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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interconversion of Escherichia coli glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] between its adenylylated and unadenylylated forms has been used to verify the prediction derived from a theoretical analysis of the steady-state functions of a model for a monocyclic interconvertible enzyme cascade system [Stadtman, E. R. & Chock, P. B. (1977) Proc. Natl. Acad. Sci. USA 74, 2761-2770]. Because glutamine and alpha-ketoglutarate are multifunctional effectors and because three active enzyme complexes are involved in both adenylylation and deadenylylation of glutamine synthetase, at least 28 constants are required to describe the glutamine synthetase monocyclic cascade. Of these, 22 constants were determined experimentally and 6 were estimated via computer curve fitting. Despite the complexity, when both adenylylation and deadenylylation reactions are functioning, the number of adenylyl groups bound per mole of enzyme, n, assumes a steady-state level as is predicted by the model. This n value is determined by the mole fraction of P(IIA)-given by ([P(IIA)]/([P(IIA)] + [P(IID)])-and the ratio of glutamine to alpha-ketoglutarate (P(IID) and P(IID) are the unmodified and the uridylylated forms of the P(II) regulatory protein). In the presence of 0.5 mM glutamine and 2 mM alpha-ketoglutarate, the value of n increases as a nearly hyperbolic function in response to increasing mole fractions of P(IIA). When the constant level of alpha-ketoglutarate is gradually increased to 40 muM, the hyperbolic function converts slowly to a parabolic function. When the P(IIA) mole fraction was maintained at 0.6 and alpha-ketoglutarate levels were varied from 1 mM to 4 muM, an 800-fold increase in signal amplification was observed with respect to glutamine activation. In addition, because glutamine activates the adenylylation and inhibits the deadenylylation reaction, a sensitivity index of 2.1 (corresponding to a Hill number of 1.5) was obtained for the variation of n values in response to increasing glutamine concentration.
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PMID:Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model. 2 22

Intact avian liver mitochondria were shown to synthesize glutamine from glutamate in the absence of exogenous ATP and ammonia. With L-[U-14C]glutamate as the substrate, there was an approximate 1:1 stoichiometry between glutamate deaminated (as measured by the release of 14CO2 due to alpha-keto-[14C]glutarate oxidation) and glutamate amidated. With L-[15N]glutamate as the substrate, the isolated glutamine was shown by low and high resolution mass spectrometry of its phenylisothiocyanate derivative to contain 15N in both the alpha-amino and amide groups. Thus, for each mole of glutamate taken up, approximately 0.5 mol is deaminated and the other 0.5 mol serves as a substrate for glutamine synthetase previously localized in these mitochondria (Vorhaben, J. E., and Campbell, J. W. (1972) J. Biol. Chem. 247,2763). The permeability of L-glutamine to intact avian liver mitochondria was studied by a rapid centrifugation technique. Efflux as well as influx of L-glutamine were both rapid and appeared to occur by a passive, energy-independent process. These results indicate that the mitochondrial glutamine synthetase present in uricotelic species represents the primary ammonia detoxication reaction in that ammonia released intramitochondrially during amino acid catabolism is converted to glutamine for efflux to the cytosol where it may serve as a substrate for purine (uric acid) biosynthesis.
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PMID:Avian mitochondrial glutamine metabolism. 124 54

The reversibility of adenylylation of glutamine synthetase from E. coli by adenylyltransferase was demonstrated. Several positive effectors (Gln, 2-hydroxyethyl-S-cysteine, Trp and Met) stimulate the back reaction in the same manner as the forward reaction. The apparent Michaelis constant for PP(i) is 2.2 mM at pH 7.35. The pH optimum of the back reaction is 6.5-7 while the pH optimum of the forward reaction is 7.6. The apparent equilibrium constant in the presence of 10 mM Mg(2+) at pH 7.36 is 8.5 in favor of adenylylated glutamine synthetase and PP(i). The equilibrium constant is strongly dependent from pH and from Mg(2+) concentration. There is a difference of about 0.5 to 1 kcal/mole free energy between the adenylyl-O-tyrosine bond and the pyrophosphate bond of adenosine triphosphate (ATP). It follows from these considerations that the adenylyl-O-tyrosine bond is an "energy-rich phosphate bond."
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PMID:Reversibility of the ATP:glutamine synthetase adenylyltransferase reaction. 491 Aug 53

The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.
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PMID:Ammonia assimilation and synthesis of alanine, aspartate, and glutamate in Methanosarcina barkeri and Methanobacterium thermoautotrophicum. 612 78

