Glutamine Synthetase Reaction Research Articles

Nitrogen Metabolism of an Anoxygenic Filamentous Phototrophic Bacterium Oscillocholris trichoides Strain DG-6

—The possible nitrogen sources for Osc. trichoides DG6, a typical strain of the Oscillochloridaceae family, are ammonium, N2, glutamate, asparagine, glycine, and glutamine. The assimilation of molecular nitrogen occurs with the participation of nitrogenase, the structural gene of which, nifH, is located in the gene cluster which also includes the genes of the nifD and nifK nitrogenase subunits and the auxiliary nifB gene. Considering that nifHBDK clusters have been also annotated in the genomes of other members of the Oscillochloridaceae family, including uncultured and candidate taxa, it can be assumed that the ability to fix nitrogen is a property immanent for this entire family. The pathways for assimilating ammonium in the cells grown using different nitrogen sources may differ. Osc. trichoides DG6 growing in a medium containing ammonium assimilated it with the participation of glutamate dehydrogenase, which is determined by a single gene. The expression product of this gene has dual functionality and can be used to implement the reaction with both NAD and NADP. With the growth of Osc. trichoides DG6 on a medium with glutamate as the only nitrogen source all the enzymes necessary for the implementation of the GS‑GOGAT pathway were found in the cells. However, for the glutamine synthetase reaction, ammonium, which was absent in the growth medium, was required. The source of ammonium may be glutamate metabolized through glutamate dehydrogenase.

Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain.

Glutamate is present in the brain at an average concentration—typically 10–12 mM—far in excess of those of other amino acids. In glutamate-containing vesicles in the brain, the concentration of glutamate may even exceed 100 mM. Yet because glutamate is a major excitatory neurotransmitter, the concentration of this amino acid in the cerebral extracellular fluid must be kept low—typically µM. The remarkable gradient of glutamate in the different cerebral compartments: vesicles > cytosol/mitochondria > extracellular fluid attests to the extraordinary effectiveness of glutamate transporters and the strict control of enzymes of glutamate catabolism and synthesis in well-defined cellular and subcellular compartments in the brain. A major route for glutamate and ammonia removal is via the glutamine synthetase (glutamate ammonia ligase) reaction. Glutamate is also removed by conversion to the inhibitory neurotransmitter γ-aminobutyrate (GABA) via the action of glutamate decarboxylase. On the other hand, cerebral glutamate levels are maintained by the action of glutaminase and by various α-ketoglutarate-linked aminotransferases (especially aspartate aminotransferase and the mitochondrial and cytosolic forms of the branched-chain aminotransferases). Although the glutamate dehydrogenase reaction is freely reversible, owing to rapid removal of ammonia as glutamine amide, the direction of the glutamate dehydrogenase reaction in the brain in vivo is mainly toward glutamate catabolism rather than toward the net synthesis of glutamate, even under hyperammonemia conditions. During hyperammonemia, there is a large increase in cerebral glutamine content, but only small changes in the levels of glutamate and α-ketoglutarate. Thus, the channeling of glutamate toward glutamine during hyperammonemia results in the net synthesis of 5-carbon units. This increase in 5-carbon units is accomplished in part by the ammonia-induced stimulation of the anaplerotic enzyme pyruvate carboxylase. Here, we suggest that glutamate may constitute a buffer or bulwark against changes in cerebral amine and ammonia nitrogen. Although the glutamate transporters are briefly discussed, the major emphasis of the present review is on the enzymology contributing to the maintenance of glutamate levels under normal and hyperammonemic conditions. Emphasis will also be placed on the central role of glutamate in the glutamine-glutamate and glutamine-GABA neurotransmitter cycles between neurons and astrocytes. Finally, we provide a brief and selective discussion of neuropathology associated with altered cerebral glutamate levels.

