Expression of glutamine synthetase in macrophages.

We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages.     

Expression of carbamoylphosphate synthetase I and glutamine synthetase in hepatic organoids reconstructed by rat small hepatocytes and hepatic nonparenchymal cells

The objective of this study was to investigate the expression of carbamoylphosphate synthetase I (CPS) and glutamine synthetase (GS) in small hepatocyte colonies and whether the heterogeneous expression of the enzymes could be induced during the maturation of small hepatocytes. Small hepatocytes isolated from an adult rat liver were cultured and proliferated to form colonies. The expression of CPS and GS was examined using immunocytochemistry and immunoblotting. In this culture more than 99% of morphologically hepatic cells were positive for CPS and all small hepatocytes were negative for GS at day 5. CPS-positive cells dramatically decreased with time in culture, whereas GS-positive ones appeared and their number increased in the colonies. Two to 3 weeks after plating, colonies with rising and piled-up cells appeared and the number of such colonies reached about 25% of all colonies at day 30. In most rising and piled-up cells in colonies both proteins were strongly expressed, whereas many small hepatocytes in monolayer colonies did not express either protein. When small hepatocytes in monolayer colonies were overlayed with Matrigel, the cells gradually piled up and both CPS and GS proteins were dramatically induced. The expression of CPS and GS in small hepatocytes may interact with the extracellular matrix because the rising and piled-up cells appear to be induced by the extracellular matrix produced by hepatic nonparenchymal cells.     

Expression and purification of glutamine synthetase cloned from Bacteroides fragilis

A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified; it had an apparent subunit Mr of approximately 75,000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.     

Regulation of glutamine synthetase. V. Partial purification and properties of glutamine synthetase from Bacillus licheniformis

The glutamine synthetase of Bacillus licheniformis has been obtained at about 15% purity. Sucrose gradient centrifugation gave a molecular weight value of approximately 612,000. Both l- and d-glutamate can be utilized as substrates in the biosynthetic reaction, although the l isomer was five times more active. The requirement for adenosine triphosphate (ATP) can be partially replaced by guanosine or inosine triphosphates, but not by cytidine or uridine triphosphates. The Mn(++) was required for activity, and the requirement cannot be satisfied with Mg(++). Maximal activity of the biosynthetic reaction was observed when ATP and Mn(++) were present in equimolar amounts. An excess of either reactant gave less activity. However, other purine and pyrimidine nucleotides, when added in combination with ATP, can partially substitute for ATP in attaining the equimolar ratio of nucleotide to Mn(++). A complex of ATP and Mn(++) is the preferred form of substrate. The B. licheniformis enzyme catalyzes the glutamyl transfer reaction but at a much slower rate than the Escherichia coli glutamine synthetase. Either adenosine diphosphate (ADP) or ATP can activate the glutamotransferase, although ADP is more active.     

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase

The relationships of five feedback inhibitors for the Bacillus licheniformis glutamine synthetase were investigated. The inhibitors were distinguishable by differences in their competitive relationship for the substrates of the enzyme. Mixtures of l-glutamine and adenosine-5'-monophosphate (AMP) or histidine and AMP caused synergistic inhibition of glutamine synthesis. Histidine, alanine, and glycine acted antagonistically toward the l-glutamine inhibition. Alanine acted antagonistically toward the glycine and histidine inhibitions. Independence of inhibitory action was observed with the other pairs of effectors. Possible mechanisms by which the inhibitors may interact to control glutamine synthesis are discussed. The low rate of catalysis of the glutamyl transfer reaction by the B. licheniformis glutamine synthetase can be attributed to the fact that l-glutamine serves both as a substrate and an inhibitor for the enzyme. Effectors which act antagonistically toward the l-glutamine inhibition stimulated glutamotransferase activity. The stimulation was not observed when d-glutamine was used as substrate for the glutamyl transfer reaction.     

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.     

Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli.

The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy. The defective E. coli gene ( glnAE ) was still present in the transductant since it could be transduced. In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy. Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B. subtilis or E. coli glutamine synthetase antigen; only the former was detected. Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B. subtilis, with no evidence for adenylylation. The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E. coli.     

Properties and regulation of glutamine synthetase from Rhodospirillum rubrum.

Glutamine synthetase from Rhodospirillum rubrum was purified and characterized with respect to its pH optimum and the effect of Mg2+ on its active and inactive forms. Both adenine and phosphorus were incorporated into the inactive form of the enzyme, indicating covalent modification by AMP. The modification could not be removed by phosphodiesterase. Evidence for regulation of the enzyme by oxidation was obtained. Extracts from oxygen-treated cells had lower specific activities than did extracts from cells treated anaerobically. Glutamine synthetase activity was found to decrease in the dark in phototrophically grown cells; activity was recovered on re-illumination.     

Cross-linking with diimidates of glutamine synthetase from Bacillus stearothermophilus.

