Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids]

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.     

Regulation of the activity of mung bean (Phaseolus aureus) glutamine synthetase by amino acids and nucleotides.

The activity of glutamine synthetase isolated from the germinated seedlings of Phaseolus aureus was regulated by feedback inhibition by alanine, glycine, histidine, AMP, and ADP. When glutamate was the varied substrate, alanine, histidine, and glycine were partial noncompetitive, competitive, and mixed-type inhibitors, respectively. The type of inhibition by these amino acids was confirmed by fractional inhibition analysis. The adenine nucleotides, AMP and ADP, completely inhibited the enzyme activity and were competitive with respect to ATP. Multiple inhibition analyses revealed the presence of separate and nonexclusive binding sites for the amino acids and mutually exclusive sites for adenine nucleotides. Cumulative inhibition was observed with these end products.     

Export of amino acids to the phloem in relation to N supply in wheat

The effect of different N supply on amino acid export to the phloem was studied in young plants of wheat (Triticum aestivum L. cv. Klein Chamaco), using the exudation in EDTA technique. Plants were grown in a growth cabinet in pots with sand, and supplied with nutrient solutions of different NO₃^? concentrations. When plants were grown for 15 days with nutrient solutions containing 1.0, 3.0, 5.0, 10.0, 15.0 or 20.0 mM KNO₃, the exudation rate of sugars from the phloem was unaffected by N supply, but sugars accumulated in the leaf tissue when the N supply was limiting for growth. On the other hand, the rate of exudation of amino acids was proportional to the NO₃^? concentration in the nutrient solution. When the supply of N to plants grown for 15 days with 15.0 mM NO₃^? was interrupted, the exudation of sugars was again unaffected, but there was a fast decrease in the amount of amino acids exudated, and of the concentration of amino acids and nitrogen in the tissues. Also, when 10-day-old plants grown without N were supplied with 15.0 mM NO₃^?, there was a sharp increase in the exudation of amino acids, without changes in the amount of sugar exudated. The rate of exudation of amino acids to the phloem was independent of the concentration of free amino acids in the leaves in all three types of experiment. Asp was the most abundant amino acid in the leaf tissue, while Glu was the one most abundant in the phloem exudate. Asp and Ala were exported to the phloem at a rate lower than expected from their leaf tissue concentrations, indicating some discrimination. On the contrary, Glu showed a preferential export at low N supply. It is concluded that the rate of amino acid export from the leaf to the phloem is dependent on the N available to the plant. This N is used for synthesis of leaf protein when the supply is low, exported to the phloem when supply is adequate, and accumulated in the storage pool when supply is above plant demand.     

Universality of phloem transport in seed plants

Since Münch in the 1920s proposed that sugar transport in the phloem vascular system is driven by osmotic pressure gradients, his hypothesis has been strongly supported by evidence from herbaceous angiosperms. Experimental constraints made it difficult to test this proposal in large trees, where the distance between source and sink might prove incompatible with the hypothesis. Recently, the theoretical optimization of the Münch mechanism was shown to lead to surprisingly simple predictions for the dimensions of the phloem sieve elements in relation to that of fast growing angiosperms. These results can be obtained in a very transparent way using a simple coupled resistor model. To test the universality of the Münch mechanism, we compiled anatomical data for 32 angiosperm and 38 gymnosperm trees with heights spanning 0.1-50 m. The species studied showed a remarkable correlation with the scaling predictions. The compiled data allowed calculating stem sieve element conductivity and predicting phloem sap flow velocity. The central finding of this work is that all vascular plants seem to have evolved efficient osmotic pumping units, despite their huge disparity in size and morphology. This contribution extends the physical understanding of phloem transport, and will facilitate detailed comparison between theory and field experiments.     

The role of cytosolic glutamine synthetase in wheat

The role of glutamine synthetase (GS; EC 6.3.1.2) was studied in wheat. GS isoforms were separated by HPLC and the two major leaf isoforms (cytosolic GS1 and chloroplastic GS2) were found to change in content and activity throughout plant development. GS2 dominated activity in green, rapidly photosynthesising leaves compared to GS1 which was a minor component. GS2 remained the main isoform in flag leaves at the early stages of grain filling but GS1 activity increased as the leaves aged. During senescence, there was a decrease in total GS activity which resulted largely from the loss of GS2 and thus GS 1 became a greater contributor to total GS activity. The changes in the activities of the GS isoforms were mirrored by the changes in GS proteins measured by western blotting. The changes in GS during plant development reflect major transitions in metabolism from a photosynthetic leaf (high GS2 activity) towards a senescencing leaf (relatively high GS1 activity). It is likely that, during leaf maturation and subsequently senescence, GS1 is central for the efficient reassimilation of ammonium released from catabolic reactions when photosynthesis has declined and remobilisation of nitrogen is occurring. Preliminary analysis of transgenic wheat lines with increased GS1 activity in leaves showed that they develop an enhanced capacity to accumulate nitrogen in the plant, mainly in the grain, and this is accompanied by increases in root and grain dry matter. The possibility that the manipulation of GS may provide a means of enhancing nitrogen use in wheat is discussed.     

