Multiple forms of glutamine synthetase in Bacillus brevis AG 4

Glutamine synthetase in Bacillus brevis AG 4, a Gram-positive spore forming bacteria, has been found to exist in multiple molecular forms. It was purified to electrophoretic homogeneity by single-step Blue Sepharose affinity chromatography. The native enzyme has a molecular weight of 600,000 with subunits of 50,000. The enzyme samples purified from different stages of growth differed in Mg2+ sensitivity and other kinetic properties. Four different enzyme samples selected on the basis of Mg2+ sensitivity showed distinct mobilities at pH 6.3 on PAGE using discontinuous buffer system. A correlation amongst Mg2+ sensitivity, electrophoretic mobility, and kinetic properties was highly suggestive of multiple forms of glutamine synthetase in Bacillus brevis arising due to modification.     

林肯链霉菌谷氨酰胺合成酶活力调节的研究

对不同氮源生长条件下林肯链霉菌无细胞粗提液中谷氨酰胺合成酶 (GS)的研究结果表明 ,高浓度NH+4阻遏了GS的生物合成。从不同氮源生长条件下林肯链霉菌中分离纯化了GS ,其性质没有差别。以受腺苷化调节的产气克雷伯氏菌GS作对照 ,林肯链霉菌GS没有明显的氨休克作用 ,经蛇毒磷酸二酯酶处理后 ,其活力没有变化。这些结果都说明林肯链霉菌GS不存在腺苷化共价修饰这一调节方式。反馈抑制作用是林肯链霉菌GS的一种重要的调节方式 ,这种抑制作用是以累积的方式进行的 ,这表明各种抑制剂对GS作用位点不同 ,各种抑制剂对GS的抑制作用是相互独立的。由此推测 ,林肯链霉菌GS是一种变构酶。     

Purification of the glutamine synthetase II isozyme of Drosophila melanogaster and structural and functional comparison of glutamine synthetases I and II

Glutamine synthetase II was purified from Drosophila melanogaster adults. It was completely separable from the isozyme glutamine synthetase I by means of DEAE chromatography. The complete enzyme has an apparent molecular weight of 360,000. After two-dimensional electrophoresis it gave a single molecular species with an apparent molecular weight of 42,000. Structural analysis of the two isozymes showed that they are different both in subunit molecular weight and in isoelectric point. Peptide maps of the purified subunits showed considerable dissimilarity. Glutamine synthetase II is more active than glutamine synthetase I in the transferase assay, while the opposite is true in the biosynthetic assay. The kinetic parameters were determined, showing again noteworthy differences between the two isozymes. We therefore conclude that two forms of glutamine synthetase are present in Drosophila, with different primary structures, different kinetic behavior, and the possibility of different functional properties.     

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.

The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N₂-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

     

Purification and characterization of glutamine synthetase from the unicellular cynabacterium Anacystis nidulans

Glutamine synthetase from the unicellular cynabacterium Anacystis nidulans was found associated with the membrane fraction of cell-free extracts. The enzyme could be solubilized by treatment of the cell membranes with the detergent alkyltrimethylammoniun and was purified to electrophoretical homogeneity by using affinity chromatography on 2′,5′-ADP-Sepharose. The molecular weight of the native enzyme was approx. 575000 but only a single protein band of 47 kDa was detected after sodium dodecyl sulphate gel electrophoresis, which implies a native enzyme complex with twelve identically sized subunits. Values for apparent Michaelis constant of the purified enzyme for ammonium, glutamate and ATP were 20, 5000 and 700 μM, respectively. Alanine behaved as an inhibitor of both activities (transferase and biosynthetic) of glutamine synthetase, whereas aspartate, leucine and lysine inhibited the biosynthetic activity of the enzyme, and glycine and serine only inhibited the transferase activity. Glutamate analogs, such as hydroxylysine, methionine sulfone, methionine sulfoximine and phosphinothricin, which inhibited ammonium uptake in vivo, behaved as potent inhibitors of glutamine synthetase in vitro. A. nidulans glutamine synthetase was inhibited by p-hydroxymercuribenzoate, the effect being reversed by treatment with dithioerythritol, dithiothreitol or mercaptoethanol.     

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M_r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the K_m ATP is 1.1 mm but the K_m for l-glutamate is not altered. The effect of pH on the K_m for ammonium indicates that NH₃ rather than NH₄⁺ may be the true substrate. At 10 mm Mg²⁺, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg²⁺ concentrations produce progressively more acidic optima while lower Mg²⁺ concentrations raise the pH optimum. The rate-response curve for Mg²⁺ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (K_i, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K_i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.     

