alanine, glutamate, glutamine and total amino acids from soleus muscles of suckling rats was studied. In all cases, the net output of metabolites was considerably higher in young animals than in 30 day old controls. Glutamine efflux of 1 day old pups was very low, probably due to lack of effectivity of the glutamine synthesizing system. The considerable output of amino acids in the younger animals was partly due to depletion of internal amino acid pools.     

Cerebral Cortex Ammonia and Glutamine Metabolism During Liver Insufficiency-Induced Hyperammonemia in the Rat

Hyperammonemia has been suggested to induce enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis and accumulation, and finally net glutamine release into the blood stream, but this has never been confirmed in liver insufficiency models. Therefore, cerebral cortex ammonia- and glutamine-related metabolism was studied during liver insufficiency-induced hyperammonemia by measuring plasma flow and venous-arterial concentration differences of ammonia and amino acids across the cerebral cortex (enabling estimation of net metabolite exchange), 1 day after portacaval shunting and 2, 4, and 6 h after hepatic artery ligation (or in controls). The intra-organ effects were investigated by measuring cerebral cortex tissue ammonia and amino acids 6 h after liver ischemia induction or in controls. Arterial ammonia and glutamine increased in portacaval-shunted rats versus controls, and further increased during liver ischemia. Cerebral cortex net ammonia uptake, observed in portacaval-shunted rats, increased progressively during liver ischemia, but net glutamine release was only observed after 6 h of liver ischemia. Cerebral cortex tissue glutamine, gamma-aminobutyric acid, most other amino acids, and ammonia levels were increased during liver ischemia. Glutamate was equally decreased in portacaval-shunted and liver-ischemia rats. The observed net cerebral cortex ammonia uptake, cerebral cortex tissue ammonia and glutamine accumulation, and finally glutamine release into the blood suggest that the rat cerebral cortex initially contributes to net ammonia removal from the blood during liver insufficiency-induced hyperammonemia by augmenting tissue glutamine and ammonia pools, and later by net glutamine release into the blood. The changes in cerebral cortex glutamate and gamma-aminobutyric acid could be related to altered ammonia metabolism.     

The Role of Ammonium Assimilating Enzymes in Lentil Roots and Nodules

Activities of ammonium assimilating enzymes glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as the amino acid content were higher in nodules compared to roots. Their activities increased at 40 and 60 d after sowing, with a peak at 90 d, a time of maximum nitrogenase activity. The GS/GOGAT ratio had a positive correlation with the amino acid content in nodules. Higher activities of AST than ALT may be due to lower glutamine and higher asparagine content in xylem. The data indicated that glutamine synthetase and glutamate synthase function as the main route for the assimilation of fixed N, while NADH-dependent glutamate dehydrogenase may function at higher NH₄ ⁺ concentration in young and senescing nodules. Enzyme activities in lentil roots reflected a capacity to assimilate N for making the amino acids they may need for both growth and export to upper parts of the plant. This revised version was published online in July 2006 with corrections to the Cover Date.     

GLUTAMINE UPTAKE AND METABOLISM BY THE ISOLATED TOAD BRAIN: EVIDENCE PERTAINING TO ITS PROPOSED ROLE AS A TRANSMITTER PRECURSOR