The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.     

Immunocytolocalization of glutamine synthetase in mesophyll and phloem of leaves ofSolanum tuberosum L.

Summary Localization of glutamine synthetase inSolanum tuberosum leaves was investigated by techniques of Western tissue printing and immunogold electron microscopy. Anti-GS antibodies used in immunolocalization recognize two peptides (45 kDa and 42 kDa) on Western blots. Antibody stained tissue prints on nitrocellulose membranes allowed low resolution localization of GS. Immunostaining was most evident in the adaxial phloem of the leaf midribs and petiole veins. High-resolution localization of glutamine synthetase by immunogold electron microscopy revealed that this enzyme occurs in both the chloroplasts and the cytosol ofS. tuberosum leaf cells. However, GS was specifically associated with the chloroplasts of mesophyll cells and with the cytoplasm of phloem companion cells. The evidence for cell-specific localization of chloroplast and cytosolic GS presented here agrees with the recently reported cell-specific pattern of expression of GUS reporter gene, directed by promoters for chloroplast and cytosolic GS form in tobacco transgenic plants. These data provide additional clues to the interpretation of the functional role of these different isoenzymes and its relationship with their specific localization.Abbreviations BSA bovine serum albumin - EM electron microscope - GOGAT glutamate synthase - GS glutamine synthetase - GUS -glucuronidase - IgG immunoglobulin - PBS phosphate buffer saline - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Regulation of glutamine synthetase I fromRhizobium meliloti

Glutamine synthetase I fromRhizobium meliloti was found to be inhibited by adenosine 5-monophosphate, alanine, glycine, carbamyl phosphate, cytidine 5-triphosphate, tryptophan, histidine, and glucosamine-6-phosphate. Each inhibitor was independent in its action and the effect was cumulative when more than one inhibitor was added.     

高等植物谷氨酰胺合成酶研究进展

谷氨酰胺合成酶（GS）是参与高等植物氮同化过程的关键酶。介绍了高等植物谷氨酰联合成酶及其同工酶的分布、性质、生理作用及分子生物学等方面的研究进展。     

Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase

The relationships of five feedback inhibitors for the Bacillus licheniformis glutamine synthetase were investigated. The inhibitors were distinguishable by differences in their competitive relationship for the substrates of the enzyme. Mixtures of l-glutamine and adenosine-5'-monophosphate (AMP) or histidine and AMP caused synergistic inhibition of glutamine synthesis. Histidine, alanine, and glycine acted antagonistically toward the l-glutamine inhibition. Alanine acted antagonistically toward the glycine and histidine inhibitions. Independence of inhibitory action was observed with the other pairs of effectors. Possible mechanisms by which the inhibitors may interact to control glutamine synthesis are discussed. The low rate of catalysis of the glutamyl transfer reaction by the B. licheniformis glutamine synthetase can be attributed to the fact that l-glutamine serves both as a substrate and an inhibitor for the enzyme. Effectors which act antagonistically toward the l-glutamine inhibition stimulated glutamotransferase activity. The stimulation was not observed when d-glutamine was used as substrate for the glutamyl transfer reaction.     

Regulation of glutamine synthetase in the blue-green alga Anabaena flos-aquae

Glutamine synthetase (GS) was isolated from log phase cells and purified to a single protein as evidenced by gel electrophoresis. Protamine and ammonium sulfate precipitation and chromatography on DEAE-cellulose and Bio-Gel resulted in 380-fold purification. The enzyme was most sensitive to alanine (85% inhibition at 0.1 mM) but was also inhibited by glycine, arginine and serine. Combinations of inhibitory amino acids or nucleotides (AMP, ADP, ATP) exhibited cumulative inhibition. Cooperative inhibition was noted with CTP and any single nucleotide. Inhibition by CTP alone was uncompetitive with respect to glutamine. The enzyme was also regulated by the energy charge of the cell.     

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants

Summary We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH₃. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.