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PubMed1997

Molecular characterization of the glnA gene encoding glutamine synthetase from the edible mushroom Agaricus bisporus.

The gene encoding glutamine synthetase (glnA) was isolated from an Agaricus bisporus H39 recombinant lambda phage library. The deduced A. bisporus glutamine synthetase amino acid sequence contains 354 residues. The amino acid sequence is very similar to that derived from the gene coding for glutamine synthetase of the yeast Saccharomyces cerevisiae. The open reading frame is interrupted by four introns. Northern analysis revealed that transcription of the gene is repressed upon addition of ammonium to the culture but the repression was not as strong as that of the gene encoding NADP+ -dependent glutamate dehydrogenase (gdhA). Enzyme activities are low in the presence of ammonium, glutamine and albumin and do not correlate with the mRNA levels revealed by Northern analysis. This suggests that glutamine synthetase expression in A. bisporus is also post-transcriptionally regulated by the nitrogen source.

Agaricus↗
PubMed2003

Improved method for determination of ammonia and nitrite oxidation activities in mixed bacterial cultures.

A simple and reliable method to measure the activity of ammonia and nitrite oxidisers in mixed bacterial cultures was developed. The developed method differentiates between the ammonia and nitrite oxidisers by consecutive injection of NO2- and NH4+. The main advantage of this method is that it avoids the use of metabolic inhibitors for ammonia or nitrite oxidisers, as used by other methods. Moreover, it allows measuring of the short-term effect of an inhibitor on both the ammonia and nitrite oxidisers in one test under controlled environmental conditions (pH, temperature). The developed method was applied to determine the inhibitory effects of salt (NaCl up to 15 g Cl/l) on an enriched culture of nitrifying bacteria. The results of the method demonstrate its potential to accurately determine the individual activities of nitrite and ammonia oxidisers.

Ammonia↗
PubMed1996

Posttranslational regulation of nitrogenase activity by fixed nitrogen in Azotobacter chroococcum.

Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32P]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.

Adenosine Diphosphate Ribose↗
PubMed1994

Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes.

Glutamine synthetase brings nitrogen into metabolism by condensing ammonia and glutamate, with the aid of ATP, to yield glutamine, ADP, and inorganic phosphate. Here we present five crystal structures of GS complexed with each of two substrates, Glu and AMPPNP (an ATP analog), with a transition-state analogue, L-methionine-S-sulfoximine, and with each of two products, Gln and ADP. GS of the present study is from Salmonella typhimurium, has Mn2+ bound, and is fully unadenylylated. Protein-metal-substrate interactions and small but significant conformational changes induced by substrate binding are defined by Fourier maps. On the basis of these maps, we propose a tentative structure-based enzymatic mechanism of glutamine synthesis with these steps: (1) ATP binds first at the top of the funnel-shaped active site cavity, adjacent to the n2 Mn2+; Arg 359 moves toward the Glu binding site. (2) Glu binds adjacent to the n1 Mn2+ at the bottom of the active site near a flexible loop (residues 324-328). As proposed earlier by Meister and others, Glu attacks the gamma-phosphorus atom of ATP to produce gamma-glutamyl phosphate and ADP. (3) The presence of ADP (but not ATP) moves Arg 339 toward the Pi site, perhaps stabilizing the gamma-glutamyl phosphate, and moves Asp 50' of the adjacent subunit toward a putative ammonium ion site, enhancing binding of this third substrate. Deprotonation of the ammonium ion, perhaps by Asp 50', permits the resulting active species, ammonia, to attack the gamma-glutamyl phosphate, forming a tetrahedral intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Imidodiphosphate↗
PubMed2002

A novel HMG A-like protein binds differentially to the AT-rich regions located in the far distal and proximal parts of a soybean glutamine synthetase gene (GS15) promoter.

