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Consideration of Glutamine Synthetase as a Multifunctional Protein
Published in James F. Kane, Multifunctional Proteins: Catalytic/Structural and Regulatory, 2019
Recently, mutants of S. typhimurium resistant to the growth inhibition of methionine sulfoximine have been isolated and characterized.22,23 The glutamine synthetase activities from these mutants are resistant to inhibition by methionine sulfoximine, have reduced γ-glutamyl transferase activities and higher apparent Km values for glutamate and ammonia but not for ATP.23 These results are consistent with a mutation that alters the active site of glutamine synthetase and simultaneously changes the affinities for the L-methionine-SR-sulfoximine, glutamate, and ammonia. The characterization of these and similar mutants should provide a genetic approach for the mapping of substrate binding sites that will augment the information from physical studies.
Glutathione Synthesis
Published in Robert A. Greenwald, CRC Handbook of Methods for Oxygen Radical Research, 2018
A satisfactory approach to the production of glutathione deficiency consists of inhibiting γ-glutamylcysteine synthetase. Such inhibition can be produced by buthionine sulfoximine.25-29 Although methionine sulfoximine inhibits γ-glutamylcysteine synthetase,17 this compound also inhibits glutamine synthetase and therefore produces convulsions.30-33 Replacement of the methyl group of methionine sulfoximine by the more bulky n-butyl moiety leads to a compound that does not interact with glutamine synthetase. Thus, mice treated with buthionine sulfoximine do not experience convulsions.26 After treatment with buthionine sulfoximine, the glutathione levels of the liver, kidney, plasma, pancreas, and muscle decline rapidly.27 The turnover of glutathione in these tissues is sufficiently rapid so that depletion occurs within a short period of time. The rate of decline of the level of glutathione in tissues reflects its rate of utilization, which is about equal to its rate of transport from the cells. The decrease in the plasma glutathione level after treatment with buthionine sulfoximine reflects the substantial interorgan transport of glutathione. Plasma glutathione is derived mainly from the liver and, therefore, after inhibition of glutathione synthesis in the liver, the plasma level falls. Buthionine sulfoximine has been successfully used to deplete the glutathione levels of cells grown in tissue culture.29
Cyclic Nucleotide Regulation of Glutamine Metabolism in Skeletal Muscle
Published in Elling Kvamme, Glutamine and Glutamate in Mammals, 1988
C. M. Maillet, A. M. Pujaras Crane, A. J. Garber
Using a variety of inhibitors of alanine aminotransferase, such as aminooxyacetate or cycloserine, inhibition of alanine aminotransferase reduces alanine release from the skeletal muscle by approximately 80%.5,6 Concomitant with such inhibition of alanine aminotransferase, the release of other, ordinarily poorly released amino acids such as asparate is increased. Similar results have also been obtained for inhibition of glutamine synthetase using methionine sulfoximine.6 Inhibition of glutamine synthetase resulted in markedly reduced glutamine synthesis and release from the skeletal muscle and a corresponding increase in alanine release.
Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells
Published in mAbs, 2023
Emma Kelsall, Claire Harris, Titash Sen, Diane Hatton, Sarah Dunn, Suzanne Gibson
An AstraZeneca CHO host, a derivative of CHO-K1, was maintained in CD CHO medium (Life Technologies, Cat# 10743029) supplemented with 6 mM L‐glutamine (Life Technologies, Cat# 25030–081). Stably transfected CHO cells were grown in an AstraZeneca production medium supplemented with methionine sulfoximine (MSX; Sigma – Aldrich, Cat# M5379). The cultures were grown in a humidified incubator at 36.5°C, 6% CO2 with agitation at 140 rpm as required. For fed-batch culture, cells were cultured in AstraZeneca production medium as above without MSX over 11–14 days. The medium was supplemented with bolus additions of an AstraZeneca nutrient feed over the course of the culture period. Glucose and lactate were monitored throughout the fed-batch process using a YSI (2900D, YSI Inc). Cell count and viability analysis was performed using a Vi-CELL XR cell viability analyzer (Beckman Coulter, USA). Cell culture medium was clarified by centrifugation, and mAb titers were quantified by protein-A HPLC affinity chromatography on an Agilent 1260 Infinity series (Agilent Technologies, CA) by comparing the peak size from each sample with a calibration curve.
Dysregulated metabolism: A friend-to-foe skewer of macrophages
Published in International Reviews of Immunology, 2023
Keywan Mortezaee, Jamal Majidpoor
Increasing the production of succinate is important for regulation of macrophage polarization. Succinate is a known regulator of pro-inflammatory responses, mediated through HIF-1α stabilization and suppression of anti-inflammatory gene expression profile. Glutamine synthetase is an enzyme related to acid-base homeostasis and nitrogen metabolism. M2 macrophages under starvation induce the activity of glutamine synthase for production of glutamine. Blockade of glutamine synthase causes M2-to-M1 skewing of macrophages and the resultant tumor regression. Incubation of M2 macrophages with the glutamine synthase inhibitor methionine sulfoximine causes succinate accumulation and increased glucose utilization, thereby promoting metabolic rewiring toward attaining M1-like phenotype. Succinate is contributed to HIF-1α stabilization, so it is expected that blockade of glutamine synthase will cause HIF-1α activation, as it is attested [84]. Due to the interference between PKM2 activity with succinate accumulation and glycolysis for M1 polarization, a suggested strategy could be targeting PKM2.
The effect of serotonin on penicillin-induced epileptiform activity
Published in International Journal of Neuroscience, 2019
Mehmet Taskiran, Abdulkadir Tasdemir, Nusret Ayyildiz, Mustafa Ayyildiz, Erdal Agar
5-HT is one of the well-known neurotransmitters that has general physiological functions including control of appetite, sleep, learning and memory and behavior [6]. Serotonin is produced by enterochromaffin cells in the gastrointestinal tract and serotonergic neurons in the central nervous system [7]. Bonnycastle et al. were the first to suggest that there may be a relationship between serotonin and epilepsy about six decades ago [8]. It is known that 5-HT reuptake blockers and 5-HTP suppress generalized and focal seizures [9,10]. Fluoxetine, a selective 5-HT reuptake blocker, increased the density of the GABA receptor and protected against lithium-pilocarpine-induced seizures in rats [11]. Similarly, increase in brain serotonin levels caused a decrease in seizure susceptibility [12–14]. Serotonin levels were significantly reduced following methionine sulfoximine (MSO) and also inhibition of the decrease in serotonin levels by coadministration of 5-HTP delayed the onset of MSO-induced seizures in mice [15].