Purification and characterization of thioredoxin from the N2-fixing cyanobacterium Anabaena cylindrica

Thioredoxin has been purified to homogeneity from the cyanobacterium Anabaena cylindrica. The protein consists of a single polypeptide chain with a relative molecular mass of about 11 680 which has two cysteine residues (residues 31 and 34) in the sequence-Cys-Gly-Pro-Cys- and an isoelectric point at pH 4.55. The N-terminal amino acid sequence of 39 residues shows distinct homologies with the sequences of Escherichia coli and Corynebacterium nephridii thioredoxins. Anti-(A. cylindrica thioredoxin) antiserum was used to quantify the thioredoxin which constituted about 0.22% of the soluble protein in cell-free extracts of N2-fixing, NO3- -grown or NH4+-grown A. cylindrica. Activation of fructose-1,6-bisphosphatase of A. cylindrica, activation of glutamine synthetase and NADP+-dependent malate dehydrogenase of the green alga Scenedesmus obliquus but not of A. cylindrica, and deactivation of glucose-6-P dehydrogenase of the cyanobacterium Anabaena variabilis were all achieved using the same thioredoxin species. No other thioredoxin species were detected in extracts of A. cylindrica when examined for the activation of these enzymes.     

Enzymes involved in ammonia assimilation in the fungus Acremonium persicinum

Abstract Acremonium persicinum grown in batch culture with ammonium tartrate as the nitrogen source possessed an NADP⁺-dependent glutamate dehydrogenase and a glutamine synthetase. Glutamate synthase was not detected under the culture conditions used. Kinetic studies of the NADP⁺-dependent glutamate dehydrogenase at 25°C and pH 7.6 revealed an apparent K _m of 3.2 × 10⁻⁴ M for 2-oxoglutarate and an apparent K _m of 1.0 × 10⁻⁵ M for ammonium ions, with corresponding apparent V _max values of 0.089 and 0.13 μmol substrate converted/min/mg of protein, respectively. Glutamine synthetase was measured by the γ-glutamyl transferase reaction at 30°C and pH 7.55. This transferase reaction of glutamine synthetase had a higher rate at 30°C than at 25°C or 37°C.     

An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli

The P_II protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that P_II, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild-type strain. We describe a new operon, glnK amtB , which encodes a homologue of P_II and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as P_II, reacted with polyclonal P_II antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli P_II. Like P_II, purified GlnK can activate the adenylylation of glutamine synthetase in vitro , and, in vivo , the GlnK protein is uridylylated in a glnD -dependent fashion. Unlike P_II, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a σ^N (σ⁵⁴) RNA polymerase-binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two-component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on P_II. Possible regulatory implications of this conditional redundancy of P_II are discussed.     

An additional PII in Escherichia coli: a new regulatory protein in the glutamine synthetase cascade

Abstract The P_II protein in the glutamine synthetase cascade transduces the nitrogen signal, as sensed by uridylyltransferase, both to the NRII/NRI two-component system and to adenylyltransferase, to regulate the activity of glutamine synthetase. Here we describe the amplification of a chromosomal DNA fragment from Escherichia coli which contains the sequence of a P_II homologue. The derived amino acid sequence of this DNA fragment is 67% identical to E. coli P_II. It contains the conserved tyrosine residue which is known to be the site of uridylylation in P_II. E. coli is the first organism in which two different P_II proteins have been detected.     

The isocitrate dehydrogenase from cyanobacteria

The present communication describes the properties of isocitrate dehydrogenase in crude extracts from the unicellular Anacystis nidulans and from heterocysts and vegetative cells of Nostoc muscorum and Anabaena cylindrica. The activity levels of this enzyme are much higher in heterocysts than in vegetative cells of N. muscorum and A. cylindrica. Isocitrate dehydrogenase is virtually inactive in vegetative cells of A. cylindrica. The enzyme is negatively regulated by the reduction charge and scarcely affected by oxoglutarate in the three cyanobacteria. The inhibition by ATP and ADP is competitive with respect to isocitrate and NADP+ in A. cylindrica and N. muscorum and noncompetitive in A. nidulans. Isocitrate dehydrogenase from the three cyanobacteria seems to be a hysteretic enzyme. All the experimental data suggest that the major physiological role of isocitrate and the isocitrate dehydrogenase in heterocysts is not to generate reducing equivalents for N2-fixation. Oxoglutarate formed by the enzyme reaction is likely required for the biosynthesis of glutamate inside the heterocysts. Thioredoxin preparations from spinach chloroplasts or from A. cylindrica activate isocitrate dehydrogenase from either heterocysts or vegetative cells of A. cylindrica. Activation is completed within seconds and requires dithiothreitol besides thioredoxin. The thioredoxin preparation which activates isocitrate dehydrogenase also activates NADP+-dependent malate dehydrogenase from spinach chloroplasts or heterocysts of A. cylindrica. Isocitrate dehydrogenase from A. cylindrica is deactivated by oxidized glutathione. It is speculated that isocitrate dehydrogenase and thioredoxin play a role in the differentiation of vegetative cells to heterocysts.     