Abstract— Hemisections of toad brains, when incubated in a physiological medium containing no glutamine. released considerable amounts of this amino acid into the medium. When glutamine was included in the medium at a concentration of 0.2 mm the net efflux from the tissue was reduced but not totally prevented. Although there was no net uptake of glutamine, the tissue did accumulate [U-¹⁴C]glu-tamine and some of this labelled glutamine was rapidly metabolized to glutamate, GABA and aspartate. The precursor-product relationship for the metabolism of glutamine to glutamate differed from the classic single compartment model in that the specific radioactivity of glutamate rose very quickly to approx one-tenth that of glutamine, but increased slowly thereafter. These data suggest that the [¹⁴C]glutamine was taken up into two metabolically distinct compartments and/or that some of the [¹⁴C]glutamine was converted to [¹⁴C]glutamate during the uptake process. The uptake of [¹⁴C]glutamine was diminished when the tissue was incubated in a non-oxygenated medium or when Na⁺ was omitted (substituted with sucrose) and K⁺ was concomitantly elevated. However, on a relative basis, the incorporation of radioactivity into glutamate and GABA was increased by these incubation conditions. The metabolism of glutamine to aspartate was greatly depressed when the tissue was not oxygenated. The glutamate formed from [U-¹⁴C]glutamine taken up by the tissue was converted to GABA at a faster rate than was glutamate derived from [U-¹⁴C]glucose. [U-¹⁴C]gly-cerol or exogenous [U-¹⁴C]glutamate. This suggests that glutamine was metabolized to GABA selectively; i.e. on a relative basis, glutamine served as a better source of carbon for the synthesis of GABA than did glucose, glycerol or exogenous glutamate. When the brain hemisections were incubated in the normal physiological medium with or without glutamine. there was very little efflux of glutamate, GABA or aspartate from the tissue. However when NaCl was omitted from the medium (substituted with sucrose) and K⁺ was elevated to 29 miu. a marked efflux of these three amino acids into the medium did occur, and over a period of 160min, the content of each amino acid in the tissue was depleted considerably. When glutamine (0.2 mm ) was included in the Na⁺ deficient-high K.⁺ medium, the average amount of glutamate, GABA and aspartate in the tissue plus the medium was greater than when glutamine was not included in the medium. Such data indicate that CNS tissues can utilize glutamine for a net synthesis of glutamate, GABA and aspartate. The results of this study provide further evidence in support of the concept that the functional (transmitter) pools of glutamate and GABA are maintained and regulated in part via biosynthesis from glutamine. One specific mechanism instrumental in regulating the content of glutamate in nerve terminals may be a process of glutamine uptake coupled to deamidation.     

Pathway of ammonia assimilation in illuminated Lemna minor

Plants of duckweed (Lemna minor) were grown under constant illumination and with a controlled supply of ammonium-N so as to maintain a constant low concentration. In two kinetic experiments (differing in illumination and N level) with ¹⁵N-ammonia, plants were periodically harvested and their free amino acids analysed for ¹⁵N abundance. Attempts were then made to fit the data by computer simulation models. Only models which had at least two or more intracellular compartments gave adequate fits. Two two-compartment models were tested fully. Both had in compartment 1 the glutamine synthetase-glutamate synthase cycle and in compartment 2 a second site of glutamine synthesis. In one model the glutamate for compartment 2 was derived by transport from compartment 1; in the second model it was synthesized from ammonia by glutamate dehydrogenase at a rate equivalent to 10% of the total N uptake. This second model was rejected after it was found that plants previously treated with methionine sulphoximine and aza-serine (inhibitors of the glutamate synthase cycle) were unable to incorporate ¹⁵N. In spite of wide differences in labelling pattern between the two experiments the first model gave acceptable fits to both when different pool sizes were allowed for. Operation of the glutamate synthase cycle was confirmed by the correspondence between model and data for labelling of glutamine amide, glutamine amino and glutamic acid. Consideration of enzyme distributions suggested that compartment 1 (the glutamate synthase system) is the chloroplasts and compartment 2 the cytosol. Analysis of asparagine and neutral amino acids made it possible to construct balance sheets for N uptake in the two experiments. They suggest that all glutamine synthesized in the chloroplast is used for glutamate and asparagine synthesis and that the cytosol enzyme meets the need of the cell for glutamine per se. The high turnover rates for asparagine indicate that this compound is an important intermediate even under steady state conditions, and carries between 20 and 50% of the products of N assimilation.     

