Presence Of Glutamine Synthetase Research Articles

Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites.

Plasmodium falciparum (Pf) is a major cause of human malaria and is transmitted by infected Anopheles mosquitoes. The initial asymptomatic infection is characterized by parasite invasion of hepatocytes, followed by massive replication generating schizonts with blood‐infective merozoites. Hepatocytes can be categorized by their zonal location and metabolic functions within a liver lobule. To understand specific host conditions that affect infectivity, we studied Pf parasite liver stage development in relation to the metabolic heterogeneity of fresh human hepatocytes. We found selective preference of different Pf strains for a minority of hepatocytes, which are characterized by the particular presence of glutamine synthetase (hGS). Schizont growth is significantly enhanced by hGS uptake early in development, showcasing a novel import system. In conclusion, Pf development is strongly determined by the differential metabolic status in hepatocyte subtypes. These findings underscore the importance of detailed understanding of hepatocyte host‐Pf interactions and may delineate novel pathways for intervention strategies.

Dynamics of the intrinsically disordered inhibitor IF7 of glutamine synthetase in isolation and in complex with its partner

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS is regulated, among other mechanisms, by protein-protein interactions with a 65-residue-long, intrinsically disordered protein (IDP), named IF7. IDPs explore diverse conformations in their free states and, in some cases, in their molecular complexes. We used both nuclear magnetic resonance (NMR) at 11.7 T and small angle X-ray scattering (SAXS) to study the size and the dynamics in the picoseconds-to-nanosecond (ps-ns) timescale of: (i) isolated IF7; and (ii) the IF7/GS complex. Our SAXS findings, together with MD results, show: (i) some of the possible IF7 structures in solution; and, (ii) that the presence of IF7 affected the structure of GS in solution. The joint use of SAXS and NMR shows that movements of each amino acid of IF7 were uncorrelated with those of its neighbors. Residues of IF7 with the largest values of the relaxation rates (R1, R2 and ηxy), in the free and bound species, were mainly clustered around: (i) the C terminus of the protein; and (ii) Ala30. These residues, together with Arg8 (which is a hot-spot residue in the interaction with GS), had a restricted mobility in the presence of GS. The C-terminal region, which appeared more compact in our MD simulations of isolated IF7, seemed to be involved in non-native contacts with GS that help in the binding between the two macromolecules.

A195 GLUTAMINE SYNTHETASE IN ENDOTHELIAL CELLS OF THE BLOOD-BRAIN BARRIER: NEW TARGET FOR THE TREATMENT OF HEPATIC ENCEPHALOPATHY?

The liver plays a major role in regulating ammonia levels in the blood. Therefore, in liver disease the loss of hepatic function leads to hyperammonemia and increased brain ammonia and consequently hepatic encephalopathy (HE). Ammonia-lowering strategies remain the mainstay therapeutic strategy. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. Therefore, the aim is to evaluate GS expression in endothelial cells of the BBB. Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS expression by western blot using brain lysates as positive control and by immunohistochemistry (co-localized with caveolin-1, a marker for ECs). In addition, to understand the effect of ammonia on GS, ECs were exposed to 1mM of ammonia chloride for 48h. Finally, ECs were subjected to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) or sham-operated controls. We have characterized this BDL model and found both systemic oxidative stress and inflammation, in addition to hyperammonemia. ECs expressed GS mRNA, protein and activity. However, expression of GS was lower compared to brain lysate control samples (p<0.05). GS expression in CMV showed similar results to brain but GS activity was less (p<0.05). Using immunohistochemistry, GS was detected in ECs and in vessels of brain from naïve rats. When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS was decreased when compared to sham-operated controls (p<0.01). These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme compared to that found in the brain, could explain why GS has never been reported in these cells. Interestingly, ammonia stimulates GS in ECs, but its activity is decreased in the presence of other pathogenic factors in plasma from cirrhotic rats such as oxidative stress and inflammation. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may be implicated in the pathogenesis of HE. We anticipate increasing GS in ECs of the BBB could become a new therapeutic target for HE. CIHR

Glutamine Synthetase in Endothelial Cells of the Blood–brain Barrier: Less But Not Insignificant?

