The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.     

Transport-related amino acid metabolism in germinating barley grains

When eight [¹⁴C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During further transport in the seedling there was a partial transfer of label from glutamine to asparagine, particularly in the shoot. None of the amino acids used supplied carbon for the synthesis of sucrose, glucose or fructose. Glutamine synthetase activity was particularly high in the scutellum during the period of rapid amino acid transport.     

The regulation of ammonia assimilating enzymes in Lemma minor

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     

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.     

Characterization of shuttle mechanisms for the transport of reducing equivalents into mitochondria

The malate-aspartate, fatty acid, and α-glycerophosphate shuttles for the transport of reducing equivalents into mitochondria were reconstituted, using isolated hepatic mitochondria and the extramitochondrial components of the shuttles. Clofibrate and thyroxin increased, while propylthiouracil treatment decreased, the activity of mitochondrial α-glycerophosphate dehydrogenase. Despite these changes, the activity of the reconstituted α-glycerophosphate shuttle was similar in mitochondria from control rats and those from rats treated with clofibrate and propylthiouracil. There was an increase in the activity of the shuttle using mitochondria from thyroxin-treated rats. Rotenone caused 60–90% inhibition of this shuttle, suggesting that rotenone-sensitive NADH dehydrogenase participates in the pathway of oxidation of extramitochondrial hydrogen. Palmitate, oleate, and octanoate were equally effective in reconstituting a cyclic fatty acid shuttle. The shuttle was inhibited by various compounds affecting mitochondrial metabolism, including oligomycin, dinitrophenol, cyanide, rotenone, atractyloside, and α-bromopalmitate. Carnitine and several dicarboxylic and tricarboxylic acids which stimulate fatty acid elongation, augmented fatty acid shuttle activity. The malate-aspartate shuttle was inhibited by cycloserine, amino-oxyacetic acid, and hydrazine, and stimulated by pyridoxal phosphate, at the same concentrations which affected the activities of cytoplasmic and mitochondrial glutamic oxalacetic transaminase. This shuttle was inhibited by uncouplers, antimycin, azide, cyanide, rotenone, amobarbital, oligomycin, and several inhibitors of anion transport including iodobenzylmalonate and avenaciolide. The reconstituted shuttle is sufficiently active to provide about 70–80% of the oxalacetate required for maximal rates of gluconeogenesis. Extrapolations based on the rates of mitochondrial oxidation of acetaldehyde and the activity of the microsomal ethanol oxidizing system suggest that any one of the shuttles could account for the rate of ethanol metabolism in vitro by the alcohol dehydrogenase pathway.     

Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.     

Uricoteley:its nature and origin during the evolution of tetrapod vertebrates

The hepatic mechanism for detoxication of ammonia formed during amino acid gluconeogenesis in uricotelic vertebrates requires the intramitochondrial synthesis of glutamine by glutamine synthetase. This glutamine then serves as a precursor of uric acid in the cytosol. The evolutionary development of uricoteley thus required the localization of glutamine synthetase in liver mitochondria. The mechanism for the mitochondrial import of glutamine synthetase in uricotelic vertebrate liver is not yet known. Tortoises, extant relatives of the stem reptiles, possess both the ureotelic and uricotelic hepatic systems. It therefore seems likely that the genetic events allowing the mitochondrial localization of glutamine synthetase in liver occurred in the amniote amphibian ancestors of the stem reptiles. The selection of ureoteley by the theropsids and of uricoteley by the sauropsids were major events in the divergence and subsequent evolution of these two lines. Once established in the sauropsid line, uricoteley has persisted through to the higher reptiles, crocodilians, and birds. Uricoteley was in part responsible for the radiation of the archosaurs during the Triassic as a water-conserving mechanism in the adult, thereby allowing them to invade the arid environments of that period. Contrary to dogma, uricoteley was probably of minor significance in the development of the cleidoic egg. Neither mammalian nor avian embryonic liver tissues catabolize amino acids to any great extent, so it is inappropriate to attribute to them a kind of "waste" nitrogen metabolism.     