Regulation of ammonium uptake and metabolism by nitrogen fixing bacteria. III. Clostridium pasteurianum

Addition of ammonium salts to N₂ fixing continuous cultures of Clostridium pasteurianum caused immediate stop of nitrogenase synthesis, while the levels of glutamine synthetase, glutamate dehydrogenase and asparagine synthetase remained constant. No evidence for an interconversion of the glutamine synthetase was found. The activities of glutamate synthase in crude extracts were inversely related to the nitrogenase levels. The intracellular glutamine pool rapidly expanded during nitrogenase repression and decreased as fast during derepression while the pool sizes of all other amino acids were not strongly related to the rate of nitrogenase formation. These investigations suggest glutamine as corepressor of nitrogenase synthesis.     

Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica.

The addition of exogenous L-methionine-DL-sulphoximine (MSO) to N₂-fixing cultures of the blue-green alga Anabaena cylindrica results in over half of the newly fixed NH₃ being released into the medium. MSO also inhibits glutamine synthetase (GS) activity, has negligible effect on alanine dehydrogenase activity, and glutamate dehydrogenase activity under N₂-fixing conditions is negligible. In the presence of MSO, intracellular pools of glutamate and glutamine decrease, those of aspartate and alanine + glycine show little change, and the NH₃ pool increases. MSO alleviates the inhibitory effect of exogenous NH₄⁺ on nitrogenase synthesis and heterocyst production. The results suggest that in N₂-fixing cultures of A. cylindrica the primary NH₃ assimilating pathway involves GS, and probably glutamate synthase (GOGAT), and that the repressor of nitrogenase synthesis and heterocyst production is not NH₄⁺ but is GS, GOGAT, or a product of their reactions.     

A common transport system for methionine, L-methionine-DL-sulfoximine (MSX), and phosphinothricin (PPT) in the diazotrophic cyanobacterium Nostoc muscorum

We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues l-methionine-dl-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in ³⁵S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX- and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX- and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.     

Effect of glutamine on growth and heterocyst differentiation in the cyanobacterium Anabaena variabilis.

Mutants of the cyanobacterium Anabaena variabilis that were capable of increased uptake of glutamine, as compared with that in the parental strains, were isolated. Growth of these mutants and their parental strains was measured in media containing N2, ammonia, or glutamine as a source of nitrogen. All strains grew well with any one of these sources of fixed nitrogen. Much of the glutamine taken up by the cells was converted to glutamate. The concentrations of glutamine, glutamate, arginine, ornithine, and citrulline in free amino acid pools in glutamine-grown cells were high compared with the concentrations of these amino acids in ammonia-grown or N2-grown cells. All strains capable of heterocyst differentiation, including a strain which produced nonfunctional heterocysts, grew and formed heterocysts in the presence of glutamine. However, nitrogenase activity was repressed in glutamine-grown cells. Glutamine may not be the molecule directly responsible for repression of the differentiation of heterocysts.     

EFFECT OF MALTOSE RELEASE ON UPTAKE AND ASSIMILATION OF AMMONIUM BY SYMBIOTIC CHLORELLA (CHLOROPHYTA)1

When incubated at pH 4–5, Chlorella freshly isolated from symbiosis with Hydra viridissima PALLAS 1766 (green hydra) release large amounts of photosynthetically fixed carbon in the form of maltose, and assimilation of inorganic N is inhibited. Physiological responses to N starvation of the cultured 3N813A strain of maltose-releasing Chlorella differed from those caused by 48 h of maltose release induced by low pH. N starvation increased rates of ammonium assimilation at pH 7.0 in light or darkness, and ammonium assimilation in darkness stimulated cell respiration. In contrast, cells pretreated at pH 5.0 to induce maltose release were unable to take up ammonium at pH 7.0 unless supplied with an external carbon source such as bicarbonate, acetate, or succinate, and rates of uptake were similar to control cells. Freshly isolated symbionts displayed a similar dependency. Rates of ammonium uptake by cells pretreated at pH 5.0 were reduced in darkness and did not stimulate cell respiration. N-starved cells supplied with ammonium also showed a large short-term increase in glutamine pools at the expense of glutamate, as might be expected if large amounts of ammonium were rapidly assimilated via glutamine synthetase/glutamate synthase, whereas after long-term maltose release cells showed only a small increase in glutamine when supplied with ammonium. Furthermore, maltose release caused a fall in pool sizes of a number of amino acids, including glutamine and glutamate, and also caused a decrease in pool sizes of 2-oxoglutarate and phospho-enol-pyruvate, which are required for ammonium assimilation into amino acids. Cells stimulated to synthesize and release maltose may be unable to assimilate ammonium and synthesize amino acids because of diversion of fixed carbon from N metabolism. We estimate that 40–50% affixed C is required for maximal maltose synthesis, whereas up to 30% fixed C is required for ammonium assimilation. These results are discussed in the context of host regulation of symbiotic algal growth.     