In soybean (Glycine max L.) ammonium provided externally or as the result of symbiotic nitrogen fixation stimulates the transcription of GS15, a gene encoding cytosolic glutamine synthetase. Strong constitutive positive expression (SCPE), silencer-like and organ-specific elements, located respectively in the distal, the central and the proximal region of the promoter are required to control the ammonium responsiveness of the gene expression [Tercé-Laforgue et al. (1999) Plant Mol. Biol. 39: 551]. It was hypothesized that the correct spatial conformation of the promoter permitted the cooperative action of these three cis-acting elements. Further investigations were therefore required to ascertain this hypothesis. A nodule nuclear protein, binding to a 66 bp AT-rich DNA fragment containing a 13 bp AT-rich repeated sequence (AT-1) and located just downstream of the SCPE element, was identified using a gel retardation assay. A cDNA clone likely to code for this protein was isolated using the yeast one-hybrid system. It encodes a novel DNA binding protein (AT-1SNBP) similar to HMG A proteins but exhibiting a higher molecular weight. AT-1SNBP appears to be encoded by a single gene that is expressed in roots, root nodules and leaves of soybean. Since two other 13 bp AT-rich repeated sequences (AT-2 and AT-3) were localized in the organ-specific element, we have quantified the binding affinity of AT-1SNBP to these sequences. We demonstrate that AT-1SNBP binds differentially to DNA fragments containing AT-1, AT-2 and AT-3 and that its binding affinity depends on the presence of adjacent sequences. This result suggests that AT-1SNBP may be an architectural protein involved in maintaining the spatial conformation of the GS15 promoter, thus facilitating the interaction between the distal and proximal regulatory elements.

AT Rich Sequence↗
PubMed2006

Effects of PII deficiency on expression of the genes involved in ammonium utilization in the cyanobacterium Synechocystis sp. Strain PCC 6803.

The Synechocystis sp. strain PCC 6803 mutant deficient in PII protein (the glnB gene product) was found to express glutamine synthetase activity at levels several times higher than the wild-type strain. There was no significant difference in nitrate reductase activity levels between the two strains, and the nitrite reductase levels were somewhat lower in the mutant than in the wild-type strain. The higher glutamine synthetase activity in the mutant was ascribed to higher expression levels of the glutamine synthetase genes (glnA and glnN), which belong to the regulon controlled by NtcA, a Crp-family transcription regulator. Examination of the effects of PII deficiency on other NtcA-regulated genes revealed that the transcript levels of amt1 (encoding an ammonium permease) and gifB (encoding an inhibitor of glutamine synthetase) were increased, whereas that of gifA (a homolog of gifB, encoding another glutamine synthetase inhibitor) was decreased, with those of nirA, nrtC, icd, sigE (rpoD2-V), nblA and ntcA being unaffected. Unlike the Synechococcus elongatus strain PCC 7942, induction or repression of the NtcA-regulated genes proceeded normally in the PII-deficient mutant upon nitrogen depletion. The altered steady-state expression levels of glnA, glnN, amt1, gifA and gifB in the PII-deficient mutant suggested that Synechocystis sp. strain PCC 6803 has a mechanism for regulation of the subset of the NtcA-regulated genes related directly to ammonium assimilation.

Bacterial Proteins↗
PubMed2004

Deletion of the Gibberella fujikuroi glutamine synthetase gene has significant impact on transcriptional control of primary and secondary metabolism.

In Gibberella fujikuroi, the gibberellin (GA) and bikaverin biosynthesis are under control of nitrogen metabolite repression. However, the signalling components acting upstream of AREA are still unknown. We investigated the role of glutamine synthetase (GS) both as an enzyme and as a possible regulator in the nitrogen regulation system. We cloned and replaced the GS-encoding gene, glnA-Gf. The mutants grow with a phenotype different from the wild type in the presence of glutamine. They were unable to express nitrogen-repressed GA and bikaverin biosynthetic genes even under nitrogen starvation conditions. Complementation with the glnA-Gf wild-type copy fully restored GS activity, the expression of secondary metabolism genes, and the production of GAs and the red pigment, bikaverin. In order to find more target genes of GS, differential cDNA-screening and differential hybridization of macroarrays were performed using cDNA from the wild type and DeltaglnA mutant as probes. Several genes were dramatically up- or downregulated in the mutant. Among them are genes involved in N- and C-catabolism, and in transcriptional and translation control. Some of these genes are also under AREA control. Treatment with the GS inhibitor l-methionine sulphoximine resulted in similar expression patterns as in the glnA mutant with ammonium as nitrogen source, whereas glutamine can overcome the up- or downregulation of most but not all of the target genes. These findings suggest that not only glutamine, but also GS itself might play an important role in nitrogen metabolite repression.

Gene Expression Profiling↗
PubMed1984

Alterations in internal partitioning of carbon in soybean plants in response to nitrogen stress.