Cerebral metabolic disturbances in the brain during acute liver failure: from hyperammonemia to energy failure and proteolysis

Several observations suggest that patients with fulminant hepatic failure may suffer from disturbances in cerebral metabolism that can be related to elevated levels of arterial ammonia. One effect of ammonia is the inhibition of the rate limiting TCA cycle enzyme alpha-ketoglutarate dehydrogenase (alphaKGDH) and possibly also pyruvate dehydrogenase, but this has been regarded to be of no quantitative importance. However, recent studies justify a revision of this point of view. Based on published data, the following sequence of events is proposed. Inhibition of alphaKGDH both enhances the detoxification of ammonia by formation of glutamine from alpha-ketoglutarate and reduces the rate of NADH and oxidative ATP production in astrocytic mitochondria. In the astrocytic cytosol this will lead to formation of lactate even in the presence of sufficient oxygen supply. Since the aspartate-malate shuttle is compromised, there is a risk of depletion of mitochondrial NADH and ATP unless compensatory mechanisms are recruited. One likely compensatory mechanism is the use of amino acids for energy production. Branched chain amino acids, like isoleucine and valine can supply carbon skeletons that bypass the alphaKGDH inhibition and maintain TCA cycle activity. Large-scale consumption of certain amino acids can only be maintained by cerebral proteolysis, as has been observed in these patients. This hypothesis provides a link between hyperammonemia, ammonia detoxification by glutamine production, cerebral lactate production, and cerebral catabolic proteolysis in patients with FHF.     

Effect of Hyperammonemia and Methionine Sulfoximine on the Kinetic Parameters of Blood-Brain Transport of Leucine and Phenylalanine

The activity of the blood-brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood-brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+ = 2.5 mmol X kg-1 X h-1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia-infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia-infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia-induced increase in the activity of transport of large neutral amino acids across the blood-brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.     

The pathway of nitrogen assimilation in plants

The major route of nitrogen assimilation has been considered for many years to occur via the reductive amination of α-oxoglutarate, catalysed by glutamate dehydrogenase. However, recent work has shown that in most bacteria an alternative route via glutamine synthetase and glutamine: 2-oxoglutarate aminotransferase (glutamate synthase) operates under conditions of ammonia limitation. Subsequently the presence of a ferredoxin-dependent glutamate synthase in green leaves and green and blue-green algae, and a NAD(P)H and ferredoxin-dependent enzyme in roots and other non-green plant tissues, has suggested that this route may also function in most members of the plant kingdom. The only exceptions are probably the majority of the fungi, where so far most organisms studied do not appear to contain glutamate synthase. Besides the presence of the necessary enzymes there is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.     

Altered fatty acid metabolism and composition in cultured astrocytes under hyperammonemic conditions

In vitro ¹H- and ¹³C-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.     

The Location of Glutamine Synthetase in Leaf Cells and its Role in the Reassimilation of Ammonia Released in Photorespiration

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.     

Avian mitochondrial glutamine metabolism.

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.     

Ammonia assimilation by Aspergillus nidulans: [15N]ammonia study

15N kinetic labelling studies were done on liquid cultures of wild-type Aspergillus nidulans. The labelling pattern of major amino acids under 'steady state' conditions suggests that glutamate and glutamine-amide are the early products of ammonia assimilation in A. nidulans. In the presence of phosphinothricin, an inhibitor or glutamine synthetase, 15N labelling of glutamate, alanine and aspartate was maintained whereas the labelling of glutamine was low. This pattern of labelling is consistent with ammonia assimilation into glutamate via the glutamate dehydrogenase pathway. In the presence of azaserine, an inhibitor of glutamate synthase, glutamate was initially more highly labelled than any other amino acid, whereas its concentration declined. Isotope also accumulated in glutamine. Observations with these two inhibitors suggest that ammonia assimilation can occur concurrently via the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways in low-ammonia-grown A. nidulans. From a simple model it was estimated that about half of the glutamate was synthesized via the glutamate dehydrogenase pathway; the other half was formed from glutamine via the glutamate synthase pathway. The transfer coefficients of nine other amino acids were also determined.     