Ammonium formation and assimilation in P(SARK)∷IPT tobacco transgenic plants under low N

Wild Type (WT) and transgenic tobacco plants expressing isopentenyltransferase (IPT), a gene encoding the enzyme regulating the rate-limiting step in cytokinins (CKs) synthesis, were grown under limited nitrogen (N) conditions. We analyzed nitrogen forms, nitrogen metabolism related-enzymes, amino acids and photorespiration related-enzymes in WT and P_SARK∷IPT tobacco plants. Our results indicate that the WT plants subjected to N deficiency displayed reduced nitrate (NO₃⁻) assimilation. However, an increase in the production of ammonium (NH₄⁺), by the degradation of proteins and photorespiration led to an increase in the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle in WT plants. In these plants, the amounts of amino acids decreased with N deficiency, although the relative amounts of glutamate and glutamine increased with N deficiency. Although the transgenic plants expressing P_SARK∷IPT and growing under suboptimal N conditions displayed a significant decline in the N forms in the leaf, they maintained the GS/GOGAT cycle at control levels. Our results suggest that, under N deficiency, CKs prevented the generation and assimilation of NH₄⁺ by increasing such processes as photorespiration, protein degradation, the GS/GOGAT cycle, and the formation of glutamine.     

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.     

Glutamine synthetase and nitrogen cycling in colonies of the marine diazotrophic cyanobacteria Trichodesmium spp.

We examined freshly collected samples of the colonial planktonic cyanobacterium Trichodesmium thiebautii to determine the pathways of recently fixed N within and among trichomes. High concentrations of glutamate and glutamine were found in colonies. Glutamate and glutamine uptake rates and concentrations in cells were low in the early morning and increased in the late morning to reach maxima near midday; then uptake and concentration again fell to low values. This pattern followed that previously observed for T. thiebautii nitrogenase activity. Our results suggest that recently fixed nitrogen is incorporated into glutamine in the N2-fixing trichomes and may be passed as glutamate to non-N2-fixing trichomes. The high transport rates and concentrations of glutamate may explain the previously observed absence of appreciable uptake of NH4+, NO3-, or urea by Trichodesmium spp. Immunolocalization, Western blots (immunoblots), and enzymatic assays indicated that glutamine synthetase (GS) was present in all cells during both day and night. GS appeared to be primarily contained in cells of T. thiebautii rather than in associated bacteria or cyanobacteria. Double immunolabeling showed that cells with nitrogenase (Fe protein) contained levels of the GS protein that were twofold higher than those in cells with little or no nitrogenase. GS activity and the uptake of glutamine and glutamate dramatically decreased in the presence of the GS inhibitor methionine sulfoximine. Since no glutamate dehydrogenase activity was detected in this species, GS appears to be the primary enzyme responsible for NH3 incorporation.     

Metabolic changes associated with adaptation of plant cells to water stress

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from ¹⁵N labeling kinetics after substituting [¹⁵N]ammonium and [¹⁵N]nitrate for the ¹⁴N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and γ-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and γ-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and γ-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low ¹⁵N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.     