The Effect of pH and Free Mg2+ on ATP Linked Enzymes and the Calculation of Gibbs Free Energy of ATP Hydrolysis

The apparent equilibrium constants, K′, of biochemical reactions containing substrates which bind [Mg2+] unequally can be significantly altered by changes in free intracellular [Mg2+]. Intracellular free [Mg2+] can be estimated by measurements of [citrate]/[isocitrate], a ratio known to vary with tissue free [Mg2+]. The combined equilibrium constant for glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and triose phosphate isomerase for the three reactions (K(GG-TPI)′) was corrected using new binding constants for dihydroxyacetone-phosphate and 3-phosphoglycerate. The result of this calculation is demonstrated in the calculation of the free energy of ATP hydrolysis. In addition, the dependence of the equilibrium constant for the glutamine synthetase reaction on pH and free [Mg2+] was demonstrated. Furthermore, a theory linking the ΔG′ value of mitochondrial complex I−II and the cytosolic ΔG′ value of ATP hydrolysis is discussed with evidence from previous publications.

Metabolism of [ 13N]ammonia in rat lung

Bolus injection of [ 13N]ammonia into the femoral vein of pentobarbital-anesthetized rats was followed by rapid clearance from the blood and first-pass extraction of nearly 30% by the lungs. Of the label present in the lungs at 6 s after injection (about 27% of the dose), more than 20% was in metabolized form. Of the label present in the lungs at 2 min after injection (about 10% of the dose), 18–25% was in ammonia, about 75% was in glutamine (amide) and less than 1% was in glutamate and aspartate. Thus, despite the presence of significant amounts of glutamate dehydrogenase, the overwhelming route for metabolism of ammonia entering the rat lung in vivo was the glutamine synthetase reaction. Lung tissue that was removed 6 s after intravenous injection of [ 13N]ammonia and incubated in Krebs–Ringer glucose medium at 37 °C for 20 min, showed a significant increase (more than one-third), compared to unincubated lung tissue in the quantity of label in glutamine. Between 6 s and 2 min after injection, during which time the total 13N content of the lungs decreased by more than 60%, the maintenance of a quasi-steady state in the concentration of labeled glutamine suggested a short-term balance between formation from extracted ammonia and loss of glutamine into the circulation. Our data support the concept that the lungs are a source of circulating glutamine in the rat. Despite the large fractional extraction of blood-borne [ 13N]ammonia by the lungs, only minute amounts of tracer (0.2–0.6 ppm of the injected dose) were detected in the expired air within the first 5 min after administration of [ 13N]ammonia to anesthetized rats, so that pulmonary excretion was not a significant pathway of ammonia elimination. The present findings emphasize the importance of the lungs in the maintenance of whole-body nitrogen homeostasis and suggest the use of [ 13N]ammonia and 13N-labeled amino acids as non-invasive probes in the study of normal and diseased lung metabolism.

The in vivo Nitrogen Isotope Discrimination Among Organic Plant Compounds.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Escherichia coli γ-Glutamylcysteine Synthetase: TWO ACTIVE SITE METAL IONS AFFECT SUBSTRATE AND INHIBITOR BINDING

Gamma-glutamylcysteine synthetase (gamma-GCS, glutamate-cysteine ligase), which catalyzes the first and rate-limiting step in glutathione biosynthesis, is present in many prokaryotes and in virtually all eukaryotes. Although all eukaryotic gamma-GCS isoforms examined to date are rapidly inhibited by buthionine sulfoximine (BSO), most reports indicate that bacterial gamma-GCS is resistant to BSO. We have confirmed the latter finding with Escherichia coli gamma-GCS under standard assay conditions, showing both decreased initial binding affinity for BSO and a reduced rate of BSO-mediated inactivation compared with mammalian isoforms. We also find that substitution of Mn2+ for Mg2+ in assay mixtures increases both the initial binding affinity of BSO and the rate at which BSO causes mechanism-based inactivation. Similarly, the specificity of E. coli gamma-GCS for its amino acid substrates is broadened in the presence of Mn2+, and the rate of reaction for some very poor substrates is improved. These results suggest that divalent metal ions have a role in amino acid binding to E. coli gamma-GCS. Electron paramagnetic resonance (EPR) studies carried out with Mn2+ show that E. coli gamma-GCS binds two divalent metal ions; Kd values for Mn2+ are 1.1 microm and 82 microm, respectively. Binding of l-glutamate or l-BSO to the two Mn2+/gamma-GCS species produces additional upfield and downfield X-band EPR hyperfine lines at 45 G intervals, a result indicating that the two Mn2+ are spin-coupled and thus apparently separated by 5 A or less in the active site. Additional EPR studies in which Cu2+ replaced Mg2+ or Mn2+ suggest that Cu2+ is bound by one N and three O ligands in the gamma-GCS active site. The results are discussed in the context of the catalytic mechanism of gamma-GCS and its relationship to the more fully characterized glutamine synthetase reaction.

