Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Control of glutamate dehydrogenase from Lemna minor by divalent metal ions

The NADH and NAD⁺ dependent reactions catalyzed by glutamate dehydrogenase (GDH) from sterile cultures of Lemna minor are completely inactivated by EDTA. The activities of both reactions can be fully restored by addition of Ca²⁺ and to a lesser extent Mn²⁺, Zn²⁺, Sr²⁺ or La³⁺, whereas Mg²⁺ reactivates only the NAD⁺ dependent reaction. Activation of the NADH reaction by Ca²⁺ has been studied by using partially purified, EDTA pretreated, and Mg²⁺ saturated GDH preparations. Saturation kinetic curves with Ca²⁺ were always sigmoidal, whereas saturation plots for the 3 substrates of the aminating reaction at various fixed Ca²⁺ concentrations showed normal Michaelis kinetics. However, a pronounced substrate inhibition at low Ca²⁺ levels was found, particularly with NH₄⁺ and NADH. Product inhibition studies revealed unchanged enzyme substrate binding characteristics for NADH and 2-oxoglutarate in the Ca²⁺ free enzyme. A drastic alteration was established for the third substrate NH₄⁺. The kinetic data suggest that Ca²⁺ governs an equilibrium between a catalytically inactive (Ca²⁺ free) and an active (Ca²⁺ saturated) enzyme form. Inactivation by removal of Ca²⁺ is related to an alteration in the binding characteristics or binding sequence of the substrate NH₄⁺.     

Control of methionine synthesis by lysine in Lemna minor

When Lemna minor was cultured in the presence of 0.25 mM l-lysine, the concentration of free methionine and formyl and methyl tetrahydrofolate (THFA) were decreased. l-lysine, l-homoserine, l-threonine and l-methionine at concentrations up to 8 mM did not affect N¹⁰-formyl THFA synthetase (E.C. 6.3.4.3) and N⁵,N¹⁰-methylene THFA reductase (E.C. 1.1.1.68). In contrast, serine hydroxymethyltransferase (E.C. 2.1.2.1) activity was inhibited by lysine. This inhibition gave a sigmoidal curve when plotted for a range of l-lysine or THFA concentrations. Exogenous lysine also reduced the incorporation of glycine [¹⁴C] and serine [3-¹⁴C] into free and protein methionine. Lysine, which is known to control synthesis of homocysteine in L. minor, may also regulate production of C-1 units for methionine synthesis by inhibition of serine hydroxymethyltransferase.     

Changes in the levels of enzymes involved in ammonia assimilation during the development of Phaseolus vulgaris seedlings.Effects of exogenous ammonia

Seeds of Phaseolus vulgaris L. cv. White Kidney were germinated and grown either in a nitrogen-free or in an ammonia-supplied medium. The changes in the soluble protein concentration and in the levels of glutamine synthetase (GS, EC 6.3.1.2), NADH–glutamate synthase (NADH-GOGAT, EC 1.4.1.14), ferredoxin-glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2), both NADH- and NAD⁺-dependent, were examined in cotyledons and roots during the first 10 days after sowing. Soluble protein declined rapidly in the cotyledons and increased slightly in the roots. GS activity was initially high both in cotyledons and roots but subsequently decreased during seedling growth. Exogenous ammonia hardly affected GS activity. High levels of NADH-GOGAT were present both in cotyledons and roots during the first days of germination. The activity then gradually declined in both organs. In contrast, Fd-GOGAT in cotyledons was initially low and progressively increased with seedling development. In roots, the levels of Fd-GOGAT were higher in young than in old seedlings. Supply of ammonia to the seedlings increased the levels of NADH-GOGAT and Fd-GOGAT both in cotyledons and roots. NADH-GDH (aminating) activity gradually increased during germination. In contrast, the levels of NAD⁺-GDH (deaminating) activity were highest during the first days of germination. Exogenous ammonia did not significantly affect the activities of GDH.     

Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant

In the wild-type of Corynebacterium glutamicum, the specific activity of glutamate dehydrogenase (GDH) remained constant at 1.3 U (mg protein)–1 when raising the ammonia (NH4) concentration in the growth medium from 1 to 90 mM. In contrast, the glutamine synthetase (GS) and glutamate synthase (GOGAT) activities decreased from 1.1 U (mg protein)–1 and 42 mU (mg protein)–1, respectively, to less than 10 % of these values at NH4 concentrations > 10 mM suggesting that under these conditions the GDH reaction is the primary NH4 assimilation pathway. Consistent with this suggestion, a GDH-deficient C. glutamicum mutant showed slower growth at NH4 concentrations 10 mM and, in contrast to the wild-type, did not grow in the presence of the GS inhibitor methionine sulfoximine. © Rapid Science Ltd. 1998     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.     