Regulation and properties of the glutamine synthetase purified from Photobacterium phosphoreum

Glutamine synthetase from a marine enterobacterium, Photobacterium phosphoreum, was purified to homogeneity from cells grown in glycerol-yeast extract medium. The purified enzyme had a molecular weight of approximately 670,000 and a subunit size of 56,000, i.e. larger than that of the enzyme from E. coli. Regulation of the glutamine synthetase activity by adenylylation/deadenylylation was demonstrated on snake venom phosphodiesterase treatment. The state of adenylylation appeared to influence both the biosynthetic and gamma-glutamyltransferase activities of P. phosphoreum glutamine synthetase similar to in the case of the E. coli enzyme. The enzyme activity was controlled by adenylylation and possibly in combination with feedback inhibition by alanine, serine, and glycine, metabolites which are especially effective in inhibiting P. phosphoreum glutamine synthetase. When either Mn2+ or Mg2+ was added to the relaxed (divalent cation-free) enzyme, similar UV-difference spectra were obtained for the enzyme, indicating that the conformational states induced by these cations were also similar. The profile of these spectra varied from those published for E. coli, and three peaks were four 1 at 282.5, 288.5, and 298 nm.     

谷氨酰胺高效酶法转化条件研究

谷氨酰胺合成酶是生物体氮代谢的中心酶之一,在消耗ATP的情况下,谷氨酰胺合成酶催化由谷氨酸和NH4+向谷氨酰胺的转化,Toch ikura提出了将酵母发酵与纯化酶结合生产谷氨酰胺(G ln)的方法,本实验通过建立酶法合成L-G ln与酵母酒精发酵的能量偶联体系,研究了在此偶联体系中各因素对谷氨酰胺酶转化效率的影响,为工业上利用酶法生产G ln提供理论依据。     

Purification and characterization of glutamine synthetase from Rhodomicrobium vannielii

Abstract Glutamine synthetase (GS) from the purple non-sulfur bacterium Rhodomicrobium vannielii has been purified to electrophoretic homogeneity by affinity chromatography. Molecular weight and catalytic properties of the enzy,e are similar to those described for other species of Rhodospirillaceae. However, the enzyme from this organism appears to be antigenically different from the glutamine synthetases of other species of Rhodospirillaceae studied.     

Regulation of Mycobacterium smegmatis glutamine synthetase by adenylylation

Glutamine synthetase from a Gram-positive acid-fast bacterium, Mycobacterium smegmatis, was purified to homogeneity from cells grown with glycerol-bouillon medium. Electron micrographs of the enzyme revealed a dodecameric arrangement of its subunits in two superimposed hexagonal rings, similar to the structure of glutamine synthetase of Escherichia coli. Disc electrophoresis in the presence of sodium dodecyl sulfate indicated a subunit molecular weight of 56,000. The sedimentation coefficient of the native enzyme was estimated to be 19.4S by ultracentrifugation in a sucrose gradient. Like the E. coli enzyme, the glutamine synthetase from M. smegmatis is regulated by adenylylation/deadenylylation. This conclusion was based on studies of the effect of snake venom phosphodiesterase treatment on the catalytic and spectral properties of the isolated enzyme. The AMP released from the enzyme by the phosphodiesterase was identified by thin-layer chromatography. Despite the structural similarity of both enzymes, striking differences were found between the catalytic properties of M. smegmatis and E. coli glutamine synthetases. The divalent cation specificity of the M. smegmatis enzyme was not altered by adenylylation of the enzyme, and deadenylylation of the enzyme caused a significant increase in the specific activities for both biosynthetic and transfer reactions with either Mg2+ or Mn2+.     

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg²⁺ in the biosynthetic assay and Mn²⁺ in the transferase assay. Under physiological conditions, Co²⁺ produces a large stimulatory effect on the Mg²⁺-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K_i values of 3 and 13 mm, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.     

林肯链霉菌谷氨酰胺合成酶的酶学性质

在分离纯化的基础上,报道了pH、温度和金属离子对林肯链霉菌(Streptomyceslincolnensis)Z-512谷氨酸胺合成酶(GS)活力的影响及GS底物专一性的研究结果.在动力学性质的研究中,发现林肯链霉菌GS在生物合成反应系统中,对底物NH_4CI的饱和曲线不遵守米氏方程.Hill作图呈两相曲线.在NH_4CI浓度低的情况下,Hill系数大于1,具有正协同效应;当NH_4CI浓度增加到一定程度时,Hill系数小于1,具有负协同效应.这说明NH_4CI不仅作为林肯链霉菌GS的底物,而且作为一种效应物调节GS的活性.林肯链霉菌GS对底物Glu及ATP的饱和曲线遵守米氏方程.在不同的激活离子存在下,GS对Glu、ATP的Km值也不同.     

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.     

Inhibition of glutamine synthetase activity by manganous ions in a cytosol extract of rat liver.