Light mediated regulation of nitrate assimilation in Synechococcus leopoliensis

Synechococcus leopoliensis was cultivated in a light/dark regime of 12:12 h. After onset of the illumination (2 h), the specific activity of nitrite reductase, glutamine synthetase and isocitric dehydrogenase increased; that of glucose-6-phosphate dehydrogenase decreased and that of nitrate reductase and NAD- (NADP) glutamate dehydrogenase remained nearly unchanged.This stimulation of the enzymes in vivo was also observed in vitro. Also, when extracts from darkened cells were incubated with thioredoxin and dithioerythriol enzyme activities increased in the same amount as obtained in vivo. In addition, glucose-6-phosphate dehydrogenase and isocitric dehydrogenase were stimulated by Mn²⁺ and Mg²⁺ in the assay mixture. Glutamine synthetase activity was enhanced only by Mg²⁺ while Mn²⁺ was inhibitory.The results are discussed with respect to the regulation of nitrogen metabolism by light.Abbreviations GS glutamine synthetase - GOGAT glutamate-oxoglutarate-aminotransferase - TR thioredoxin - DTE dithioerythritol - LD change from light to dark     

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.     

Effects of water stress on enzymes of ammonia assimilation in root nodules of alfalfa (Medicago sativa)

Protein content and activities of the enzymes glutamine synthetase (EC 6.3.1.2), NADH-glutamate synthase (EC 1.4.1.14), NADH-glutamate dehydrogenase (reductive amination (EC 1.4.1.2) and NAD⁺-glutamate dehydrogenase (oxidative deamination) (EC 1.4.1.2) from the plant fraction of root nodules of alfalfa ( Medicago sativa L. cv. Aragon) were determined under water stress. Only NADH-glutamate synthase activity was inhibited during drought. The results indicate that the glutamine synthetase/NADH-glutamate synthase cycle was fully operational in alfalfa nodules of control or even mildly stressed plants when N₂-fixation was not inhibited, but that the coupling between glutamine synthetase and NADH-glutamate synthase was lost as drought progressed. Patterns of glutamine synthetase and NADH-/NAD⁺-gluta-mate dehydrogenase activities reflect changes in ammonia content of nodules and/or availability of carbon substrates, and indicate that nodules maintain sufficient enzyme activity for ammonia assimilation throughout water stress.     

Glutamine synthetase isoforms in leaves of a C4 plant: Sorghum vulgare

In Sorghum vulgare L. (cv. INRA 450) leaves, two isoforms of glutamine synthetase GS₁ and GS₂ were identified by DEAE Sephacel chromatography and polyac-rylamide gel electrophoresis. GS₁ was present both in etiolated and green leaves and its activity remained constant during the greening process. In green leaves another isoform GS₂ was identified that was responsible for a 33% increase in total glutamine synthetase activity after light exposure. The two isoforms differed in their heat stability but exhibited similar pH optima and Km values for L-glutamate. The total glutamine synthetase activity was equally distributed between mesophyll and bundle sheath cells; moreover in the two types of cells both GS₁ and GS₂ were present in the proportions of 67% and 33% of the total respectively. In mesophyll cells the two isoforms had a different subcellular distribution: GS₂ was localized in the chloroplast and GS₁ in the cytosol.     

