Purification and characterization of glutamine synthetase from Rhodomicrobium vannielii

Abstract Glutamine synthetase (GS) from the purple non-sulfur bacterium Rhodomicrobium vannielii has been purified to electrophoretic homogeneity by affinity chromatography. Molecular weight and catalytic properties of the enzy,e are similar to those described for other species of Rhodospirillaceae. However, the enzyme from this organism appears to be antigenically different from the glutamine synthetases of other species of Rhodospirillaceae studied.     

Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups

Abstract: The structure of glutamine synthetase (GS) enzymes from diverse bacterial groups fall into three distinct classes. GSI is the typical bacterial GS, GSII is similar to the eukaryotic GS and is found together with GSI in plant symbionts and Streptomyces , while GSIII has been found in two unrelated anaerobic rumen bacteria. In most cases, the structural gene for GS enzyme is regulated in response to nitrogen. However, different regulatory mechanisms, to ensure optimal utilization of nitrogen substrates, control the GS enzyme in each class.     

Evolutionary relationships of bacterial and archaeal glutamine synthetase genes

Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII. Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria. GSIII genes have been found in only a few bacterial species. Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred. In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii. Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-a and GSI-. GSIa-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles). GSI--type genes occur in all other bacteria. GSI-- and GSI--type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter. Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it. The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision. The S. solfataricus GSI gene has a shorter GSI--type insertion, but like GSI-a-type genes, lacks conserved sequences about the adenylylation site. We suspect that the similarity of GSI- genes from Euryarchaeota and several bacterial species does not reflect a common phylogeny but rather lateral transmission between archaea and bacteria.Correspondence to: J.R. Brown 1073     

Azospirillum brasilense glutamine synthetase: influence of the activating metal ions on the enzyme properties

The kinetic properties of the Mg²⁺-activated and Mn²⁺-activated glutamine synthetase (GS) of Azospirillum brasilense in the biosynthetic reaction were studied. The Mg²⁺-supported and Mn²⁺-supported GSs in an average state of adenylylation varied in pH optimum, maximum activity, saturation functions for ammonium and glutamate, affinity to substrates, and in the Me²⁺-ATP ratio required for the optimal enzyme activity. Seventeen other cations were tested for the maintenance of GS activity. The level of the latter and the kinetic behavior of the GS in A.brasilense is suggested to depend essentially on the concentrations of Mg²⁺, Mn²⁺ and Co²⁺, as well as on their ratio     

Chloroplastic glutamine synthetase from tobacco leaves: a glycosylated protein

The amino acid and carbohydrate content of chloroplastic glutamine synthetase from tobacco leaves has been analysed. The enzyme subunit contanins 5% carbohydrate, mainly represented by glucosamine, galactosamine, glucose, galactose and mannose residues. The enzyme subunit displayed a single band of molecular mass 44000 Da after sodium dodecyl sulphate (SDS) electrophoresis. However, when isoelectrofocussing electrophoresis was performed, four subunits were evident differing by their charge. Furthermore, the four different subunits stained positively when tested with periodic acid Shiff reagent, showing that sugars and amino sugars were present within all the subunits.     

Glutamine synthetase and glutamine synthetase-like protein from human brain: purification and comparative characterization

Glutamine synthetase (GS; EC 6.3.1.2), a key enzyme of glutamate metabolism, and another enzyme possessing high hydroxylamine-L-glutamine transferase activity comparable to that of GS and termed GS-like protein (GSLP) were purified from human brain concurrently. In two-dimensional electrophoresis, GS subunits migrate to at least six different positions (44 +/- 1 kDa, pl = 6. 4-6.7), whereas GSLP subunits migrate to at least four different positions (54 +/- 1 kDa, pl = 5.9-6.2). Dependences of enzymatic activity in the transferase reaction on concentrations of Mn(2+) and Mg(2+) for GS and GSLP are different. High immunological cross-reactivity between GS and GSLP was observed in ELISA. Nevertheless, antisera were raised to GS and GSLP, and a method was developed for the separate detection of GS and GSLP in brain extracts by enzyme-chemiluminescent amplified (ECL) immunoblotting. The distribution of GS and GSLP immunoreactivities between soluble protein and crude mitochondrial fractions indicates tighter association with the particulate fraction for GSLP than for GS. The results from activity measurements suggest that the hydroxylamine-L-glutamine transferase activity measured routinely in protein extracts from brain is the sum of GS and GSLP activities. Similarly, immunoreactivity evaluated by ELISA is a sum of immunoreactivities of GS and GSLP. The relative contributions of GS and GSLP to the total immunoreactivity can be evaluated by ECL-immunoblotting.     

Immunogold localization of glutamine synthetase in soybean leaves,roots, and nodules

Summary Immunogold labelling was used to detect the cellular and sub-cellular distribution of glutamine synthetase (GS) in nodulatedGlycine max var. maple arrow. The protein was detected in thin sections of tissue embedded in LR white acrylic resin by employing two polyclonal antibody preparations, one active chloroplastic GS, the other against the cytosolic form of the enzyme. In the mature leaf tissue, GS was visualized only in the chloroplasts, exclusively within the stroma matrix; in the root cortical tissue, the enzyme was distributed homogenously in the cytosol but with a slight preferential localization associated with certain endomembranes, whereas in the root nodules both cytosolic and plastidial compartments were labelled in infected and uninfected cells. Particular to the infected cells, the bacteroids' inner matrix reacted slightly to the GS antibody and a strong signal was preferentially localized on the bacteroids' outer envelope membranes. In general, GS was more concentrated in nodules as estimated by gold particle distribution, whether in the cytosol, plastids or on the bacteroid envelope membranes, than in either root tissue or leaf tissue. Although the cytoplasmic labelling density in nodules was similar in uninfected and infected cells, certain structural features in the latter (abundant cytosol, numerous GS-positive bacteroids and GS-reactive proplastids) contribute to a more enzyme-rich type than its uninfected counterpart.Abbrevation GS glutamine synthetase     

Metabolic characteristics of recombinant Chinese hamster ovary cells expressing glutamine synthetase in presence and absence of glutamine

To elucidate the metabolic characteristics of recombinant CHO cells expressing glutamine synthetase (GS) in the medium with or without glutamine, the concentrations of extra- and intracellular metabolites and the activities of key metabolic enzymes involved in glutamine metabolism pathway were determined. In the absence of glutamine, glutamate was utilized for glutamine synthesis, while the production of ammonia was greatly decreased. In addition, the expression of recombinant protein was increased by 18%. Interestingly, the intracellular glutamine maintained almost constant, independent of the presence of glutamine or not. Activities of glutamate-oxaloacetate aminotransferase (GOT), glutamate-pyruvate aminotransferase (GPT), and glutamate dehydrogenase (GDH) increased in the absence of glutamine. On the other hand, intracellular isocitrate and the activities of its downstream isocitrate dehydrogenase in the TCA cycle increased also. In combination with these two factors, a 8-fold increase in the intracellular α-ketoglutarate was observed in the culture of CHO-GS cells in the medium without glutamine.     

Purification and properties of glutamine synthetase from the archaebacterium Halobacterium salinarium

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the halophilic archaebacterium Halobacterium salinarium. The enzyme was purified 300-fold to homogeneity with 30% yield. By gel filtration and SDS gel electrophoresis, it was shown that the enzyme has a native molecular weight of 495,000 and a subunit molecular weight of 62,000. This indicates an octameric quaternary structure. The amino acid composition and the isoelectric point of 4.9 are similar to other GSs. The enzyme shows highest stability in 4 M NaCl or KCl and at temperatures up to 45°C. Lower salt concentrations or higher temperatures lead to rapid and irreversible denaturation. By low concentrations of Mg²⁺ or Mn²⁺, the salt dependence was decreased and the thermostability increased. Mg²⁺ or Mn²⁺ are essential cofactors. The two resulting activities show differences in pH and salt concentrations required for optimal activity, different K _m-values and different sensitivity to inhibition by amino acids. The enzyme is not adenylylated like the GS from some eubacteria but cytidylylated. The covalently bound CMP increases Mn²⁺-and Mg²⁺-dependent activities at a different extent.     