Background and Aim: The liver plays a major role in regulating ammonia levels in the blood. Therefore, during liver disease, the loss of hepatic function leads to hyperammonemia and consequently increased brain ammonia and hepatic encephalopathy (HE). Reducing ammonia neurotoxicity through systemic ammonia-lowering strategies remains the mainstay therapeutic strategy for HE. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which during the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (astrocytes) but has never been thoroughly explored in the endothelial cells of the BBB. Methods: Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS activity and protein (brain lysate was used as positive control). GS was also evaluated by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, GS activity was assessed in ECs exposed to 1 mM of ammonium chloride for 48 h. Finally, GS activity and protein expression were evaluated in ECs submitted to plasma from 6-week bile-duct ligated (BDL) rats or sham-operated controls for 72 h. Results: ECs expressed mRNA, protein and activity of GS. However, EC's expression (normalized to g/protein) of GS was lower compared to brain lysate control samples (P < 0.05). GS protein expression in CMV showed similar results to that of brain but GS activity was significantly less in CMV (P < 0.05). Using immunohistochemistry, GS was detected in ECs and in CMV from naïve rats. When ECs were exposed to ammonia (1 mM), an increase in GS activity was demonstrated (P < 0.05). However, when exposed to conditioned medium from BDL rats, GS activity and protein expression were decreased when compared to sham-operated controls (P < 0.05). Conclusion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme in CMVs compared to brain, could reason why GS has never been reported in these cells. Interestingly, ammonia stimulates GS activity in ECs, but GS activity is decreased following treatment with plasma from BDL rats. This suggests other factors such as oxidative stress and inflammation, could inhibit GS activity. We speculate that a downregulation of GS in the BBB allows for a faster entry of ammonia into the brain and therefore may play a significant role in the onset of HE. We anticipate upregulating GS in ECs of the BBB could become a new therapeutic target for HE. The authors have none to declare.

Molecular and biochemical modifications of liver glutamine synthetase elicited by daytime restricted feeding.

The circadian clock system in the liver plays important roles in regulating metabolism and energy homeostasis. Restricted feeding schedules (RFS) become an entraining stimulus that promotes adaptations that form part of an alternative circadian clock known as the food entrained oscillator (FEO). The aim of this study was to evaluate the daily variations of glutamine synthetase (GS) in liver under a daytime RFS. Hepatic GS properties were analysed at 3-h intervals over a 24-h period in adult male Wistar rats maintained in a 12:12 h light–dark cycle. RFS group: food access for 2-h in light phase, during 3 weeks. AL group: feeding ad libitum. Fa group: acute fast (21 h). Fa–Re group: acute fast followed by refed 2 h.mRNA expression was measured by RT-qPCR, protein presence by Western-blot and immunohistochemistry, enzyme activity by a spectrophotometric assay, and glutamine by high pressure liquid chromatography. Restricted feeding schedule induced circadian rhythmicity inmRNA levels of GS and the loss of the rhythmic pattern in mitochondrial GS activity. GS activity in liver homogenates displayed a robust rhythmic pattern in AL that was not modified by RFS. The presence of GS and its zonal distribution did not show rhythmic pattern in both groups. However, acute Fa and Fa–Re diminished GS protein and activity in liver homogenates. Hepatic glutamine concentrations showed a 24-h rhythmic pattern in both groups, in an antiphasic pattern. In conclusion, daytime RFS influences the liver GS system at different levels, that could be part of rheostatic adaptations associated to the FEO, and highlight the plasticity of this system.

Identification and cellular location of glutamine synthetase in human sperm

Glutamine synthetase (GS) catalyzes the de novo synthesis of glutamine, an amino acid that has been shown to influence sperm motility in mammals. To date, no information is available about GS content in human sperm. In this study, we have characterized the presence and cellular location of GS in fresh human normozoospermic samples. We have detected a single band corresponding to GS by Western blot. Confocal analysis has revealed GS immunoreactivity in the post-acrosomal head region. Moreover, double-labeling experiments with either F-actin or calicin have demonstrated GS confinement in the post-acrosomal region of the perinuclear theca. These data have been validated by a post-embedding ultra-structural study. The presence of GS in the post-acrosomal region of the perinuclear theca suggests that human sperm can carry out in glutamine synthesis.