The activation of ovine brain glutamine synthetase by Mn(II) or Mg(II) was studied by steady-state kinetics. The metal ion concentration was varied at several fixed concentrations of ATP, and vice versa, and the resultant kinetic curves were analyzed according to the method of London and Steck [London, W. P., & Steck, T. L. (1969) Biochemistry 8, 1767-1779]. The data for Mg(II) indicated optimal activation at Mg:ATP = 2:1, whereas that for Mn(II) occurred at Mn:ATP = 1:1. This was interpreted as indicating formation of Mg . E . Mg . ATP as the subunit complex of optimum activity with Mg(II) (pHopt 7.5). With Mn(II) (pHopt 5.0), the complex of optimum activity may be either E . Mn . ATP or Mn . E . Mn . ATP, where the Mn . E complex is very tight. So that the latter two possibilities could be distinguished, titrations of the enzyme with Mn(II) were performed, electron paramagnetic resonance being used to determine free Mn(II). Four moles of Mn(II) ions was found to bind per mole of octameric enzyme, with an apparent Kd congruent to 0.54 microM. Addition of either HCl or Nd(III) ions to Chelex-treated enzyme results in the release of 3.7 +/- 0.2 Mn(II) ions. Thus, it appears that four Mn(II) are very tightly bound per octamer and that four more Mn(II) can bind tightly. Neither Mg(II) nor Ca(II) at 50 mM can displace Mn(II) from the Mn4 . E complex, but Mn(II) still binds tightly to apoenzyme or Mn4E in the presence of 50 mM Mg(II). As one proceeds from apo-E to Mn4 . E to Mn4 . E . Mn4 (+/- ATP), the intensity of the fluorescence emission of protein tryptophan residues decreases strongly and successively. The specific activities of the apo-E, Mn4 . E, and Mn4 . E . Mn4 complexes were found to be 0, 50, and 200 units/mg, respectively. If apoenzyme is added to a continuous coupled assay system with Mg(II), buffer, and with or without Mn(II) present, a time-dependent activation is observable with t1/2 congruent to 0.5-1.0 min. The total intracellular concentration of Mn(II) in ovine brain tissue was determined to be 1.9-2.6 microM, whereas the free [Mn(II)] was below 0.5 microM. Since the enzyme binds Mn(II) in preference to other divalent ions, it appears that the enzyme may exist as a manganoenzyme in vivo.
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PMID:Glutamine synthetase from ovine brain is a manganese(II) enzyme. 612 92

The kinetic properties of glutamine synthetase (EC 6.3.1.2) isolated from pea chloroplasts and purified according to the previously developed procedure have been investigated. The pH optimum for the enzymatic reaction in the presence of Mg2+ and Mn2+ are 7.5-7.6 and 5.5, respectively. The corresponding values of the activation energy per enzyme monomer (Mr = 60 000) are equal to 2900 and 1190 cal/mole. With Mg2+ the values of Km(app.) for NH4+, NH2OH, L-glutamate (+NH4+), L-glutamate (+NH2OH), ATP(+NH4+ and NH2OH) and Mg-ATP (+NH4+ and NH2OH) are 0.64, 17.5, 5.6, 7.0, 1.3 and 0.74 mM, respectively.
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PMID:[Kinetic characteristics of glutamine synthetase from the chloroplasts of pea leaves]. 613 4

Although glutamine is a major carbon source for mammalian cells in culture, its chemical decomposition or cellular metabolism leads to an undesirable excess of ammonia. This limits the shelf-life of glutamine-supplemented media and may reduce the cell yield under certain conditions. We have attempted to develop a less ammoniagenic medium for the growth of BHK-21 cells by a mole-to-mole substitution of glutamine by glutamate. This results in a medium that is thermally stable but unable to support an equivalent growth yield. However, supplementation of the glutamate-based medium with asparagine (3 mM) and a minimal level of glutamine (0.5 mM) restored the original growth capacity of the cultures. Substitution of the low level of glutamine with the glutamine dipeptides, ala-gln (1 mM), or gly-gln (3 mM) resulted in an equivalent cell yield and in a thermally stable medium. The ammonia accumulation in cultures with glutamate-based medium was reduced significantly (>60%). Factors mediating growth and adaptation in medium substituted with glutamate were also investigated. The maximum growth capacity of the BHK-21 cells in glutamate-based medium (without glutamine) was achieved after a period of adaptation of 5 culture passages from growth in glutamine-based cultures. Adaptation was not influenced by increases in glutamate uptake which was constitutively high in BHK cells. Adaptation was associated with changes in the activities of enzymes involved in glutamate or glutamine metabolism. The activities of glutamine synthetase (GS) and alanine aminotransferase (ALT) increased significantly and the activity of phosphate-activated glutaminase (PAG) decreased significantly. The activity of glutamate dehydrogenase (GDH) showed no significant change after adaptation to glutamate. These changes resulted in an altered metabolic profile which included a reduced ammonia production but an increased alanine production. Alanine production is suspected of being an alternative route for removal of excess nitrogen.
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PMID:The adaptation of BHK cells to a non-ammoniagenic glutamate-based culture medium. 1039 67