Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M. Lam et al. 1995, Plant Cell 7: 887-898). We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tremula x P. alba) via Agrobacterium tumefaciens. Transformed poplar lines were selected by their ability to grow on selective medium containing kanamycin. The presence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis. Transgene expression was detected in all selected poplar lines at the mRNA level. The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angiosperm translational machinery and that GS1 subunits are assembled in functional holoenzymes. Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees. Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months. Our results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.
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PMID:Expression of a conifer glutamine synthetase gene in transgenic poplar 1059 28

The residue Cys-92 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase is a highly conserved residue in prokaryotic and eukaryotic glutamine synthetase genes. This cysteine residue was previously proposed as a good candidate for being essential for enzyme activity. We have examined through heterologous expression in Escherichia coli and site-directed mutagenesis the functional importance of this residue. We have found that the thiol group of Cys-92 is not essential either for glutamine synthetase biosynthetic or transferase enzyme activities. The characteristic inhibition by p-hydroxymercuribenzoate (a specific sulphydryl reagent) was not substantially altered as a consequence of replacement of Cys-92 by Ala. Immunoreactivity of the glutamine synthetase mutant protein, examined both under native and denaturing conditions, was similar to the wild-type, indicating that no significant conformational changes were produced as a consequence of the introduced mutation. However, the mutant enzyme C92A was considerably less stable than the wild-type. These results indicate that Cys-92 is not an essential residue for enzyme activity but it is important for stability of the glutamine synthetase protein.
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PMID:Site-directed mutagenesis of Cys-92 from the alpha-polypeptide of Phaseolus vulgaris glutamine synthetase reveals that this highly conserved residue is not essential for enzyme activity but it is involved in thermal stability. 1072 18

A Fasciola hepatica cDNA clone of 779 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 582 bp which encoded a 194-amino-acid-residue polypeptide (M(r) 21,723 Da) showing a high degree of homology to thioredoxin peroxidases. This putative antioxidant protein gene was expressed in Escherichia coli as a GST fusion protein. The recombinant fusion protein showed in vitro antioxidant properties and protected rabbit muscle enolase and E. coli glutamine synthetase from inactivation by nonenzymatic Fe(3+)/O(2)/DTT or Fe(3+)/O(2)/ascorbate metal-catalyzed oxidation systems.
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PMID:Fasciola hepatica: heterologous expression and functional characterization of a thioredoxin peroxidase. 1086 19

The glutamine synthetase (GS)-impaired mutants of Nostoc muscorum, isolated as ethylenediamine (EDA) resistant phenotype, were characterized for glnA linked processes to investigate the molecular mechanism of GS impairment. The GS enzyme was purified to homogeneity from the wild and mutant strains of N. muscorum employing ion-exchange followed by affinity chromatography. The final yield and specific activity of the enzyme were low in the mutant strains as compared to the wild type. The molecular weight of the native enzyme and the subunit size of GS polypeptide in all the strains were identical. Compared to the wild type, the GS purified from the mutant strains showed refractory response to its structural analogues like methionine sulfone (MSO), methionine sulfoximine (MSX), EDA, and metabolic substrates, such as ammonium and glutamine. The Km and Vmax for the substrates of transferase and biosynthetic reactions were altered in the mutant strains. The immunological assays performed with partially purified IgG molecules raised against wild-type GS revealed quantitative and qualitative heterogeneity for cross reactivity among the wild-type and EDA-resistant strains. Southern hybridization of genomic DNA in all the strains of N. muscorum restricted with different enzymes showed identical pattern with Anabaena 7120 glnA probe and localization of complete gene for GS on 7.5 kb HindIII fragment. Northern blot analysis indicated differential levels of GS specific mRNA and abundance of glnA transcripts of 1.7 and 1.9 kb in the mutant strains. Results suggested that point mutation(s) inflicted in glnA gene of N. muscorum are responsible for differential production of catalytically modified GS in EDA-resistant phenotypes.
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PMID:Molecular characteristics of glnA linked mutations in the nitrogen-fixing cyanobacterium Nostoc muscorum. 1181 52

Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37 degrees C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309 + or - 35 nkat mg(-1)) and GS1b (1,166 + or - 65 nkat mg(-1)) enzymes were obtained by decreasing the expression temperature to 10 degrees C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.
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PMID:Functional expression of two pine glutamine synthetase genes in bacteria reveals that they encode cytosolic holoenzymes with different molecular and catalytic properties. 1215 43

The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 [Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228], encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells. A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit. The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts. The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity. Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction. Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf. Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves. GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation. The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.
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PMID:Immunocharacterization of Vitis vinifera L. ferredoxin-dependent glutamate synthase, and its spatial and temporal changes during leaf development. 1217 46

Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration.
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PMID:Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells. 1223 92

The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide.
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PMID:Regulation of Glutamate Dehydrogenase and Glutamine Synthetase in Avocado Fruit during Development and Ripening. 1223 22

Nitrogen assimilation in the methanogenic archaeon Methanococcus maripaludis is regulated by transcriptional repression involving a palindromic 'nitrogen operator' repressor binding sequence. Here we report the isolation of the nitrogen repressor, NrpR, from M. maripaludis using DNA affinity purification. Deletion of the nrpR gene resulted in loss of nitrogen operator binding activity in cell extracts and loss of repression of nif (nitrogen-fixation) and glnA (glutamine synthetase) gene expression in vivo. Genetic complementation of the nrpR mutation restored all functions. NrpR contained a putative N-terminal winged helix-turn-helix motif followed by two mutually homologous domains of unknown function. Comparison of the migration of NrpR in gel-filtration chromatography with its subunit molecular weight (60 kDa) suggested that NrpR was a tetramer. Several lines of evidence suggested that the level of NrpR itself is not regulated, and the binding affinity of NrpR to the nitrogen operator is controlled by an unknown mechanism. Homologues of NrpR were found only in certain species in the kingdom Euryarchaeota. Full length homologues were found in Methanocaldococcus jannaschii and Methanothermobacter thermoautotrophicus, and homologues lacking one or more of the three polypeptide domains were found in Archaeoglobus fulgidus, Methanopyrus kandleri, Methanosarcina acetivorans, and Methanosarcina mazei. NrpR represents a new family of regulators unique to the Euryarchaeota.
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PMID:A novel repressor of nif and glnA expression in the methanogenic archaeon Methanococcus maripaludis. 1249 67

cDNA of human peroxiredoxin VI, one of the recently discovered novel antioxidant proteins, was expressed in Escherichia coli cells. The expression product was obtained in water-soluble form and purified by a two-step chromatographic procedure using DEAE-Sepharose and Sephacryl S-200. According to CD data, the polypeptide chain of the recombinant human peroxiredoxin VI contains approximately 40% alpha-helical region and 30% beta-structure, which is the same as for native rat peroxiredoxin VI. The protective properties of the recombinant protein determined as its ability to prevent the inactivation of glutamine synthetase from E. coli in a model oxidation system were comparable with the protective properties of native rat peroxiredoxin VI.
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PMID:Recombinant human peroxiredoxin VI: preparation and protective properties in vitro. 1249 19


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