Glutamine synthetase activity in a cytosol extract of liver was inhibited non-competitively by Mn2+ ions. The apparent Ki for Mn2" in the presence of phosphate was 8 micro M. Inhibition of glutamine synthetase by intracellular Mn2+ may contribute to the very low rates of glutamine synthesis observed in perfused liver and isolated hepatocytes.     

Molecular and regulatory properties of glutamine synthetase from the phototrophic bacterium Rhodopseudomonas capsulata E1F1.

The glutamine synthetase of the phototrophic bacterium Rhodopseudomonas capsulata E1F1 was purified to homogeneity by a procedure which used a single affinity chromatography step. Like enzymes from other photosynthetic procaryotes, native glutamine synthetase from R. capsulata E1F1 was found to be a dodecameric protein of approximately 660 kilodaltons with identical subunits of about 55 kilodaltons each. The Stokes radius and S20,w of the native enzyme were 8.35 nm and 19.20, respectively. The enzyme exhibited different aggregation states with detectable oligomers of 1, 2, 3, 4, 6, 8, 10, and 12 subunits. Disaggregation of the glutamine synthetase occurred after the native protein was subjected to electrophoresis in polyacrylamide gels, as well as occurring spontaneously at low ionic strength. Glutamine synthetase from R. capsulata E1F1 was regulated by an adenylylation-deadenylylation mechanism, and the adenylylation state of the protein depended on the nitrogen source, growth phase, and light intensity. Ammonia repressed glutamine synthetase, whereas glycine, serine, alanine, valine, and aspartate were noncompetitive inhibitors of the glutamine synthetase biosynthetic activity.     

Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Glutamine synthetase from bean nodules can be separated into two isoforms, GS_n1 and GS_n2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, Dye-Matrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GS_n1 and GS_n2 have been purified to homogeneity. Subunit structure analysis using two-dimensional polyacrylamide gel electrophoresis revealed that GS_n1 was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GS_n2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GS_n1. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GS_n2 was but one of the isozymes, and GS_n1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric.     

Tissue and Cellular Distribution of Glutamine Synthetase in Roots of Pea (Pisum sativum) Seedlings

The effect of nitrate application on glutamine synthetase activity in roots of pea (Pisum sativum L.) seedlings (2 weeks old) was studied. Separation of organelles from root fragments by sucrose density-gradient centrifugation revealed that both nitrite reductase and glutamine synthetase activities increased in root plastids as a response to nitrate application and that no such response was induced by ammonium application. Glutamine synthetase activity was also found to increase in plastids with distance from apex in nitrate-treated plants, the highest specific activity being located in the fourth 1-centimeter segment. Separation by SDS-PAGE and characterization by Western blotting showed that cytosolic glutamine synthetase contains one subunit polypeptide (28 kilodaltons) and that plastid glutamine synthetase contains both the 38-kilodalton subunit and a heavier subunit. When nitrate was present in the nutrient solution, the heavier subunit increased in abundance in protein fractions obtained from purified root plastids.     

Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate.

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.     

Inactivation of glutamine synthetases by an NAD:arginine ADP-ribosyltransferase

Glutamine synthetase from ovine brain has a critical arginine residue at the catalytic site (Powers, S. G., and Riordan, J.F. (1975) Proc. Natl. Acad. Sci. U.S. A. 72, 2616-2620). This enzyme is now shown to be a substrate for a purified NAD:arginine ADP-ribosyltransferase from turkey erythrocyte cytosol that catalyzes the transfer of ADP-ribose from NAD to arginine and purified proteins. The transferase catalyzed the inactivation of the synthetase in an NAD-dependent reaction; ADP-ribose and nicotinamide did not substitute for NAD. Agmatine, an alternate ADP-ribose acceptor in the transferase-catalyzed reaction, prevented inactivation of glutamine synthetase. MgATP, a substrate for the synthetase which was previously shown to protect that enzyme from chemical inactivation, also decreased the rate of inactivation in the presence of NAD and ADP-ribosyltransferase. Using [32P]NAD, it was observed that approximately 90% inactivation occurred following the transfer of 0.89 mol of [32P]ADP-ribose/mol of synthetase. The erythrocyte transferase also catalyzed the NAD-dependent inactivation of glutamine synthetase purified from chicken heart; 0.60 mol of ADP-ribose was transferred per mol of enzyme, resulting in a 95% inactivation. As noted with the ovine brain enzyme, agmatine and MgATP protected the chicken synthetase from inactivation and decreased the extent of [32P]ADP-ribosylation of the synthetase. These observations are consistent with the conclusion that the NAD:arginine ADP-ribosyltransferase modifies specifically an arginine residue involved in the catalytic site of glutamine synthetase. Although the transferase can use numerous proteins as ADP-ribose acceptors, some characteristics of this particular arginine, perhaps the same characteristics that are involved in its function in the catalytic site, make it a favored ADP-ribose acceptor site for the transferase.     

Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L

Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule-enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined.