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1.
The Location of Glutamine Synthetase in Leaf Cells and its Role in the Reassimilation of Ammonia Released in Photorespiration 总被引:4,自引:2,他引:2
WALLSGROVE ROGER M.; KEYS ALFRED J.; BIRD IVAN F.; CORNELIUS MARTIN J.; LEA PETER J.; MIFLIN BENJAMIN J. 《Journal of experimental botany》1980,31(4):1005-1017
The localization of glutamine synthetase within the cells ofbarley and pea leaves has been reinvestigated using either amechanical technique or rupturing of isolated protoplasts torelease the cellular organelles, and both differential and densitygradient centrifugation to separate them. In no case could wefind evidence for any significant association between glutaminesynthetase and the mitochondria; our results suggest that theenzyme is present in the chloroplast and the cytoplasm of bothspecies. Experiments with isolated mitochondria from spinachalso failed to provide any suggestion that these organellesmight contain glutamine synthetase. Thus there is no evidenceto support the hypothesis, published by others, that mitochondriareassimilate ammonia, released from glycine oxidation, by meansof their own glutamine synthetase. Further experiments werecarried out to see if glutamate dehydrogenase present in themitochondria could reassimilate ammonia under conditions inwhich the electron transport chain to oxygen was blocked. Althoughthere was some evidence for a small amount of assimilatory glutamatedehydrogenase activity under these conditions it was dependenton adding relatively high concentrations of ammonia and wasinsufficient to sustain the rate of recycling of NAD requiredfor glycine oxidation. The results were thus considered to becompatible with the operation of the photorespiratory nitrogencycle as previously published. 相似文献
2.
The ability of isolated pea-shoot mitochondria conditioned to incorporate ammonia into glutamate to reassimilate endogenously produced ammonia from glycine transformation was investigated. In the presence of 1 mM to 20 mM glycine less than 15% of the ammonia liberated was found to be incorporated into glutamate. Thus, a prominent role of mitochondrial glutamate dehydrogenase in the reassimilation of intramitochondrially produced ammonia can be excluded.Abbreviation GDH
Glutamate dehydrogenase (L-glutamate: NAD+ oxidoreductase (deaminating), EC 1.4.1.2) 相似文献
3.
Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts 总被引:13,自引:7,他引:6
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Intact mitochondria were prepared from spinach (Spinacia oleracea L. var. Kyoho) leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. Assays of several marker enzymes showed that the final mitochondrial preparations obtained are nearly free from other contaminating organelles, e.g. chloroplasts, peroxisomes, and endoplasmic reticulum. These mitochondria oxidized malate, glycine, succinate, and NADH, tightly coupled to oxidative phosphorylation with high values of ADP to O ratio as well as respiratory control ratio. The rate of NADH oxidation was 331 nmoles O2 per milligram mitochondrial protein per minute, which is comparable to that obtained by highly purified potato or mung bean mitochondria. However, the activity of glutamine synthetase was barely detectable in the isolated mitochondrial fraction. This finding rules out a hypothetical scheme (Jackson, Dench, Morris, Lui, Hall, Moore 1971 Biochem Soc Trans 7: 1122) dealing with the role of the mitochondrial glutamine synthetase in the reassimilation of NH3, which is released during the step of photorespiratory glycine decarboxylation in green leaf tissues, but it is consistent with the photosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea, Wallsgrove, Miflin 1978 Nature (Lond) 275: 741), in which NH3 reassimilation occurs outside the mitochondria. 相似文献
4.
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. 相似文献
5.
Channeling of ammonia from glutaminase to carbamoyl-phosphate synthetase in liver mitochondria 总被引:2,自引:0,他引:2
A J Meijer 《FEBS letters》1985,191(2):249-251
In isolated rat-liver mitochondria the rate of citrulline synthesis from glutamine does not respond to changes in the ammonia concentration in the extramitochondrial fluid. This suggest that ammonia, produced in the mitochondria via glutaminase, is directly channeled to carbamoyl-phosphate synthetase. 相似文献
6.