The activation of glutamine synthetase from the cyanobacterium Anabaena cylindrica by thioredoxin

Abstract The present communication defines the conditions under which thioredoxin activates glutamine synthetase from Anabaena cylindrica . Effects are obtained at pH values around neutrality, and the activation is affected by Mg²⁺ in the assays. The thioredoxin systems from A. cylindrica and spinach are functionally interchangeable in the activation of glutamine synthetase. The enzyme is efficiently activated by thioredoxin_m and also by thioredoxin_f, but at much higher concentrations. Thioredoxin_m has previously been shown to activate NADPH-dependent malate dehydrogenase and isocitrate dehydrogenase from cyanobacteria. It is speculated that thioredoxin_m plays a role in the differentiation of vegetative cells to heterocysts.     

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants

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

Role of glutamine as a direct co-repressor of glutamine synthetase in Rhodobacter capsulatus E1F1

High performance liquid chromatography (HPLC) has been used to determine the internal levels of amino acids in Rhodobacter capsulatus E1F1 cells, subjected to different treatments and nutritional conditions. Glutamine synthetase activity and enzyme concentration correlated negatively with the level of glutamine, suggesting that glutamine per se acts as a co-repressor in the enzyme synthesis. Moreover, addition of the specific inhibitor L-methionine-D,L-sulfoximine, that produced an increase in enzyme concentration, specifically promoted a depletion of intracellular glutamine.     

Proteasome accessory factor A (PafA) transferase activity makes sense in the light of its homology with glutamine synthetase

The Pup-proteasome system (PPS) is a prokaryotic tagging and degradation system analogous in function to the ubiquitin-proteasome system (UPS). Like ubiquitin, Pup is conjugated to proteins, tagging them for proteasomal degradation. However, in the PPS, a single Pup-ligase, PafA, conjugates Pup to a wide variety of proteins. PafA couples ATP hydrolysis to formation of an isopeptide bond between Pup and a protein lysine via a mechanism similar to that used by glutamine synthetase (GS) to generate glutamine from ammonia and glutamate. GS can also transfer the glutamyl moiety from glutamine to a hydroxyl amine in an ATP-independent manner. Recently, the ability of PafA to transfer Pup from one protein to another was demonstrated. Here, we report that such PafA activity mechanistically resembles the transferase activity of GS. Both PafA and GS transferase activities are ATP-independent and proceed in two catalytic steps. In the first step catalyzed by PafA, an inorganic phosphate is used by the enzyme to depupylate a Pup donor, while forming an acyl phosphate Pup intermediate. The second step consists of Pup conjugation to the new protein, alongside the release of an inorganic phosphate. Detailed experimental analysis, combined with kinetic modeling of PafA transferase activity, allowed us to correctly predict the kinetics and magnitude of Pup transfer between two targets, and analyze the effects of their affinity to PafA on the efficiency of transfer. By deciphering the mechanism of the PafA transferase reaction in kinetic detail, this work provides in-depth mechanistic understanding of PafA, a key PPS enzyme.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Some properties of glutamine synthetase from Anabaena cylindrica

Some properties of the biosynthetic and -glutamyltransferase activities of glutamine synthetase (EC 6.3.1.2) from Anabaena cylindrica are described, including requirement for divalent cations, pH optimum and K_m for substrates. The -glutamyl-transferase reaction was inhibited by L-glutamate, ammonia and ATP. The inhibition by L-glutamate and ammonia was competitive for L-glutamine and non-competitive for hydroxylamine. Both the biosynthetic and the -glutamyltransferase activities of the desalted enzyme were much more sensitive to inactivation by treatments such as urea, hydroxylamine and incubation at 50° C than the preparation which contained a divalent cation. The effects of some substrates of these reactions on protection against thermal denaturation and hydroxylamine were examined. An interpretation of these results in terms of the sequence of binding of substrates both in the biosynthetic and the -glutamyltransferase reactions are discussed.     

Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

 Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28–30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76±0.78 mg·g⁻¹ wet tissue in normal unexposed rats; 15.82±2.30 mg·g⁻¹ wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure. Glutamine synthetase activity in muscle was significantly higher in the 14-day exposed group (4.32 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹) in comparison to normal (1.53 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹); this parameter had decreased by 40% following 21 days of exposure. These results suggest that since no dramatic changes in the levels of protein were observed in the muscle and liver, there is an alteration in glutaminase and glutamine synthetase activity in order to maintain nitrogen metabolism in the initial phase of hypoxic exposure. Received: 30 March 1998 / Revised: 18 November 1998 / Accepted: 25 November 1998