Cytosolic glutamine synthetase is present in the phloem sap of rice (Oryza sativa L.)

The presence of glutamine synthetase (GS) in the rice sieve tube was examined. Proteins in the rice phloem sap from leaf sheaths were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes and immunoblotted with anti-GS1 antibody. A cross-reacting band, thought to be GS1, was detected in the phloem sap. Moreover, the phloem sap contained a significant amount of GS transferase activity. Previous studies have shown that the concentrations of substrates and cofactors in the rice phloem sap are sufficient for cytosolic GS reaction. These data suggest that physiologically active GS1 is present in rice phloem sap, which might convert glutamate to glutamine in vivo.

Zonation of alanine metabolism in the bivascularly perfused rat liver

Zonation of alanine metabolism was investigated in the bivascularly perfused rat liver, a technique in which a selective area of the periportal region can be reached via the hepatic artery. Bivascular liver perfusion was done in both the antegrade and retrograde modes. Predominance of a given metabolic parameter in the periportal or perivenous area was deduced from comparisons of the changes caused by alanine infusion into the hepatic artery in antegrade and retrograde perfusion. Confirming previous notions, glutamine synthesis predominated in the perivenous area, however, the contribution of the periportal area was significant. Gluconeogenesis and the associated extra oxygen consumption were more pronounced in the periportal region. The capacity of urea synthesis in the periportal region was relatively small as indicated by the ratios of urea to glucose production, which were lower in this region. Ammonia in the periportal region was considerably above the mean ammonia production of whole the liver parenchyma. The overflows of pyruvate and lactate were considerably smaller in the periportal region. The distribution of alanine metabolism seems to reflect mainly zonation of the fates of the carbon skeleton (mainly gluconeogenesis). The production of glutamine in the periportal area is in agreement with recent reports about the presence of glutamine synthetase in Kupffer and endothelial cells.

Glutamine synthetase in brain: effect of ammonia

Glutamine synthetase (GS) in brain is located mainly in astrocytes. One of the primary roles of astrocytes is to protect neurons against excitotoxicity by taking up excess ammonia and glutamate and converting it into glutamine via the enzyme GS. Changes in GS expression may reflect changes in astroglial function, which can affect neuronal functions. Hyperammonemia is an important factor responsible of hepatic encephalopathy (HE) and causes astroglial swelling. Hyperammonemia can be experimentally induced and an adaptive astroglial response to high levels of ammonia and glutamate seems to occur in long-term studies. In hyperammonemic states, astroglial cells can experience morphological changes that may alter different astrocyte functions, such as protein synthesis or neurotransmitters uptake. One of the observed changes is the increase in the GS expression in astrocytes located in glutamatergic areas. The induction of GS expression in these specific areas would balance the increased ammonia and glutamate uptake and protect against neuronal degeneration, whereas, decrease of GS expression in non-glutamatergic areas could disrupt the neuron-glial metabolic interactions as a consequence of hyperammonemia. Induction of GS has been described in astrocytes in response to the action of glutamate on active glutamate receptors. The over-stimulation of glutamate receptors may also favour nitric oxide (NO) formation by activation of NO synthase (NOS), and NO has been implicated in the pathogenesis of several CNS diseases. Hyperammonemia could induce the formation of inducible NOS in astroglial cells, with the consequent NO formation, deactivation of GS and dawn-regulation of glutamate uptake. However, in glutamatergic areas, the distribution of both glial glutamate receptors and glial glutamate transporters parallels the GS location, suggesting a functional coupling between glutamate uptake and degradation by glutamate transporters and GS to attenuate brain injury in these areas. In hyperammonemia, the astroglial cells located in proximity to blood-vessels in glutamatergic areas show increased GS protein content in their perivascular processes. Since ammonia freely crosses the blood–brain barrier (BBB) and astrocytes are responsible for maintaining the BBB, the presence of GS in the perivascular processes could produce a rapid glutamine synthesis to be released into blood. It could, therefore, prevent the entry of high amounts of ammonia from circulation to attenuate neurotoxicity. The changes in the distribution of this critical enzyme suggests that the glutamate–glutamine cycle may be differentially impaired in hyperammonemic states.