Kinetic and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120 总被引:10,自引:0,他引:10
A number of biochemical parameters of glutamine synthetase (EC 6.3.1.2) isolated from the cyanobacterium Anabaena 7120 were determined. Apparent Michaelis constants for glutamate and ATP were found to be 2.1 and 0.32 mM, respectively; that for ammonia was found to be below 20 microM, significantly lower than that reported for glutamine synthetases from other species. Serine, alanine, glycine, cysteine, aspartic acid, methionine sulfone, and methionine sulfoximine were found to inhibit the enzyme. The enzyme is controlled neither by adenylylation nor by feedback inhibition by glutamine, mechanisms found in some other prokaryotes. It must therefore be regulated by a different mechanism, possibly a combination of feedback by alanine, serine, and glycine, metabolites which are especially effective in inhibiting Anabaena glutamine synthetase. 相似文献
7.
Hepatocyte heterogeneity in glutamine and ammonia metabolism and the role of an intercellular glutamine cycle during ureogenesis in perfused rat liver 总被引:12,自引:0,他引:12
D H?ussinger 《European journal of biochemistry》1983,133(2):269-275
1. The metabolism of glutamine and ammonia was studied in isolated perfused rat liver in relation to its dependence on the direction of perfusion by comparing the physiological antegrade (portal to caval vein) to the retrograde direction (caval to portal vein). 2. Added ammonium ions are mainly converted to urea in antegrade and to glutamine in retrograde perfusions. In the absence of added ammonia, endogenously arising ammonium ions are converted to glutamine in antegrade, but are washed out in retrograde perfusions. When glutamine synthetase is inhibited by methionine sulfoximine, direction of perfusion has no effect on urea synthesis from added or endogenous ammonia. 3. 14CO2 production from [1-14C]glutamine is higher in antegrade than in retrograde perfusions as a consequence of label dilution during retrograde perfusions. 4. The results are explained by substrate and enzyme activity gradients along the liver lobule under conditions of limiting ammonia supply for glutamine and urea synthesis, and they are consistent with a perivenous localization of glutamine synthetase and a predominantly periportal localization of glutaminase and urea synthesis. Further, the data indicate a predominantly periportal localization of endogenous ammonia production. The results provide a basis for an intercellular (as opposed to intracellular) glutamine cycling and its role under different metabolic conditions. 相似文献
8.
Summary
Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS
Glutamine synthetase
- GDH
Glutamate dehydrogenase
- GOGAT
Glutamate synthase 相似文献
9.
Keys AJ 《Photosynthesis research》2006,87(2):165-175
Photorespiration involves the conversion of glycine to serine with the release of ammonia and CO2. In C3 terrestrial higher plants the flux through glycine and serine is so large that it results in the production of ammonia at a rate far exceeding that from reduction of new nitrogen entering the plant. The photorespiratory nitrogen cycle re-assimilates this ammonia using the enzymes glutamine synthetase and glutamine:2-oxoglutarateaminotransferase. 相似文献
10.
Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456-462]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg2+, MgATP, and L-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and L-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1-7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5-10% ethylene glycol. The apparent Km values for MgATP, L-glutamate, and ammonia (NH4+-NH3) are 0.7, 11.0, and 0.015 mM, respectively. Mg2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn2+ cannot replace Mg2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent Km for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine-N-oxide. Except for activation by halogen anions and the very low apparent Km for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent Km for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis. 相似文献
11.
Altered fatty acid metabolism and composition in cultured astrocytes under hyperammonemic conditions
In vitro 1 H- and 13 C-NMR spectroscopy was used to investigate the effect of ammonia on fatty acid synthesis and composition in cultured astrocytes. Cells were incubated 3 and 24 h with 5 mM ammonia in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. An increase of de novo synthesized fatty acids and the glycerol subunit of lipids was observed after 3 h treatment with ammonia (35% and 40% over control, respectively), the initial time point examined. Both parameters further increased significantly to 85% and 60% over control after 24 h ammonia treatment. Three hours incubation with ammonia increased the synthesis of diacylglycerides, while formation of triacylglycerides was decreased (40% over and 15% under control, respectively). The degradation of fatty acids was not affected by ammonia treatment. Furthermore, ammonia caused alterations in the composition of fatty acids, e.g. increased mono- and decreased polyunsaturated fatty acids (85% over and 15% under control concentrations, respectively). The decrease of polyunsaturated fatty acids was even more pronounced in isolated astrocytic mitochondria (39% lower than controls). Our results suggest ammonia-induced abnormalities in astrocytic membranes, which may be related to astrocytic mitochondrial dysfunction in hyperammonemic states. Most of the observed effects of ammonia on fatty acid synthesis and composition were ameliorated when glutamine synthetase was inhibited by methionine sulfoximine, supporting a pathological role of glutamine in ammonia toxicity. This study further emphasizes the importance of investigating the relative contribution of exogenous ammonia, effects of glutamine and of glutamine-derived ammonia on astrocytes and astrocytic mitochondria. 相似文献
12.