Glutamine synthetase [EC 6.3.2.1] from Bacillus stearothermophilus was modified with diethyl malonimidate (DEM), dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). DMA modified most epsilon-amino groups. On modification with DMA, formation of 3 to 4 cross-links/subunit resulted in a large increase in thermostability. The activity, allosteric properties and fluorescence spectrum of the enzyme were not changed on cross-linking. The SDS-polyacrylamide gel electrophoretic profiles of DEM-, DMA-, and DMS-modified enzymes suggested that the interaction berween six subunits in each of the two hexagonal rings of the protein are heterologous and are different from those between the piled subunits on different rings.     

Expression of glutamine synthetase during the anaerobic germination of Oryza sativa L.

Glutamine synthetase (GS; EC.6.3.1.2.) occurs as cytosolic (GS₁) and plastidic (GS₂) polypeptides. This paper describes the expression of GS isoenzymes in coleoptile during the anaerobic germination of rice (Oryza sativa L.) and the influence of exogenous nitrate on this. By immunoprecipitation with anti-GS serum, two polypeptides of 41- and 44-kDa were detected of which the former was predominant. After fractionation by ion-exchange chromatography, the 41 and 44 kDa bands were identified as GS₁ and GS₂, respectively. Northern blot analysis with specific probes showed the presence of mRNA for cytosolic GS but not for the plastidic form. The presence of exogenous nitrate did not alter the activity and expression of GS in the coleoptile. The role of GS during the anaerobic germination of rice seems to induce the re-assimilation of ammonia rather than the assimilation of nitrate.Abbreviations GS glutamine synthetase - GS₁ cytosolic glutamine synthetase - GS₂ platidic glutamine synthetase We are grateful to Dr. Julie V. Cullimore for providing GS anti-serum and clones. The research was supported by the National Research Council of Italy, special project RAISA, sub-project N. 2 paper N. 1586.     

Characterization of glutamine synthetase isoforms from chlorella

Ion-exchange chromatography of extracts derived from Chlorella sorokiniana mutant strain (oxygen resistant) yielded two separate activity peaks of glutamine synthetase (GS). GS_I and GS_II were purified 220- and 187-fold and have molecular weights of approximately 398,000 and 360,000, respectively. Both enzymes are composed of eight identical subunits with a subunit molecular weight of 47,000 for GS_I and 43,000 for GS_II. The amino acid composition, catalytic, and immunological properties for both enzymes are similar.     

小麦谷氨酰胺合成酶的原核表达特点

谷氨酰胺合成酶(GS)是植物氮同化的关键酶,为了研究小麦GS同工酶的结构及其表达特点,我们构建了小麦GS1、GSr、GSe、GS2和GS2前体GS2p的原核表达载体,并对表达条件进行了优化。结果表明,尽管小麦GS同工酶氨基酸序列同源性达70%–80%,蛋白质表达却各具特点。30℃诱导3 h后,GSr、GSe及GS2表达量达最大,诱导7 h后GS1表达量达最大,GS2p不表达,表达量依次为GS1(22%)GSr(15%)GS2(12%)GSe(5%);且GSe可溶性表达,GS1主要为可溶性表达,而GSr和GS2为包涵体。30℃诱导3 h,GS同工酶相对转录量为GSr(7.59)GS2(1.84)GS2p(1.66)GSe(1.46)GS1(1.00),酶蛋白质翻译水平与转录水平不一致。mRNA结构分析显示,GS同工酶翻译起始区稳定二级结构的自由能不同:GS1(14.4)GSr(17.2)GS2(22.6)GSe(25.4)GS2p(31.6),自由能越小,翻译起始区结构越不稳定,蛋白表达水平越高。GS1、GSr、GSe和GS2可溶性表达的最佳诱导条件不同,分别是30℃诱导5 h、16℃诱导15 h、37℃诱导5 h及25℃诱导7 h;可溶性表达量为GS1(20%)GSr(13%)GS2(10%)GSe(7%),酶活性为GS1GSeGS2,GSr无活性。可见,GS同工酶的基因序列决定了蛋白质在原核细胞中的表达量、状态及其活性。     

Oxidation of Neurospora crassa glutamine synthetase.

The glutamine synthetase of Neurospora crassa, either purified or in cell extracts, was inactivated by ascorbate plus FeCl3 and by H2O2 plus FeSO4. The inactivation reaction was oxygen dependent, inhibited by MnCl2 and EDTA, and stimulated in cell extracts by sodium azide. This inactivation could also be brought about by adding NADPH to the cell extract. The alpha and beta polypeptides of the active glutamine synthetase were modified by these inactivating reactions, giving rise to two novel acidic polypeptides. These modifications were observed with the purified enzyme, with cell extracts, and under in vivo conditions in which glutamine synthetase is degraded. The modified glutamine synthetase was more susceptible to endogenous phenylmethylsulfonyl fluoride-insensitive proteolytic activity, which was inhibited by MnCl2 and stimulated by EDTA. The possible physiological relevance of enzyme oxidation is discussed.