Alterations in internal partitioning of carbon were evaluated in plants exposed to limited nitrogen supply. Vegetative, nonnodulated soybean plants (Glycine max (L.) Merrill, 'Ransom') were grown for 21 days with 1.0 mM NO3- and then exposed to solutions containing 1.0, 0.1, or 0.0 mM NO3- for a 25-day treatment period. In nitrogen-limited plants, there were decreases in emergence of new leaves and in the expansion rate and final area at full expansion of individual leaves. As indicated by alterations in accumulation of dry weight, a larger proportion of available carbon in the plant was partitioned to the roots with decreased availability of nitrogen. Partitioning of reduced nitrogen to the root also was increased and, in plants devoid of an external supply, considerable redistribution of reduced nitrogen from leaves to the root occurred. The general decrease in growth potential and sink strength for nutrients in leaves of nitrogen-limited plants suggested that factors other than simply availability of nitrogen likely were involved in the restriction of growth in the leaf canopy and the associated increase in carbon allocation to the roots.

Ammonia↗
PubMed2001

Transport of NH(3)/NH in oocytes expressing aquaporin-1.

The aim of this study was to determine whether expressing aquaporin (AQP)-1 could affect transport of NH(3). Using ion-selective microelectrodes, the experiments were conducted on frog oocytes (cells characterized by low NH(3) permeability) expressing AQP1. In H(2)O-injected oocytes, exposure to NH(3)/NH (20 mM, pH 7.5) caused a sustained cell acidification and no initial increase in pH(i) (as expected from NH(3) influx), and the cell depolarized to near 0 mV. The absence of Na(+), the presence of Ba(2+), or raising bath pH (pH(B)) did not inhibit the magnitude of the pH(i) decrease or result in an initial increase in pH(i) when NH(3)/NH was added. However, after the cell was acidified (because of NH(3)/NH), raising pH(B) to 8.0 caused a slow increase in pH(i) but had no effect on membrane potential. The changes in pH(i) with raising pH(B) did not occur in the absence of NH(3)/NH. In AQP1 oocytes, exposure to NH(3)/NH usually resulted in little or no change in pH(i), and in the absence of Na(+) there was a small increase in pH(i) (the cell still depolarized to near 0 mV). However, after exposure to NH(3)/NH, raising pH(B) to 8.0 caused pH(i) to increase more than two times faster than in control oocytes. This increase in pH(i) is likely the result of increased NH(3) entry and not the result of NH transport. These results indicate that 1) the oocyte membrane, although highly permeable to NH, has a significant NH(3) permeability and 2) NH(3) permeability is enhanced by AQP1.

Ammonia↗
PubMed2000

Mechanisms of glutamate metabolic signaling in retinal glial (Müller) cells.

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina. Our results demonstrate that lactate production and release is controlled by the combined action of glutamate and NH(4)(+), both at micromolar concentrations. Transport of glutamate by a high-affinity carrier can produce in Müller cells a rapid rise of glutamate concentration. In our isolated Müller cells, glutamine synthetase (GS) converted transported glutamate to glutamine that was released. This reaction, predominant when enough NH(4)(+) is available, was limited at micromolar concentrations of NH(4)(+), and more glutamate entered then as substrate into the mitochondrial tricarboxylic acid cycle (TCA). Increased production of glutamine by GS leads to increased utilization of ATP, some of which is generated glycolytically. Methionine sulfoximine, a specific inhibitor of GS, suppressed the stimulatory effect of glutamate and NH(4)(+) on glycolysis and induced massive entry of glutamate into the TCA cycle. The rate of glutamine production also determined the amount of pyruvate transaminated by glutamate to alanine. Lactate, alanine, and glutamine can be taken up and metabolized by photoreceptor neurons. We conclude that a major function of Müller glial cells is to nourish retinal neurons and to metabolize the neurotoxic ammonia and glutamate.

Ambystoma↗
PubMed2005

Chemical denitrification for nitrogen removal from landfill leachate.

A new system that removes nitrogen from landfill leachate and other waste waters with similar properties has been proposed with nitritation (i.e. oxidation of ammonium to nitrite) of half of the influent ammonium followed by chemical denitrification with a reaction between equal amounts of ammonium and nitrite to form nitrogen gas. Chemical denitrification occurs at high concentrations and the reactions were studied in combination with a concentration step. Studied concentration methods were freezing/thawing and evaporation/drying. Chemical denitrification is well-known in inorganic chemistry and has been observed in natural systems. Studies in laboratory were focused on chemical denitrification and showed that nearly complete removal of soluble nitrogen can be obtained in evaporation/drying of water solutions or leachate with equal amounts of ammonium and nitrite. Freezing/drying was less efficient with a removal of about 50-60% at high initial concentrations. Chemical denitrification is much influenced by concentration, pH-value, temperature and some compounds in leachate have an inhibiting effect on the reaction. Factors as safety (ammonium nitrite as a salt is explosive above 60 degrees C) and possible side-reactions as formation of ammonia and nitrogen oxides must be carefully evaluated before use in full-scale. Conductivity is a suitable parameter to follow-up the chemical denitrification process.

Ammonia↗
PubMed2001

Nitrogen reduction under hydrothermal vent conditions: implications for the prebiotic synthesis of C-H-O-N compounds.

Dinitrogen is reduced in dilute hydrogen sulfide (H2S) solutions to ammonium at 120 degrees C. Experiments with dissolved dinitrogen (partial pressure 50 bar) in a 12 x 10(-3) mol/L H2S(aq) solution yield approximately 10(-5) mol/L NH4+ within 2-7 days. These yields are consistent with the equilibrium NH4+ concentration for the N-S-H system under these conditions. The formation of ammonium is catalyzed by the presence of freshly precipitated iron monosulfide. These results indicate that dinitrogen can be reduced at moderate temperatures in hydrothermal vent systems. Abiotic nitrogen reduction could have taken place within primordial hydrothermal vents, supplying some ammonia for the synthesis of C-H-O-N compounds via abiotic processes. The yield of ammonia via dinitrogen reduction by hydrogen sulfide, however, is so low that it is doubtful this process could have produced enough ammonia to sustain prebiotic hydrothermal synthesis of C-H-O-N compounds in or around vent systems.

Ammonia↗
PubMed1968

The 'neurotoxicity' of L-2,4-diaminobutyric acid.

The neurolathyrogen l-2,4-diaminobutyric acid is concentrated by liver, and liver damage can yield neurotoxicity; thus the neurotoxicity caused by this compound may be due to liver damage followed by secondary brain damage. 1. The intraperitoneal administration of toxic doses of l-2,4-diaminobutyric acid to rats resulted in hyperirritability, tremors and convulsions in 12-20hr. and increased the concentration of ammonia of blood and brain slightly and the concentration of glutamine of brain two- to three-fold. By contrast, toxic doses of l-homoarginine, l-lysine, l-leucine and ammonium acetate caused dyspnoea, extreme prostration, and in some cases coma in 15-30min., and increased the concentration of ammonia of blood significantly and the concentration of glutamine of brain slightly. These results indicate that l-2,4-diaminobutyric acid caused a chronic ammonia toxicity, whereas the other amino acids and ammonium acetate resulted in an acute ammonia toxicity. 2. Liver slices from l-2,4-diaminobutyric acid-treated animals and normal liver slices preincubated with l-2,4-diaminobutyric acid utilized ammonia and formed urea at a lower rate than control slices from normal rats. 3. l-2,4-Diaminobutyric acid inhibited competitively ornithine carbamoyltransferase of rat liver homogenates, thus demonstrating that this reaction is a primary site of toxicity for this neurolathyrogen. Although we were unable to show marked elevations of blood ammonia concentration after treatment with l-2,4-diaminobutyric acid, these results are interpreted to mean that ammonia utilization (urea synthesis) in liver is inhibited by l-2,4-diaminobutyric acid and that at least part of the neurotoxicity is due to a prolonged slight increase in body ammonia concentration.

Aminobutyrates↗
PubMed2002

Regulation of amino acid uptake in conifers by exogenous and endogenous nitrogen.

Although an accumulating amount of research clearly indicates that plants are capable of taking up exogenous amino acids, the actual importance of such organic N sources for plant N nutrition is under debate. In this study, we show that amino acid uptake by Scots pine (Pinus sylvestris L.) is significantly decreased by elevated internal NH(4)(+) levels, while it increases following exposure to exogenous amino acids. Furthermore, amino acid uptake is larger in N-deficient plants than in plants grown with a large access of N. The regulatory pattern of amino acid uptake shows important similarities to the regulation of NO(3)(-) and NH(4)(+) transport as well as to the regulation of yeast amino acid transporters. In addition, our data suggest that uptake may be regulated by factors not originating from N metabolism. The up-regulation of uptake in response to N deficiency suggests that amino acid uptake may be a significant contributor to the N economy of P. sylvestris.

Adaptation, Physiological↗