Release and refixation of ammonia during photorespiration

Photorespiratory ammonia metabolism in isolated spinach ( Spinacia oleracea L. cv. Viking II) mitochondria was measured using a selective ammonia electrode. The mitochondria showed high rates of ammonia production in the presence of glycine. The isolated mitochondria contained less than 0.02% of the glutamine synthetase activity present in the original homogenate and no significant reassimilation of the released ammonia could be observed with added glutamate or α-ketogluterate. Exogenous added glutamine synthetase did reassimilate the released ammonia. In a recombinated system, with a chlorophyll to mitochondrial protein ratio equal to the ratio in vivo, chloroplasts could very effectively reassimilate the ammonia released in the mitochondria during oxidation of glycine.     

Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.     

Genetic basis for tissue isozymes of glutamine synthetase in elasmobranchs

Tissue-specific isozymes of glutamine synthetase are present in elasmobranchs. A larger isozyme occurs in tissues in which the enzyme is localized in mitochondria (liver, kidney) whereas a smaller form occurs in tissues in which it is cytosolic (brain, spleen, etc.). The nucleotide sequence of spiny dogfish shark (Squalus acanthias) liver glutamine synthetase mRNA, derived from its cDNA, shows there are two in-frame initiation codons (AUG) at the N-terminus which will account for the size differences between the two isozymes. Initiation at the up-stream and down-stream sites would yield peptides of 45,406 and 41,869 mol. wts. representing the precursor of the mitochondrial isozyme and the cytosolic isozyme, respectively. The additional N-terminal 29 amino acids present in the mitochondrial isozyme precursor contains two putative cleavage sites based on the Arg-X-(Phe,Ile,Leu) motif. The predicted two-step processing would remove 14 of the 29 N-terminal amino acids. These 14 amino acids can be predicted to form a very strong amphipathic mitochondrial targeting signal. Their removal would yield a mature peptide of 43,680 mol. wt. The calculated mol. wts. based on the derived amino acid sequence are therefore in good agreement with previous estimates of an approximately 1.5–2-kDa difference between the M_rs of the mitochondrial and cytosolic isozymes. A model for the evolution of the mitochondrial targeting of glutamine synthetase in vertebrates is proposed. Correspondence to: J.W. CampbellThe nucleotide sequence reported will appear in GenBank under accession number U04617     

Glutamine in the Pathogenesis of Hepatic Encephalopathy: The Trojan Horse Hypothesis Revisited

Hepatic encephalopathy (HE) is major neuropsychiatric disorder occurring in patients with severe liver disease and ammonia is generally considered to represent the major toxin responsible for this condition. Ammonia in brain is chiefly metabolized (“detoxified”) to glutamine in astrocytes due to predominant localization of glutamine synthetase in these cells. While glutamine has long been considered innocuous, a deleterious role more recently has been attributed to this amino acid. This article reviews the mechanisms by which glutamine contributes to the pathogenesis of HE, how glutamine is transported into mitochondria and subsequently hydrolyzed leading to high levels of ammonia, the latter triggering oxidative and nitrative stress, the mitochondrial permeability transition and mitochondrial injury, a sequence of events we have collectively termed as the Trojan horse hypothesis of hepatic encephalopathy.     

Methionine Sulfoximine Prevents the Accumulation of Large Neutral Amino Acids in Brain of Portacaval-Shunted Rats

Portal-systemic shunting and hyperammonemia lead to an accumulation of the large neutral amino acids in brain and apparently alter transport of neutral amino acids across the blood-brain barrier. It has been proposed that portal-systemic shunting leads to a high brain concentration of glutamine, a product of cerebral ammonia detoxification, and thereby affects the transport of other neutral amino acids across the blood-brain barrier. To test this hypothesis, rats with a portacaval shunt were treated with L-methionine-dl-sulfoximine (MSO), an inhibitor of glutamine synthesis. Treatment with MSO resulted in lower concentrations of the neutral amino acids in brain of portacaval-shunted rats and a higher brain ammonia concentration, compared with untreated shunted rats. These results suggest that the accumulation of neutral amino acids in brain after portacaval shunt depends on the increased synthesis of glutamine in brain.