Effects of tabtoxin on nitrogen metabolism

Tabtoxin is a chlorosis-inducing toxin produced by the plant pathogenic bacterium Pseudomonas syringae pv. tabaci. Previous studies have indicated that tabtoxin inhibits glutamine synthetase (EC 6.3.1.2) in vitro. We report here that tabtoxin also inhibits glutamine synthetase in vivo. The main evidence was that assimilation of exogenous ¹⁵NH₃ into Asparagus sprengeri protein was rapidly inhibited in isolated cells exposed to tabtoxin. This was associated with an equivalent decline in glutamine synthetase activity in extracts of these cells and the accumulation of extracellular ammonia. Glutamine synthetase was also inhibited in leaves of Nicotiana tabacum L. cv. White Burley treated with tabtoxin and the affected tissue accumulated ammonia and became chlorotic. However, the development of symptoms and accumulation of ammonia was suppressed when the leaves were held in air containing 1% CO₂ to reduce photorespiration. This indicates that the chlorotic symptom did not result from the inhibition of nitrogen assimilation but was a consequence of the interruption of the photorespiratory nitrogen cycle.     

Glycolate metabolism in cyanobacteria

A comparative analysis of glycolate excretion in 11 cyanobacteria showed that 8 strains, although grown and assayed in air, excreted glycolate. The largest quantities were excreted by the filamentous strains Plectonema boryanum 73110 and Anabaena cylindrica (Lemm). The carbon lost by excretion was at most 9% of the net fixed carbon in air for heterocystous cyanobacteria but increased (up to 60%) in some strains under a high pO₂ (0.03 kPa CO₂ in pure O₂). A. cylindrica excreted glycolate at a maximum level of 2 and 10 μmol (mg chl a )⁻¹ h⁻¹ in air and at high pO₂, respectively. The excretion continued for several hours. Increases in light intensity and pO₂ and a shift in pH from 7 to 9 increased the amount of glycolate excreted. A. cylindrica also showed the most O₂-sensitive fixation of CO₂. In vitro activity of phosphoglycolate phosphatase (EC 3.1.3.18) was found in all strains tested, with the highest activities noted for Gloeobacter violaceus 7.82 and Gloeothece 6909 and for young cultures of A. cylindrica . The lowest activities were found in Anabaena 7120 and Anacystis nidulans 625, strains excreting no or only minor quantities of glycolate.     

Effect of atmospheric ammonia on the nitrogen metabolism of Scots pine (Pinus sylvestris) needles

Four-year-old seedlings of Scots pine ( Pinus sylvestris L.) were exposed to filtered air (FA), and to FA supplemented with NH₃ (60 and 240 μg m⁻³) in controlled-environment chambers for 14 weeks. Exposure to the higher NH₃ concentration resulted in an increased activity of glutamine synthetase (GS, EC 6.3.1.2), and an increase in the concentrations of soluble proteins, total nitrogen, free amino acids and leaf pigments in the needles. The GS activity (μmol g⁻¹ fresh weight h⁻¹) in the needle extract increased to levels 69% higher than in FA and the soluble protein concentration to levels 22% higher. Total nitrogen concentration in the needles was 42% higher than in FA, while the free amino acid concentration was 300% higher, which was caused by an increase in arginine, glutamate, aspartate and glutamine. Chlorophyll a , chlorophyll b and carotenoid concentrations were 29, 38 and 11% higher, respectively. Neither the glutamate dehydrogenase (GDH, EC 1.4.1.2) activity nor the concentrations of free NH₄⁺ and glucose in the needles were affected by exposure to NH₃. After NH₃ fumigation at 240 μg m⁻³ the starch concentration decreased by 39% relative to the FA. The results indicate that the metabolism of Scots pine acclimates to concentrations of NH₃ which are 3 to 10 times higher than the average concentration in areas with intensive stock farming. The possible mechanisms underlying acclimation to NH₃ are discussed.     

Effect of applied benzyladenine on endogenous cytokinin content during the early stages of bud development of kiwifruit

The filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) was able to utilize glutamine as the sole nitrogen source. The addition to ammonium-grown cultures of the irreversible inhibitor of glutamine synthetase activity L-methionine-D, L-sulfoximine (MSX) inhibited cell growth. Supplying glutamine to the culture restored cell growth. This re-established growth was not due to interference by glutamine of MSX uptake by the cells, since glutamine synthetase (GS, EC 6.3.1.2) activity remained completely inhibited by MSX even when glutamine was simultaneously present. Both glutamine and ammonium exerted a negative effect on nitrate reductase (NR. EC 1.7.7.2) and nitrite reductase (NiR, EC 1.7.7.1) in vivo. This negative effect was reversed by MSX. When glutamine was added to MSX-treated cells, intracellular glutamine level was high, but the activity of both reductases remained at a high level. These results suggest that the presence of the active form of glutamine synthetase is required for the in vivo prevention of nitrate assimilation caused by ammonium and glutamine.     

Modification of NO−3 metabolism in heterocysts of the N2-fixing cyanobacterium Anabaena 7120 (ATCC27893)

Abstract The utilization of NO⁻₃, NO⁻₂ and NH⁺₄ was studied in whole filaments and isolated heterocysts of Anabaena 7120 (ATCC27893). NO⁻₃- and NO⁻₂-uptake were detectable in whole filaments but not in heterocysts, whereas NH⁺₄-uptake was detectable in both. Activity of NO⁻₃-reductase was present in cell-free extracts of whole filaments but not of heterocysts, whereas activities of NO⁻₂-reductase and glutamine synthetase were present in both. NO⁻₃-uptake and reductase activities could not be induced in heterocysts even after prolonged incubation in NO⁻₃ medium. It is suggested that NO⁻₃-metabolism in heterocysts is impaired due to a selective and irreversible loss of NO⁻₃-uptake and reductase systems resulting in the abolition of competition for molybdenum cofactor (Mo-Co) and reductant between nitrogenase and NO⁻₃-reductase, and an increase in glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase levels.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Impact of gaseous nitrogen deposition on plant functioning

Dry deposition of NH₃ and NO_x (NO and NO₂) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH₃ dissolves to form NH₄⁺, whereas NO₂ dissolves to form NO₃⁻ and NO₂⁻. The latter compound can also be formed after exposure to NO. There is evidence that NH₃-N and NO_x-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO₃⁻ and NO₂⁻ can be reduced, and NH₄⁺ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH₃ increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH₃ and in vitro GDH activity. The contribution of atmospheric NH₃ and NO₂ deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates ( RGRs ). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.     

Glucocorticoid Stimulation of Glutamine Synthetase Production in Cultured Rat Glioma Cells

Abstract: A sensitive radioisotopic assay has been used to examine the kinetic properties and regulation of biosynthesis of glutamine synthetase in C-6 glioma cultures. The K_m values for glutamate, MgATP, and ammonium ion were 5mM, 14 mM, and 0.042 mM, respectively, when measured at the pH optimum of 7.2. There was an absolute requirement for a divalent metal ion, with 15 mM- Mg²⁺ being the preferred ion at pH 7.2. Activity was completely inhibited after 30 min with 8 mM-L-methionhe sulfoximine. The addition of 1 μM-cortisol to C-6 cultures caused a two to threefold increase in glutamine synthetase specific activity over a 96-h period, while dexamethasone at the same concentration elevated the level some 7-10-fold. This was specific for glucocorticoids, as other steroid hormones or catecholamines did not significantly affect glutamine synthetase specific activity. Cycloheximide (30 μM) or actinomycin D (0.01 μg/ml) blocked the hormone response. The continued presence of hormone was required in order to maintain an elevated enzyme level. The results suggest that glucocorticoids act to induce glutamine synthetase by stimulating new enzyme synthesis.     

Regional Distribution of Prostaglandins D2, E2, and F2α and Related Enzymes in Postmortem Human Brain

Abstract: The presence of prostaglandins D₂, E₂, and F_2α was demonstrated and their contents measured in various regions of postmortem human brain, pineal body, and pituitary by using specific radioimmunoassays and gas chromatography-mass spectrometry. The three prostaglandins were widely distributed in similar concentrations ranging from several hundred pg/g wet weight to about 40 ng/g wet weight. Prostaglandins D₂ and E₂ showed consistent and similar regional distributions in all six brains tested; amounts were high in pineal body, pituitary, olfactory bulb, and hypothalamus. On the other hand, prostaglandin F_2α was distributed more evenly. Prosta- glandin D synthetase and prostaglandin E synthetase activities were found in cerebrum homogenate from a single subject and were recovered from the 100,000 × g supernatant. The presence of 1 m M glutathione, reduced form, markedly stimulated the activity of prostaglandin E synthetase, but did not affect prostaglandin D synthetase activity. Activity of 15-hydroxyprostaglandin dehydrogenase was found in the cerebrum homogenate and was partially purified. This enzyme required NADP as a cofactor and copurified with prostaglandin E 9-ketoreductase.