Mouse 3T6 cells that overproduce glutamine synthetase

A mouse 3T6 subline that grows in glutamine-free medium has been cloned and exposed to a regimen of increasing concentrations of the glutamine synthetase inhibitor, methionine sulfoxime. Cells selected for resistance to 700 microM methionine sulfoxime show a 75-fold increase in glutamine synthetase activity relative to the original subclone. Immune precipitation of extracts prepared from cells pulse-labeled with L-[35S] methionine indicates that the increase in enzyme activity reflects an increase in biosynthesis of glutamine synthetase. Results obtained from in vitro translation followed by immune precipitation suggests that the methionine sulfoxime-resistant cells are highly enriched in mRNA encoding glutamine synthetase. The increase in enzyme activity is lost upon culture of the cells in nonselective medium--a finding consistent with the observation of double minute chromosomes in only the drug-resistant cells. These data strongly support the notion that methionine sulfoxime treatment has resulted in selection of cells that have amplified the gene encoding glutamine synthetase.     

Regulation of carbon and nitrogen flow by glutamate synthase in Neurospora crassa

A glycine-resistant Neurospora crassa mutant (am-132;glyr), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for NADP-dependent glutamate dehydrogenase (GDH).] This new mutation also conferred resistance to serine and methionine sulphoximine (MS), which are inhibitors of glutamine synthetase (GS). In addition, the mutant obtained grew better on ammonium than the am-132 parental strain. Resistance to glycine was not due to increased synthesis of glutamine by an altered or induced GS, nor to increased glutamate synthesis by induction of the catabolic NAD-dependent GDH, nor to NADH-dependent glutamate synthase (GOGAT), which was as sensitive to inhibitors as the GOGAT from the parental strain. The glycine-resistance mutation lowered but did not abolish the carbon flow; this resulted in a lower content of tricarboxylic acid cycle intermediates. GOGAT activity was inhibited in vitro by several organic acids and methionine sulphone (MSF). The higher growth rate of the glycine-resistant mutant on ammonium or on ammonium plus glycine, serine or MS was explained by an increased capacity of GOGAT to synthesize glutamate in vivo due to a lower content of inhibitory tricarboxylic acid cycle intermediates; the higher glutamate content overcomes the effect of the GS inhibitors and explains the MSF resistance of the mutant.     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

The response ofRhizobium meliloti to L-methionine DL-sulphoximine

A strain ofRhizobium meliloti has been shown to be capable of growth in the presence of methionine sulphoximine concentrations at least two orders of magnitude higher than that required for the complete inhibition of glutamine synthetase activity. Neither the specific growth rate, nor the nutritional requirements of the organism were affected by methionine sulphoximine in the medium.Rhizobium meliloti appeared to assimilate ammoniavia the glutamate dehydrogenase pathway during growth in the presence of methionine sulphoximine. This suggests thatRhizobium meliloti may have some regulatory mechanism controlling ammonia assimilation that is not present in other enterobacteria possessing similar enzymatic machinery     

Stimulation of bicarbonate incorporation by ammonium in nonphotosynthetic cell suspension cultures of Acer pseudoplatanus

Ammonium supplied to Acer cells incubated in Tricine buffer raised net dark incorporation of ¹⁴C-bicarbonate by 1.5 to 3.3 fold; this stimulation was not abolished by prior inhibition of glutamine synthetase by methionine sulphoximine. With cells in phosphate buffer, ammonium gave a smaller 1.15 to 1.3 fold stimulation which was abolished when glutamine synthetase was inhibited. Ammonium had no direct effect on the activities of phosphoenolpyruvate carboxylase and pyruvate kinase assayed in partially purified extracts or on the enzymes catalysing release of label.     

Regulation of nitrogenase A and R concentrations in Rhodopseudomonas capsulata by glutamine synthetase.

Nitrogen-starved purple non-sulphur bacteria have an active unregulated form of nitrogenase (nitrogenase A); however, the nitrogenase of a glutamine synthetase-negative mutant of Rhodopseudomonas capsulata, when nitrogen-starved, was predominantly inactive and required activation by Mn2+ and activating-factor protein. This regulatory form of nitrogenase has been called nitrogenase R. Treatment of wild-type cells (containing nitrogenase A) with methionine sulphoximine, an inhibitor of glutamine synthetase, converted the enzyme into nitrogenase R. Glutamine synthetase thus appears to control the intracellular concentrations of nitrogenase A and R and in this way regulates nitrogenase activity in the photosynthetic bacterium.     

Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules.

We have characterized the distinct polypeptides, primary translation products and mRNAs encoding glutamine synthetase (GS) in the various organs of pea. Western blot analysis of soluble protein has identified five distinct GS polypeptides which are expressed at different relative levels in leaves, roots and nodules of pea. Of the two GS polypeptides in leaves (44 and 38 kd), the 44-kd GS polypeptide is predominant and is localized to the chloroplast stroma. In roots, the predominant GS polypeptide is 38 kd. Upon Rhizobium infection of roots, three 37-kd GS polypeptides increase in abundance in the nodules relative to uninfected roots. cDNA clones encoding three different GS mRNAs have been characterized. Hybrid-select translation has identified three different GS primary translation products (49, 38 and 37 kd). Two cDNA clones (pGS134 and pGS341) are homologous to GS mRNAs most abundant in nodules which encode the 38- and 37-kd GS primary translation products. A third cDNA (pGS197) corresponds to a larger GS mRNA species specific to leaf poly(A) RNA, which encodes a 49-kd putative precursor to the mature chloroplast GS polypeptide. cDNA sequence analysis and Southern blot analysis of pea nuclear DNA identifies at least three genes encoding GS in pea which are related but distinct in structure and in vivo pattern of expression.     

Glutamine synthetase activity,ammonia assimilation and control of nitrate reduction in the unicellular red algaCyanidium caldarium

Addition ofl-methionine-dl-sulphoximine to cells ofCyanidium caldarium brings about a loss of glutamine synthetase activity. Concomitantly ammonia assimilation is prevented.Under physiological conditions nitrate reductase [NAD(P)H: nitrate oxidoreductase EC 1.6.6.2] is reversibly converted into an inactive enzyme upon addition of ammonia. In the presence of methionine sulphoximine, when glutamine synthetase activity is lost, nitrate reductase is no longer inactivated by ammonia. It is suggested that ammonia itself is not the actual effector of nitrate reductase inactivation.Concomitantly with the failure of nitrate reductase to undergo ammonia-inactivation, in the presence of methionine sulphoximine nitrate reduction is an uncontrolled process, thus, in media with nitrate ammonia continues to be produced and excreted into the external medium at a constant rate.Abbreviations NR Nitrate reductase - GS Glutamine synthetase - GOGAT Glutamate syntase - MSX l-methionine-dl-sulphoximine     

Ammonium generation by nitrogen-starved cultures of Chlamydomonas reinhardii

Ammonium (NH ₄ ⁺ ) assimilation by Chlamydomonas reinhardii was inhibited when cultures were incubated with methionine sulphoximine (MSO). Methionine sulphoximine inhibited glutamine synthetase acitvity in vitro in extracts from wild-type (2192) and mutant (CC419) cultures. Mutant cultures were insensitive to MSO inhibition in vivo. Nitrogen-starved, wild-type cultures excreted ammonium when they were incubated with MSO in light or in darkness. Ammonium generation was stimulated by glutamine, inhibited by CO₂ and stoichiometrically related to loss of protein. Notrogen replete cultures treated with MSO excreted ammonium in light but little was excreted in darkness. Ammonium excretion in darkness, in the presence of MSO, was enhanced by either a period of nitrogen deprivation or by the addition of acetate. Nitrogen deprivation also diminished the lag before ammonium excretion commenced.Abbreviation MSO methionine sulphoximine     

The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase

Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (>90%) and nucleotide (>80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

Multiple mechanisms by which glutamine synthetase levels are controlled in murine tissue culture cells

We report the isolation of a complimentary DNA (cDNA) clone encoding glutamine synthetase, derived from a population of methionine sulfoxime-resistant mouse GF1 fibroblasts. When GF1 cells are incubated for 48 h in the presence of the glucocorticoid hormone dexamethasone, the specific activity of glutamine synthetase (GS), assayed as glutamyltransferase activity, increases by threefold. Based on dot hybridization analysis, hormonal treatment also produces a similar increase in the level of GS mRNA. When GF1 cells or mouse Neuro 2A neuroblastoma cells are transferred from medium containing 4 mM glutamine to glutamine-free medium, glutamyltransferase activity increases by at least fivefold. However, the presence or absence or glutamine in the medium does not affect the relative level of glutamine synthetase mRNA in either cell line. With both GF1 and Neuro 2A cells, the half-time for the decline in glutamine synthetase enzyme activity on addition of glutamine to the medium is approximately 1.5 h. This rapid decline, coupled with the lack of effect of glutamine on the level of GS messenger RNA in Neuro 2A cells, renders it unlikely that neural cells alter glutamine synthetase levels in response to glutamine by a biosynthetic mechanism, as suggested by previous authors [L. Lacoste, K.D. Chaudhary, and J. Lapointe (1982) J. Neurochem. 39, 78-85].     