Manipulating the pathway of ammonia assimilation through genetic engineering and breeding: consequences to plant physiology and plant development

In this article we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation have been increased by the use of leguminous and non-leguminous plants with modified capacities for ammonium assimilation. These modifications have been achieved through genetic engineering and breeding. An improved understanding of nitrogen assimilation will be vital if improvements in crop nitrogen use efficiency are to be made to reduce the need for excessive input of fertilisers. In this review we present an overall view of past work and more recent studies on this topic. In our work, using tobacco and Lotus as model plants, glutamine synthetase and glutamate synthase activites have been altered by stimulating or inhibiting in an organ- or tissue-specific manner the expression of the corresponding genes. The physiological impact of these genetic manipulations has been studied on plants grown under different nitrogen regimes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glutamine synthetase and glutamate synthase from Sclerotinia sclerotiorum

Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) were purified from Sclerotinia sclerotiorum and some of their properties studied. The GS transferase and biosynthetic activities, as well as GOGAT activity, were sensitive to feedback inhibition by amino acids and other metabolites. GS showed a marked dependence on ADP in the transferase reaction and on ATP in the Mg²⁺-dependent biosynthetic reaction. Regulation of GS activity by adenylylation/deadenylylation was demonstrated by snake venom phosphodiesterase treatment of the purified enzyme. GOGAT required NADPH as an electron donor; NADH was inactive. GOGAT was strongly inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine. The enzyme was also markedly inhibited by o-phenanthroline, 2,2′-bipyridyl and azaserine. l-Methionine-dl-sulphoximine (MSX) and azaserine inhibited the incorporation of ¹⁵N-labelled ammonium sulphate into washed cells of S. sclerotiorum. MSX and azaserine respectively also inhibited purified GS and GOGAT activities. GDH activity was not detected in cell-extracts. Thus the GS/GOGAT pathway is the main route for the assimilation of ammonium compounds in this fungus.     

Effect of nitrogen starvation on ammonium assimilation by Chlamydomonas reinhardtii

In nitrogen-starved Chlamydomonas reinhardtii , wild type, strain 21 gr cells, consumption of nitrate, nitrite and ammonium may occur in the dark in the absence of an added carbon source. Consumption of ammonium in the dark was about 25% higher than in the light, while consumption of nitrate or nitrite in the dark was lower than in the light.
N starvation produced a linear increase with time in the intracellular level of glutamine synthetase (GS, EC 6.3.2.1) and glutamate synthase (NADH-GOGAT, EC 1.4.1.14 and ferredoxin-GOGAT, EC 1.4.7.1) activities in C. reinhardtii . The effect on GS₁ (3-fold) and NADH-GOGAT (4.5-fold) was higher than that on GS₂ (1.5-fold) and ferredoxin-GOGAT (1.5-fold).
Experiments with methylammonium, L-methionine-D, L-sulfoximine (MSX) and azaserine suggest that: 1) Ammonium itself decreases the intracellular levels of glutamine synthetase and ferredoxin-glutamate synthase activities; and 2) a metabolite resulting from ammonium assimilation by the alga may be a negative modulator of NADH-glutamate synthase activity.     

Effects of medium glutamine,glutamate, and ammonia on glutamine synthetase activity in cultured mouse astroglial cells

Mouse astroglial cells were grown during the last week of culture in either glutamine-free or glutamine-containing medium. The addition of cortisol to the glutamine-containing medium resulted in a doubling of astroglial glutamine synthetase (GS) activity. Withdrawal of glutamine from the medium resulted in a 50% elevation of GS and addition of cortisol to such a medium resulted in a further increase in GS which was not additive to glutamine withdrawal. Both in glutamine-free and glutamine-containing medium, the addition of glutamate resulted in a depression of both basal and cortisol induced GS activity. The simultaneous addition of ammonia plus glutamate to the culture medium ameliorated the glutamate mediated depressive effects on cortisol induced but not basal GS activity. Glutamine withdrawal from the culture medium resulted in an astroglial protein deficit. The addition of ammonia to the medium considerably reduced this deficit and the addition of glutamate completely eliminated this protein deficit.     

Allogibberic acid: An inhibitor of flowering in Lemna perpusilla

Allogibberic acid (I) has been identified as the compound responsible for the inhibition of flowering, increase in frond multiplication rate and decrease in frond size produced in Lemna perpusilla 6746 by autoclaved, unbuffered aqueous solutions of gibberellic acid (VII). 13-Deoxyallogibberic acid (IV), a product of autoclaving aq. GA₇ (VIII) solutions, also inhibits flowering in L. perpusilla and is about 10 times more active than allogibberic acid.     