A bioreactor with glutamine synthetase-transfected recombinant HepG2 cells exhibits ammonia removal activity without the need for added cofactors and substrates: Advantage of a cellular bioreactor over enzyme-immobilized beads

The biological efficacy of recombinant human hepatic cell line, glutamine synthetase-transfected HepG2 (GS-HepG2), was examined under various culture conditions in large-scale culture. GS-HepG2 (2×109 cells) were cultured in a circulatory flow bioreactor, and the changes in the concentrations of ammonia, glucose, glutamine, and glutamic acid were checked with normal, glutamic acid-insufficient, and glutamine-excess medium or porcine plasma. Whereas the glutamine synthetase reaction required high concentrations of Mg2+ in vitro, GS-HepG2 removed ammonia in the culture medium without the addition of Mg2+. Similarly, ammonia removal activity was observed in the medium with physiological concentrations of glutamic acid far lower than the enzymeK m. Moreover, product-induced inhibition of the enzyme activity was not observed with addition of excess glutamine to the medium. Ammonia removal activity was also detected in porcine plasma at the level equivalent to that in the culture medium. The substrates and cofactors indispensable to the glutamine synthetase reaction were likely to be generated and/or stored inside the cells. Thus, the bioreactor with recombinant cells catalyzes the reaction without the need for added cofactors and substrates and surpasses the enzyme-immobilized system to perform blood dialysis by selective removal of toxic substances.

Glutamine: Role in the Fetus and Low-birthweight Infant

After completing this article, readers should be able to: 1. Describe the glutamine-glutamate shuttle. 2. Delineate the effects of glutamine supplementation during total parenteral nutrition, chemotherapy, and radiotherapy in animals. 3. Describe the beneficial effects of glutamine supplementation in low-birthweight infants and in critically ill adults. 4. List the primary actions of glutamine in the intestine. The importance of glutamine for optimal growth of cells in culture has been known since the 1950s, when Eagle published his landmark paper in Science .1 His work demonstrated that of all the amino acids, cell growth in culture relied most on glutamine. Numerous subsequent studies have established the importance of glutamine for a variety of metabolic processes in the entire organism and are beginning to demonstrate that nutritional supplementation of this amino acid may be highly beneficial under certain circumstances. Glutamine and glutamate are structurally similar five-carbon amino acids. Glutamine is a neutral amino acid at pH 7.0 that contains an alpha-amino group and a deltaamide group. In glutamate, the amide nitrogen of glutamine is replaced by a carboxy-group, conferring to it a net negative charge at pH 7.0. This at least partially accounts for the transport of these two amino acids across cell membranes via different transport systems. The presence or absence of the amide group is responsible for different metabolic interactions of these amino acids. The array of metabolic processes in which glutamine is involved is highlighted by its relationship to its metabolites and the citric acid cycle (Fig. 1⇓ ).Glutamine, via glutamate, is readily converted to alpha-ketoglutarate, an integral component of the citric acid cycle. Deamidation of glutamine via glutaminase produces glutamate, a precursor of gamma-amino butyric acid (GABA), a neurotransmission inhibitor. Proline also is produced by the cyclization of glutamate. Proline is an important amino acid component of collagen and …

Characteristics and efficiency of glutamine production by coupling of a bacterial glutamine synthetase reaction with the alcoholic fermentation system of baker's yeast.

