Inhibition of glutamine synthetase activity by manganous ions in a cytosol extract of rat liver.
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ABSTRACT:
SUBMITTER: Joseph SK
PROVIDER: S-EPMC1161787 | biostudies-other | 1979 Nov
REPOSITORIES: biostudies-other
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Project description:The use of phosphoenolpyruvate plus pyruvate kinase as an ATP-generating system in the assay for glutamine synthetase activity via the formation of gamma-glutamylhydroxamate from glutamate and hydroxylamine with crude tissue preparations is shown to give values far in excess of the true glutamine synthetase activity of the tissue. This is due to the generation of pyruvate, which reacts with hydroxylamine to give a compound that is chromogenic with the ferric chloride reagent used for measuring gamma-glutamylhydroxamate.
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Project description:Asymptomatic valproic acid (VPA)-induced hyperammonemia in the absence of liver impairment is fairly common. However, the underlying mechanisms through which VPA causes elevation in plasma ammonia (NH4) remains under investigation. Male Sprague Dawley rats (n = 72) were randomly allocated to receive VPA 400 mg/kg, 200 mg/kg, or vehicle IP daily for either 8, 14, or 28 consecutive days. The behavioral effects of VPA were assessed. Plasma, liver, and prefrontal cortex (PFC), striatum (Str), and cerebellum (Cere) were collected 1 h post last injection and assayed for NH4 concentration and glutamine synthetase (GS) enzyme activity. Chronic VPA treatment caused attenuation of measured behavioral reflexes (p < 0.0001) and increase in plasma NH4 concentration (p < 0.0001). The liver and brain also showed significant increase in tissue NH4 concentrations (p < 0.0001 each) associated with significant reduction in GS activity (p < 0.0001 and p = 0.0003, respectively). Higher tissue NH4 concentrations correlated with reduced GS activity in the liver (r = -0.447, p = 0.0007) but not in the brain (r = -0.058, p = 0.4). Within the brain, even though NH4 concentrations increased in the PFC (p = 0.001), Str (p < 0.0001), and Cere (p = 0.01), GS activity was reduced only in the PFC (p < 0.001) and not in Str (p = 0.2) or Cere (p = 0.1). These results suggest that VPA-induced elevation in plasma NH4 concentration could be related, at least in part, to the suppression of GS activity in liver and brain tissues. However, even though GS is the primary mechanism in brain NH4 clearance, the suppression of brain GS does not seem to be the main factor in explaining the elevation in brain NH4 concentration. Further research is urgently needed to investigate brain NH4 dynamics under chronic VPA treatment and whether VPA clinical efficacy in treating seizure disorders and bipolar mania is impacted by its effect on GS activity or other NH4 metabolizing enzymes.
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Project description:L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.
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Project description:Tabtoxinine-?-lactam (T?L), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the T?L 3-(S)-hydroxy-?-lactam (3-H?L) warhead. Phosphorylation of T?L generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The T?L ?-lactam ring remains intact during enzyme inhibition, making T?L mechanistically distinct from traditional ?-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-H?L transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.
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Project description:1. A radiochemical assay for glutamine synthetase has been developed in which an ATP-regenerating system is incorporated to prevent accumulation of inhibitory amounts of ADP. It is particularly suitable for assay of the enzyme in crude tissue extracts containing high adenosine triphosphatase activity. 2. A survey of the distribution of the enzyme in tissues from normal male rats showed that activity is present in liver, brain cortex, kidney cortex, spleen, testis and retina. 3. The K(m) of the enzyme for l-glutamate is approx. 1.5x10(-2)m.
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Project description:1. The p-nitrophenyl beta-D-galactosidase asctivity in rat liver homogenates of lysosome-rich fractions was shown to be markedly affected by the ionic composition of the medium. A stimulation of the reaction rate at pH 5 was produced by most of the salts tested, which contained anions such as acetate, SO4(2-) and Cl-, and cations such as Na+, K= and Mg2+. The most pronounced effect was observed with MgCl2. Only potassium glutamate was inhibitory. 2. Five peaks of beta-galactosidase activity obtained by DEAE-cellulose chromatography were equally sensitive to changes in the ionic composition of the medium. In the presence of added NaC1, the whole rate-pH curve was displaced towards higher pH values, the optimum being shifted from 2.0-2.5 to 3.5. The stimulation at pH 5.0 appeared to be mainly due to changes in Vmax., whereas the apparent Km was slightly modified. 3. Unlike the total, the free beta-galactosidase activity remained unchanged or even declined when KC1 was added to the reaction medium.
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Project description:The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.
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