Effects of Branched-Chain L-Amino Acids, L-Phenylalanine, and L-Methionine on the Transport of L-Glutamine in Rat Brain Cortex In Vitro. Influence of Cations

Abstract: Uptake of L-glutamine (2 mM) by rat brain cortex slices against a concentration gradient is markedly inhibited (40%) by branched-chain Lamino acids (1 mM), L-phenylalanine (1 mM), or L-methionine (1 mM); that of L-asparagine (2 mM) is much less affected by these amino acids. Other amino acids investigated have little or no effect on cerebral L-glutamine uptake. The suppressions of L-glutamine uptake by the inhibitory amino acids are apparently blocked by high [K⁺], which itself has little or no effect on glutamine uptake. This abolition of suppression is partly explained by high [K⁺] retention of endogenous glutamine; in the absence of Ca²⁺ such retention disappears. The inhibitory amino acids (1 mM) also enhance the release of endogenous glutamine, exogenous glutamine with which slices have been loaded, or glutamine synthesized in the slices from exogenous glutamate. The enhanced release of endogenous glutamine is diminished by high [K⁺]. The suppression of glutamine uptake by the branched-chain amino acids is independent of the concentration of glutamine at low concentrations (0.25–0.5 mM), indicating non-competition, but is reduced with high concentration of glutamine. The inhibition by L-phenylalanine is noncompetitive. L-Glutamine (2 mM) exerts no inhibition of the cerebral uptakes of the branched-chain L-amino acids or Lphenylalanine (0.25–2 mM). The inhibitory amino acids are as active in suppressing L-glutamine uptake with immature rat brain slices as with adult, although the uptake, against a gradient, of L-glutamine in the infant rat brain is about one-half that in the adult. They are also just as inhibitory on the concentrative uptake of L-glutamine by a crude synaptosomal preparation derived from rat brain cortex. Such a nerve ending preparation takes up L-glutamine (0.25 mM), against a gradient, at about ninefold the rate at which it is taken up by cortex slices (for equal amounts of protein), and the uptake process is markedly suppressed by high [K⁺] in contrast to the effects of high [K⁺] with slices. The possible physiological and pathological consequences of the suppression of glutamine uptake are discussed.     

Cerebral Cortex Ammonia and Glutamine Metabolism in Two Rat Models of Chronic Liver Insufficiency-Induced Hyperammonemia: Influence of Pair-Feeding

Abstract: Enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis, and glutamine release into the bloodstream have been hypothesized to deplete cerebral cortex glutamate pools. We investigated this hypothesis in rats with chronic liver insufficiency-induced hyperammonemia and in pair-fed controls to rule out effects of differences in food intake. Cerebral cortex plasma flow and venous-arterial concentration differences of ammonia and amino acids, as well as cerebral cortex tissue concentrations, were studied 7 and 14 days after surgery in portacaval-shunted/bile duct-ligated, portacaval-shunted, and sham-operated rats, while the latter two were pair-fed to the first group, and in normal unoperated ad libitum-fed control rats. At both time points, arterial ammonia was elevated in the chronic liver insufficiency groups and arterial glutamine was elevated in portacaval shunt/biliary obstruction rats compared to the other groups. In the chronic liver insufficiency groups net cerebral cortex ammonia uptake was observed at both time points and was accompanied by net glutamine release. Also in these groups, cerebral cortex tissue glutamine, many other amino acid, and ammonia levels were elevated. Tissue glutamate levels were decreased to a similar level in all operated groups compared with normal unoperated rats, irrespective of plasma and tissue ammonia and glutamine levels. These results demonstrate that during chronic liver insufficiency-induced hyperammonemia, the rat cerebral cortex enhances net ammonia uptake and glutamine release. However, the decrease in tissue glutamate concentrations in these chronic liver insufficiency models seems to be related primarily to nutritional status and/or surgical trauma.