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker's yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.

Advances in the enzymology of glutamine synthesis.

Meister's proposal of a gamma-glutamyl-P intermediate in the glutamine synthetase reaction set the scene for understanding how the stepwise activation of the carboxyl group greatly increased its susceptibility toward nucleophilic attack and amide bond synthesis. Topics covered in this review include: the discovery of the enzymatic synthesis of glutamine; the role of glutamine synthetase in defining the thermodynamics of ATPases; early isotopic tracer studies of the synthetase reaction; the proposed intermediacy of gamma-glutamyl-phosphate; the mechanism of methionine sulfoximine inhibition; stereochemical mapping of the enzyme's active site; detection of enzyme reaction cycle intermediates; borohydride trapping of gamma-glutamyl-P; positional isotope exchanges catalyzed by glutamine synthetase; regulation of bacterial enzyme; and a brief account of how knowledge of the atomic structure of bacterial glutamine synthetase has clarified ligand binding interactions. Concluding remarks also address how the so-called "Protein Ligase Problem" may be solved by extending the catalytic versatility of carboxyl-group activating enzymes.

The Rabbit Kidney Tubule Simultaneously Degrades and Synthesizes Glutamate: A 13C NMR STUDY

The rabbit kidney does not readily metabolize but synthesizes glutamine at high rates by pathways that remain poorly defined. Therefore, the metabolism of variously labeled [13C]- and [14C]glutamates has been studied in isolated rabbit kidney tubules with and without acetate. CO2, glutamine, and alanine were the main carbon and nitrogenous end products of glutamate metabolism but no ammonia accumulated. Absolute fluxes through enzymes involved in glutamate metabolism, including enzymes of four different cycles operating simultaneously, were assessed by combining mainly the 13C NMR data with a new model of glutamate metabolism. In contrast to a previous conclusion of Klahr et al. (Klahr, S., Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be initiated by glutamate dehydrogenase at high rates. Glutamate dehydrogenase also operated at high rates in the reverse direction; this, together with the operation of the glutamine synthetase reaction, masked the release of ammonia. Addition of acetate stimulated the operation of the "glutamate --> alpha-ketoglutarate --> glutamate" cycle and the accumulation of glucose but reduced both the net oxidative deamination of glutamate and glutamine synthesis. Acetate considerably increased flux through alpha-ketoglutarate dehydrogenase and citrate synthase at the expense of flux through phosphoenolpyruvate carboxykinase; acetate also caused a large decrease in flux through alanine aminotransferase, pyruvate dehydrogenase, and the "substrate cycle" involving oxaloacetate, phosphoenolpyruvate, and pyruvate.

Growth of cultured rabbit renal tubular cells does not require exogenous glutamine

Following the pioneering work of Eagle et a! [1], glutamine, which is the most abundant amino acid in plasma (0.5 to 0.6 mM) [2] and an important source of energy and nitrogen for cell metabolism and growth [3], is considered to be a mandatory component of cell culture media. In renal epithelial cell cultures, it is added often at a final concentration of 2 to 5 mrvi [4—10]. However, this is not necessarily useful in primary cultures of renal proximal tubular cells from the rabbit, the most widely used species for renal cell cultures. Indeed, glutamine synthetase is present in the renal cortex of this species; in addition, the activity of phosphate-activated glutaminase, the enzyme which initiates glutamine metabolism and yields glutamate and ammonium [3], is low in the rabbit kidney [2]. Moreover, freshly isolated rabbit proximal tubules do not or hardly remove glutamine in net amounts when they are incubated in the presence of glutamine concentrations varying from 1 to 5 mtvi [11]. In an attempt to clarify this point, we cultured rabbit proximal tubular cells on a permeable collagen membrane and investigated whether the addition of glutamine to the culture medium influences cellular growth and glycolysis. The results obtained show that omission of exogenous glutamine does not alter the growth of rabbit renal proximal tubular cells in primary culture, and that the provision of glutamine by the glutamine synthetase reaction is sufficient and mandatory for their growth and DNA synthesis. Addition of glutamine, which stimulated glycolysis in freshly isolated proximal tubules, failed to do so in cultured rabbit renal cells.