Characterization of glutamine synthetase transcript, protein, and enzyme activity in the human placenta

This study characterizes the molecular mechanisms necessary for glutamine synthesis in the human placenta. RNA hybridization and protein immunoblotting were used to verify the presence of glutamine synthetase (GS) transcripts and protein, respectively. Additionally, the presence of GS was determined by immunohistochemistry. RNA hybridization demonstrated the presence of 1.8- and 2.8413 transcripts and protein immununoblotting yielded a single 49-kDa band, characteristic of GS transcripts and protein, respectively. The mean (± s.d.) specific activity of placental GS, expressed as μmol gamma-glutamyl hydroxamic acid/mg protein/h was 1.80 ± 0.59, which is comparable to other organs which are net glutamine producers. Immunohistochemical analysis indicated the presence of GS within the cytotrophoblast and mesenchyme layers of placental villi, but not in the syncytiotrophoblast. Although these results suggest that the human placenta is capable of synthesizing glutamine, the fate of glutamine produced by this organ remains speculative.

The presence of glutamine synthetase in the thylakoids of nitrogen‐fixing cyanobacterium Nostoc muscorum

AbstractThe intracellular distribution of glutamine synthetase (GS) in cell‐free extracts of Nostoc muscorum has been investigated. 64–73% of the enzyme activity was associated with the thylakoid membranes. No enzyme activity was found in cytoplasmic membranes. 92% inhibition of the enzyme activity was obtained with an antiserum raised to the thylakoids using Triton‐treated membranes, in comparison to 7.95% inhibition with the intact membranes. This suggests that the majority of the enzyme is embedded inside these membranes. This was also supported by the effect of pronase on the exposed glutamine synthetase using intact membranes.

Immunogold localization of glutamine synthetase and NADP-glutamate dehydrogenase of Laccaria laccata in Douglas fir ectomycorrhizas

Using antibodies raised against glutamine synthetase (GS) and NADP-glutamate dehydrogenase (NADP-GDH) from Laccaria laccata, we examined tissular localization of GS and NADP-GDH in symbiotic tissues of Douglas fir/L. laccata ectomycorrhizas by immunogold labeling. Thin sections of mycorrhizal roots were first treated either with an anti-GS- or antiNADP-GDH-specific antibody and then with a colloidal gold marker. Both enzymes appeared to be cytoplasmic. Our results also indicated the presence of GS in some fungal cells in dense cytoplasmic patches. It also appeared that GS is more abundant than NADP-GDH. The distribution of these nitrogen-assimilating enzymes in the fungal Hartig net and the sheath did not differ significantly. No labelling was observed in host cells.

Inflorescence Necrosis Induced from Ammonium Incubation and Deterred by α-Keto-Glutarate and Ammonium Assimilation in Pinot noir Grapevines

Inflorescence necrosis (IN) and increased tissue NH4 ÷ occurred after incubating single-node, field-grown, Pinot noir grape (Vitis vinifera L.) cuttings with one leaf and cluster in 120 mM or higher NH4 ÷ solutions of NH4NO 3 or (NH4)2SO 4. Incubation with solutions of NO3-, SO4 =, and K ÷ at the same concentrations did not induce IN. The addition of 80 mM KG, a substrate for ammonium assimilation, to an incubation solution containing 160 mM NH4 ÷ deterred IN and lowered flower or fruit NH4 ÷ to near control levels. The presence of glutamine synthetase/ glutamate synthase (GS/GOGAT) pathway of ammonium assimilation was detected in flowers, fruit, and pedicels, but not in rachis of cuttings treated with GS/GOGAT inhibitors: Le., 10 mM MSX (a GS inhibitor) or 5 mMAS (a GOGAT inhibitor). Glutamate dehydrogenase (GDH) is not involved in ammonium assimilation and detoxification in grape inflorescence. Chemical names used: a-keto-glutarate (KG), methionine sulfoximine (MSX), azaserine (AS).