The possible synthesis of citrulline, a rate limiting step for urea synthesis via the ornithine-urea cycle (OUC) in teleosts was tested both in the presence of ammonia and glutamine as nitrogen-donating substrates by the isolated liver mitochondria of ureogenic air-breathing walking catfish, C. batrachus. Both ammonia and glutamine could be used as nitrogen-donating substrates for the synthesis of citrulline by the isolated liver mitochondria, since the rate of citrulline synthesis was almost equal in presence of both the substrates. The citrulline synthesis by the isolated liver mitochondria requires succinate at a concentration of 0.1 mM as an energy source, and also requires the involvement of intramitochondrial carbonic anhydrase activity for supplying HCO3 as another substrate for citrulline synthesis. The rate of citrulline synthesis was further stimulated significantly by the isolated liver mitochondria of the fish after pre-exposure to 25 mM NH4Cl for 7 days. Due to possessing this biochemical adaptational strategy leading to the amelioration of ammonia toxicity mainly by channeling ammonia directly and/or via the formation of glutamine to the OUC, this air-breathing catfish could succeed in surviving in high external ammonia, which it faces in its natural habitat in certain seasons of the year. 相似文献
13.
Surprisingly, glutamine synthetase was recently shown to be dual targeted to chloroplasts and mitochondria in Arabidopsis leaves. It is likely that mitochondrial glutamine synthetase assimilates ammonia, which is generated in large amounts in mitochondria during photorespiration. However, ammonia assimilation is a two-step process and the second step, catalyzed by glutamate synthase, is exclusively located in plastids. Hence, a shuttle for ammonia, possibly in the form of amino acids, is required between mitochondria and plastids. We discuss two alternative shuttles, an ornithine-citrulline shuttle and a glutamine-glutamate shuttle. Both shuttles allow the safe transport of the toxic metabolite ammonium in the form of amino acids. The ornithine-citrulline shuttle also provides a means for the transport of carbon dioxide from mitochondria to plastids, but this shuttle requires more energy than the alternative glutamate-glutamine shuttle. 相似文献
14.
When adequate concentrations of phosphinothricin (a potent inhibitor of glutamine synthetase) are added to Anacystis nidulans cells suspended in nitrate medium, ammonia excretion into the medium takes place. Similarly, when phosphinothricin is added to nitrogen fixing cultures of Anabaena ATCC 33047, ammonia is also released at high rates. Methionine sulphoximine, phosphinothricin and its 2-oxo-derivative (1 mM) stimulate ammonia production and cause a sharp drop in glutamine and asparagine concentrations, when fed to leaves of Triticum, Pisum and Helianthus. Less pronounced effects were detected with the leaves of a C4 plant Zea. 相似文献
15.
The kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase has been determined at pH 7.5, 25 degrees C. With ammonia as the nitrogen source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3-, and NH3. Phosphate is then released and the second MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate, and MgADP. With glutamine as the ammonia donor, the patterns are consistent with a two-site mechanism in which glutamine binds randomly to the small molecular weight subunit producing glutamate and ammonia. Glutamate is released and the ammonia is transferred to the larger subunit. Carbamoyl-phosphate synthetase has also been shown to require a free divalent cation for full activity. 相似文献
16.
Regulation and biochemical characterization of the glutamine synthetase of azotobacter vinelandii 总被引:2,自引:0,他引:2
We have investigated the regulation of the activity and synthesis of the glutamine synthetase (l-glutamate:ammonia ligase (ADP-forming), EC (6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH+4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a 'relaxed, divalent-cation-free form which was catalytically inactive. The 'taut', active form could be restored with 1-5 mM Mg2+, Mn2+, Ca2+ or CO2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg2+ and CO2+ each supported biosynthetic catalysis, but with different substrate Km and Vmax values. L-Alanine, glycine and L-aspartate were the most potent of several inhibitors of the biosynthetic and the gamma-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co2+-supported biosynthetic activity of Al vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1-10 mM aspartate. 相似文献
17.
Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate. 总被引:5,自引:5,他引:0
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The glutamine synthetase from Bacillus licheniformis A5 was purified by using a combination of polyethylene glycol precipitation and chromatography on Bio-Gel A 1.5m. The resulting preparation was judged to be homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, equilibrium analytical ultracentrifugation, and electron microscopic analysis. The enzyme is a dodecamer with a molecular weight of approximately 616,000, and its subunit molecular weight is 51,000. Under optimal assay conditions (pH 6.6, 37 degrees C) apparent Km values for glutamate, ammonia, and manganese.adenosine 5'-triphosphate (1:1 ratio) were 3.6, 0.4, and 0.9 mM, respectively. Glutamine synthetase activity was inhibited approximately 50% by the addition of 5 mM glutamine, alanine, glycine, serine, alpha-ketoglutarate, carbamyl phosphate, adenosine 5'-diphosphate, or inosine 5'-triphosphate to the standard glutamine synthetase assay system, whereas 5 mM adenosine 5'-monophosphate or pyrophosphate caused approximately 90% inhibition of enzyme activity. Phosphorylribosyl pyrophosphate at 5 mM enhanced activity approximately 60%. We were unable to detect any physical or kinetic differences in the properties of the enzyme when it was purified from cells grown in the presence of ammonia or nitrate as sole nitrogen source. The data indicate that B. licheniformis A5 contains one species of glutamine synthetase whose catalytic activity is not regulated by a covalent modification system. 相似文献
18.
Regulatory mutations in the Klebsiella aerogenes structural gene for glutamine synthetase. 总被引:7,自引:4,他引:3
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Glutamine synthetase could be repressed several hundredfold rather than 6- to 10-fold as previously reported. Ammonia was not the primary repression signal for glutamine synthetase. Repression appeared to be mediated by a high level of glutamine and probably by a high ratio of glutamine to alpha-ketoglutarate. Mutations in glnA (the structural gene for glutamine synthetase) were seen to fall into three phenotypic groups: glutamine auxotrophs that produced no detectable glnA product; glutamine auxotrophs that produced a glnA product lacking enzymatic activity (and hence repressibility by ammonia) but were repressible under appropriate conditions; and glutamine synthetase regulatory mutants, whose glnA product was enzymatically active and not repressible under any conditions. 相似文献
19.
GMP synthetase, a class I amidotransferase, catalyzes the last step of the purine biosynthetic pathway, where ammonia from glutamine is incorporated into xanthosine 5'-monophospate to yield guanosine 5'-monnophosphate as the main product. Combined biochemical, structural, and computational studies of glutamine amidotransferases have revealed the existence of physically separate active sites connected by molecular tunnels that efficiently transfer ammonia from the glutaminase site to the synthetase site. Here, we have investigated aspects of ammonia channeling in P. falciparum GMP synthetase using biochemical assays in conjunction with 15N-edited proton NMR spectroscopy. Our results suggest that (1) ammonia released from glutamine is not equilibrated with the external medium, (2) saturating concentrations of glutamine do not obliterate the incorporation of external ammonia into GMP, and (3) ammonia in the external medium can access the thioester intermediate when the ATPPase domain is bound to substrates. Further, mutation of Cys-102 to alanine confirmed its identity as the catalytic residue in the glutaminase domain, and ammonia-dependent assays on the mutant indicated glutamine to be a partial uncompetitive inhibitor of the enzyme. 相似文献
20.
The glutamate analog, alpha-aminomethylglutaric acid, was synthetized by Michael addition of ammonia to 2-methylene glutaronitrile followed by hydrolysis of the intermediate alpha-aminomethylglutaryl nitrile; the analog cyclizes readily on heating to 2-piperidone-5-carboxylic acid. Sheep brain glutamine synthetase utilizes one isomer of DL-alpha-aminomethylglutarate at about 10% of the rate with L-glutamate. gamma-Glutamylcysteine synthetase uses both isomers of DL-alpha-aminomethylglutarate, preferentially acting on the same isomer used by glutamine synthetase. gamma-(alpha-Aminomethyl)glutaryl-alpha-aminobutyrate, prepared enzymatically with gamma-glutamylcysteine synthetase, was found to be a substrate and an inhibitor of glutathione synthetase. alpha-Aminomethylglutarate does not inhibit gamma-glutamyl cyclotransferase and gamma-glutamyl transpeptidase appreciably. When alpha-aminomethylglutarate was administered to mice, there were substantial decreases in the levels of glutamine, glutathione, glutamate, and glycine in the kidney, and of glutamine and glutamate in the liver, indicating that this glutamate analog is effective as an inhibitor of glutamine and glutathione synthesis in vivo, and suggesting that it may also inhibit other enzymes. 相似文献