In vivo inhibition of nitrogenase by hydroxylamine in Rhodospirillaceae Role of nitric oxide

Rhodobacter capsulatus strains E1F1 and B10 and Rhodobacter sphaeroides DSM 158 did not use hydroxylamine as nitrogen source for growth but metabolized it mainly through the glutamine synthetase reaction. Hydroxylamine had a high toxicity for cells growing either under phototrophic or dark-aerobic conditions. l-methionine-d,l-sulfoximine partially inhibited hydroxylamine uptake and increased the inhibition time of nitrogenase activity by this nitrogen compound. Nitric oxide was also a powerful inhibitor of nitrogenase in intact cells of R. capsulatus. Since low amounts of NO were produced from hydroxylamine, short-term inhibition of nitrogenase in the presence of this compound could be mediated in vivo by nitric oxide.

Usefulness of immobilized glutamine synthetase for the removal of ammonia and its application to the determination of creatinine in urine

A method for removing ammonia from a reaction mixture by using glutamine synthetase and its application to the automated determination of urinary creatinine using creatinine deiminase and glutamate dehydrogenase are described. The method for determining creatinine in urine is based on measuring the decrease in absorbance, at 340 nm, of NADPH produced by successive reactions of creatinine deiminase and glutamate dehydrogenase with creatinine in a reaction mixture from which ammonia has been removed by the glutamate synthetase reaction. The three enzymes were immobilized and used in column form. The method did not require any blank correction, because concentrations of up to 150 mmol l −1 of ammonia in a sample could be removed by the glutamine synthetase reaction when a concentration of 50 mmol l −1 of glutamate in a recipient buffer (Tri-HCI) was used. The method was found to give perfect linearity of the data with an accurate reaction recovery ranging from 99.5% to 100.2% for up to 18 mmol l −1 creatinine. The within-day and day-to-day relative standard deviations were 2.1% and 3.0%, respectively, for 4.1 mmol l −1 creatinine and 1.2% and 1.8%, respectively, for 18.7 mmol l −1 creatinine. The immobilized enzyme reactor unit showed good operational stability for a 2-month period, during which time it was used repeatedly (over 2000 times) for analyses. The results correlated satisfactorily with those obtained by the modified Jaffe reaction rate method.

Methionine sulfoximine intensifies cancer anorexia

Consistent anorexia was first observed 33 days after inoculating Fischer 344 rats with methylcholanthrene-induced sarcoma. Daily treatment of a similar group of rats with the glutamine synthetase inhibitor, methionine sulfoximine, elicited significant reductions of feeding by day 29 at a dose that had no effect on nontumor-bearing rats. Blood concentrations of ammonia were elevated in both groups of tumor-bearing rats and brain ammonia level was increased in the methionine sulfoximine-treated tumor-bearing rats. Forebrain concentrations of tyrosine, tryptophan, DOPAC and 5-HIAA were elevated in both groups of tumor-bearing rats. Since ammonia is detoxified through the glutamine synthetase reaction, these results suggest that blood and brain ammonia concentrations are more important than the neurochemical consequences of ammonia detoxification for the etiology of cancer anorexia.

Effect of metal ions and adenylylation state on the internal thermodynamics of phosphoryl transfer in the Escherichia coli glutamine synthetase reaction.