Observations on the subcellular distribution of the ammonium ion in maize root tissue using in-vivo 14N-nuclear magnetic resonance spectroscopy

We show that the pH dependence of the base-catalysed exchange rate of the ammonium ion provides a basis for discriminating between the cytoplasmic and vacuolar pools of ammonium in plant tissues. In vivo, ¹⁴N-nuclear magnetic resonance spectra were recorded with and without ¹H-decoupling and information on the subcellular distribution of NH ₄ ⁺ was obtained from a lineshape analysis of the ¹H-coupled spectrum. We applied this method to maize (Zea mays L.) root tissues and found that: (i), the cytoplasmic ammonium concentration was low, which was in accord with the large activity of glutamine synthetase present in the roots; and (ii), inhibition of glutamine synthetase with methionine sulphoximine increased the cytoplasmic ammonium concentration, and led to the appearance of ammonium in the xylem sap.Abbreviations GS glutamine synthetase - MSO l-methionine sulphoximine - NMR nuclear magnetic resonance - Pi inorganic phosphate On secondment to the Department of Plant Sciences, University of Oxford.We acknowledge the financial support of the Agricultural and Food Research Council. R.B. Lee also thanks the Department of Plant Sciences, University of Oxford, for hospitality.     

Advances in animal cell recombinant protein production: GS-NS0 expression system

The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.     

Kinetic and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120

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.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.     

Photorespiratory ammonia assimilation in the leaves of barley, sorghum and Moricandia arvensis

Effects of methionine sulphoximine (MSX) on glutamine synthetase activity and ammonia accumulation in the leaves of barley, sorghum and Moricandia arvensis were studied. Glutamine synthetase activity of sorghum leaves was relatively resistant to MSX treatment during initial stages. All the three plant species exhibited considerable accumulation of ammonia arising from non-photorespiratory sources, in response to MSX treatment. Minimal estimates of the rate of glycine decarboxylation were made employing MSX and isonicotinyl hydrazide together in the treatment solution. Changes in the pool sizes of amino acids involved in photorespiratory nitrogen metabolism as affected by MSX have also been investigated.     

Factors regulating amino acid release from extrasplanchnic tissues in the rat. Interactions of alanine and glutamine.

1. Factors regulating the release of alanine and glutamine in vivo were investigated in starved rats by removing the liver from the circulation and monitoring blood metabolite changes for 30 min. 2. Alanine and glutamine were the predominant amino acids released into the circulation in this preparation. 3. Dichloroacetate, an activator of pyruvate dehydrogenase, inhibited net alanine release: it also interfered with the metabolism of the branched-chain amino acids valine, leucine and isoleucine. 4. L-Cycloserine, an inhibitor of alanine aminotransferase, decreased alanine accumulation by 80% after functional hepatectomy, whereas methionine sulphoximine, an inhibitor of glutamine synthetase, decreased glutamine accumulation by the same amount. 5. It was concluded that: (a) the alanine aminotransferase and the glutamine synthetase pathways respectively were responsible for 80% of the alanine and glutamine released into the circulation by the extrasplanchnic tissues, and extrahepatic proteolysis could account for a maximum of 20%; (b) alanine formation by the peripheral tissues was dependent on availability of pyruvate and not of glutamate; (c) glutamate availability could influence glutamine formation subject, possibly, to renal control.     

Heterogeneous expression of glutamine synthetase mRNA in rat liver parenchyma revealed by in situ hybridization and Northern blot analysis of RNA from periportal and perivenous hepatocytes

Using radiolabeled specific cDNA glutamine synthetase mRNA could be detected by in situ hybridization exclusively within those few perivenous hepatocytes which stained immunocytochemically for glutamine synthetase. This localization of glutamine synthetase mRNA was recently reported by Moorman et al. [(1988) J. Histochem. Cytochem. 36, 751-755]. Biotinylated cDNA was not suitable for mRNA detection because of a very high background staining under the conditions of in situ hybridization. Dot blot and Northern blot analysis of RNA isolated from periportal and perivenous subfractions of hepatocytes also demonstrated the exclusive perivenous localization of two hybridizable glutamine synthetase mRNAs of length 2.8 and 1.6 kilobases. These results indicate that the unique heterogeneity of glutamine synthetase in rat liver parenchyma is controlled at the pretranslational level.