Effect of nitrogen sources upon the activity of l-glutamate dehydrogenase of Lemna gibba

When Lemna gibba cultures, grown on medium containing l-glutamate as the sole nitrogen source are transferred to medium in which ammonium is the only source of nitrogen, the activity of a NAD-dependent l-glutamate dehydrogenase (GDH) increases approximately 5-fold over 3 days. Upon re-transfer to glutamate medium the activity declines to its initial value after a further 6 days. The rise in activity is independent of the presence of EDTA and is not the result of an increase in the ease with which the enzyme can be extracted. p-Fluoro-dl-phenylalanine, azetidine-2-carboxylic acid and puromycin but not d-threo-chloramphenicol, erythromycin or lincomycin inhibit the increase when included in ammonium medium. These observations, together with those obtained from the use of a deuterium oxide-labelling technique, suggest that the increase in GDH activity is due to de novo synthesis on 80S ribosomes.     

Nitrogen assimilation and partitioning in two nitrogen-fixing cultivars of Phaseolus vulgaris L.

Two cultivars of Phaseolus vulgaris L., one responsive (Mexico 309) and one less-responsive (Rio Tibagi) to nodulation with Rhizobium were grown in Leonard jars in a greenhouse. Bean plants were either inoculated with a strain of Rhizobium leguminosarum bv. phaseoli (UMR-1899), a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus etunicatum) or were left non-inoculated (controls). At two harvests (21 and 28 days post-emergence), extracts containing soluble proteins and free amino acids were prepared from leaves, roots and nodules of field beans. Nodulated plants contained a significantly higher concentration of protein and amino acids in all plant parts. Nitrogen-fixing beans invested a significantly greater proportion of total N as protein-N and amino acid-N as compared to VAM or control beans. Abundant nodule-specific proteins (nodulins) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), identified and quantified using scanning densitometry. Rio Tibagi nodules contained a significantly lower concentration of glutamine synthetase (GS) subunits than did Mexico 309 nodules. Glutamate synthase (GOGAT) and GS activities were low relative to other legumes. The transferase/synthetase ratio for GS was also low indicating that the synthetase activity was caturated and was operating at maximal level in these young N₂-fixing associations. Specific nodule activity (SNA) and the level of GS were correlated (r=0.90, p<0.05) for both cultivars at both harvests. GS activity was only 8 or 24% higher than SNA in nodules of Mexico 309 or Rio Tibagi cultivars, respectively, under conditions where substrate was not limiting. This suggests that early in the functioning of this symbiosis N assimilation by GS is the rate-limiting step in N₂ fixation by these two bean cultivars, each with a different symbiotic efficiency. Phaseolus breeding programs that attempt to improve N₂ fixation in beans should identify germplasm that expresses elevated levels of nodule-specific GS or GOGAT, and this material should be used along with effective R. leguminosarum bv. phaseoli strains that have already been selected, to determine superior host-microsymciont associations.     

水稻种子萌发期间谷氨酰胺合成酶和NADH-谷氨酸合酶的变化

测定了水稻种子不同萌发时期胚乳、胚芽鞘和幼根的谷氨酰胺合成酶（GS）和依赖于NADH的谷氨酸合酶（NADH－GOGAT）活性变化。胚乳和胚芽鞘的GS活性在萌发过程中升高,幼根的GS活性则有所降低。NADH－GOGAT的活性变化趋势与GS相同。Native-PAGE活性染色表明,在萌发阶段的水稻种子胚乳和幼根里,始终只观察到一种GS活性带。但是,在水稻种子萌发3d后,在胚芽鞘中除继续检测到GS1的活性外,还可以观察到GS2的活性。蛋白质印迹显示,水稻种子胚乳中的GS（GSe)和GS1和GSra一样是一种胞质型GS。实验结果提示,这些不同组织中的GS与NADH－GOGAT构成的循环途径也许是水稻种子萌发时氨同化的主要途径。     

Lipoxygenase in Vicia faba minor

Lipoxygenase activity was demonstrated in partially purified preparations from small faba beans. The enzyme was shown to possess a pH optimum of 6·5 and was inactivated by exposure to 70° for 15 min. The K_m value for linoleic acid was calculated to be 0·57 mM. Ammonium sulphate fractionation yielded two highly active preparations, which were both active towards linoleic and linolenic acids. Neither fraction was inhibited by either cyanide or p-chloromercuribenzoate. The two fractions showed markedly differing responses to calcium ions, suggesting the presence of two lipoxygenases in faba beans. Activation of the enzyme by calcium ions was eliminated by the addition of EDTA.