Experiments were conducted to study the differences in catalytic behavior of various forms of Escherichia coli glutamine synthetase. The enzyme catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia via a gamma-glutamyl phosphate intermediate. The physiologically important metal ion for catalysis is Mg2+; however, Mn2+ supports in vitro activity, though at a reduced level. Additionally, the enzyme is regulated by a covalent adenylylation modification, and the metal ion specificity of the reaction depends on the adenylylation state of the enzyme. The kinetic investigations reported herein demonstrate differences in binding and catalytic behavior of the various forms of glutamine synthetase. Rapid quench kinetic experiments on the unadenylylated enzyme with either Mg2+ or Mn2+ as the activating metal revealed that product release is the rate-limiting step. However, in the case of the adenylylated enzyme, phosphoryl transfer is the rate-limiting step. The internal equilibrium constant for phosphoryl transfer is 2 and 5 for the unadenylylated enzyme with Mg2+ or Mn2+, respectively. For the Mn2(+)-activated adenylylated enzyme the internal equilibrium constant is 0.1, indicating that phosphoryl transfer is less energetically favorable for this form of the enzyme. The factors that make the unadenylylated enzyme most active with Mg2+ are discussed.

Metabolic Encephalopathy

Disorders of metabolism are the most common cause of coma of unknown etiology. Hyperammonemia is important as an etiologic factor in the development of hepatic encephalopathy, operating via direct and indirect mechanisms that affect the function of the limbic system. The entry of ammonia into the brain is controlled by blood-brain pH gradients, and cerebral blood flow, coupled with regional variations in the capillary surface area-permeability product. In the brain, ammonium ions may inhibit the generation of action potentials, by substituting for potassium and sodium in current generation, and interfere with the chloride pump, producing a reversible depolarizing shift of the inhibitory post synaptic equilibrium potential toward the equilibrium potential. Ammonia entering the brain is quickly trapped by the ATP-consuming glutamine synthetase reaction leading to energy depletion in the reticular activating system. Ammonia may also interfere with ATP production due to a suspected inhibition of the malate aspartate shuttle, the mechanism for moving reducing equivalents into mitochondria for oxidative phosphorylation. Ammonia also depletes glutamate, causing a potential disruption of glutamatergic neurotransmission. We recently evaluated the effects of chronic portacaval shunts (PCS) and ammonia on regional brain glucose metabolism using the 14C-deoxyglucose technique and found important direct and indirect effects on the limbic system. After a PCS, glucose metabolism was significantly increased in all 20 brain regions that were sampled, with the smallest increase in the cortex and the greatest increase in the reticular activating system. The pattern of glucose metabolism appeared to be different in the two groups, an impression that was confirmed by multivariate statistical analytical techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of non-autotrophic carbon dioxide assimilation by ammonia in cultured cells of Acer pseudoplatanus L

The assimilation of 14CO 2 by Acer pseudoplatanus cells in the dark was stimulated by the addition of either NH 4Cl or methylamine. Results were obtained demonstrating that methylamine was not metabolised to any appreciable extent by Acer cells. This suggests that the mechanism of stimulation of dark fixation by methylamine does not involve metabolism via the glutamine synthetase reaction. NH 4 + stimulation of CO 2 fixation also occurred in cells pretreated with the glutamine-synthetase inhibitor methionine sulfoximine. This further supports the conclusion that neither NH 4 + nor methylamine exerts its effect on CO 2-assimilation via a mechanism that depends upon the assimilation of NH 3 by glutamine synthetase.

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 μmol m(-2) s(-1) PAR during incubation with the leaf base in 2 mM (15)NH4Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. (15)N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO2 assimilation (8.3 and 17.4 μmol m(-1) s(-1) at 150 and 1350 μmol m(-2) s(-1) PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% (15)N. In the high light regime the amino acids contained up to 15% (15)N, whereas in low light (15)N enrichments were small (up to 6%). The kinetics of (15)N incorporation indicated that NH3 was firstly assimilated into glutamine and then into glutamate. After 15 min (15)N was also found in glycine, serine and alanine. At high light intensity nearly half of the (15)N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant (15)N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.