Purification and Characterization of a Repressor for the &lt;italic&gt;Bacillus cereus glnRA&lt;/italic&gt; Operon

We report the overexpression, purification, and properties of the regulatory protein, GlnR, for glutamine synthetase synthesis of Bacillus cereus. The protein was found to be a dimer with a molecular weight of approximately 30,000, and its subunit molecular weight was 15,000 in agreement with that (15,025) of deduced amino acid sequence of GlnR. The purified GlnR protein bound specifically to the promoter region of the glnRA operon of B. cereus and Bacillus subtilis. The binding of the GlnR protein to the DNA fragment was enhanced by the presence of glutamine synthetase, the product of glnA, of B. cereus or B. subtilis, although the affinity of the GlnR protein for DNA was not affected in the presence of glutamate, glutamine, Mg2+, Mn2+, or ammonia. These results indicate the existence of an interaction between GlnR and glutamine synthetase, and support the hypothesis that the regulation of glnA expression requires both GlnR protein and glutamine synthetase in Bacillus.

Enzymic urea assay: a new colorimetric method based on hydrogen peroxide measurement.

We describe a new enzymic colorimetric method in which urea is measured in serum by use of a single reagent mixture. Ammonia produced by urea hydrolysis, catalyzed by urease, reacts with glutamate and ATP in the presence of glutamine synthetase. The ADP so produced is assayed in reactions catalyzed sequentially by pyruvate kinase and pyruvate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 500 or 550 nm in a reaction catalyzed by horseradish peroxidase, with phenol/4-aminophenazone as the chromogen. The reaction is complete in 15 min at 37 degrees C. The standard curve is linear up to a urea concentration of 40 mmol/L. Precision is good; CVs ranged from 2.5% to 3.1%. Results by the present method compared well with those by a candidate Reference Method and are not subject to interferences from commonly used drugs and anticoagulants.

Glutamine synthetase and energy metabolism enzymes in cultivated chick neurons and astrocytes: modulation by serum and hydrocortisone

Primary cultures of astroglial cells and of neurons obtained from chick embryos were grown in culture medium with and without serum added. The expression of glutamine synthetase (GS) in the cultured nerve cells was investigated immunocytochemically and biochemically. The cellular localization of GS in cerebellar tissue sections and in cerebral cortex of chick embryos was investigated by immunohistochemical staining. In tissue sections the enzyme is only present in astrocytes and their processes; neurons and their structures do not express the enzyme. In contrast, in pure neuronal primary cultures, a high level of GS was detected by biochemical and immunochemical methods. Thus, our results clearly indicate the presence of GS in pure neuronal cell cultures and its absence in this type of cells in vivo. Removal of serum from the culture medium enhanced GS levels in primary astrocyte cultures, but was without effect on GS activity in neurons. Addition of calf serum to the culture medium induces a two-fold increase of cellular lactate dehydrogenase (LDH) activity in neurons by increasing specifically the M subunit containing isoenzymes. The sensitivity of chick astroglial cells and neurons toward the GS inducing effect of hydrocortisone and modulation of its effect by serum was also investigated. Differences in the sensitivity of the two types of nerve cells in culture toward the GS inducing effect of hydrocortisone, and the effect of serum could be demonstrated.

Glutamine production by the isolated perfused rat heart during ammonium chloride perfusion

Myocardial levels of ammonia, glutamate, and glutamine and the release of glutamate and glutamine were studied in the isolated perfused rat heart during perfusion with ammonium chloride, epinephrine, and conditions of anoxia or ischaemia. Perfusion for 15 min with effective ammonium chloride concentrations of 0.53, 0.71, and 2.06 mmol/l resulted in glutamine production of 1.34, 0.95, and 4.41 mmol with 15 min-1/200 dry weight compatible with the presence of glutamine synthetase in rat myocardium. Myocardial ammonium content was unchanged by perfusion with 0.53 and 0.71 mmol/l ammonium chloride, but was increased by 1.36 mumol with 15 min-1/200 mg dry weight by perfusion with 2.06 mmol/l ammonium chloride. Increased myocardial contents of ammonia and glutamine were not accompanied by depression of left ventricular pressure. Perfusion with epinephrine (0.20 mug/ml) resulted in an increased myocardial content of glutamine. Anoxia or ischaemia resulted in no changes in ammonia content, and no changes in glutamine or glutamate production. The net release of glutamine into the perfusate was about 10 times the net release of glutamate.