Ammonia Ligase Research Articles

Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture.

The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells.

Purification and comparison of glutamine synthetase from chicken liver, brain and neural retina

1. 1. Glutamine synthetase ( l-glutamate: ammonia ligase, EC 6.3.1.2) was isolated from chicken liver, brain and neural retina. 2. 2. The specific activities of the purified enzyme preparations from the three different sources were similar. They were composed of subunits of the same molecular weight (43 K) and were immunologically indistinguishable. 3. 3. Slight differences were detectable among them in relation to the amino acid compositions and regulation of their activities by the several effectors tested.

Investigation of binding sites in the complex pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyl transferase enzyme of Neurospora crassa

The pyr-3 gene of Neurospora crassa codes for the bifunctional enzyme pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase (carbon dioxide: ammonia ligase (ADP-forming, carbamate-phospborylating)/carbamoylphosphate: l-aspartate carbamoyltransferase), EC 6.3.4.16/EC 2.1.3.2). We describe the investigation of substrate- and product-binding sites of the enzyme by affinity chromatography, using the ligands aspartate, glutamate, and adenosine 5′-diphosphate, and investigate the channelling of carbamoyl phosphate, the product of the first function and substrate of the second, through the pathway. For this latter aspect of the investigation, two new enzyme assays were devised and are described. The results of the competition studies on carbamoyl phosphate-binding are consistent with the existence of two different binding sites within the enzyme for this metabolic intermediate, one for it as the product of the first step and the other for it as the substrate of the second.

Micrococcal nuclease cleavage of nucleotide linked to glutamine synthetase yields phosphotyrosine at the ligation site.

The activity of micrococcal nuclease was studied on a novel substrate, denatured adenylylated glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2], which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP. The products of the digestion were adenosine and O-phosphotyrosylglutamine synthetase. The Km of the macromolecular substrate with the nuclease was 1/40 that of the synthetic substrate, nitrophenyl-pdT, which is commonly used for assay of the enzyme. Native adenylylated glutamine synthetase was not deadenosylated by micrococcal nuclease under the conditions that permit rapid deadenosylation of denatured glutamine synthetase. Failure to attack native glutamine synthetase is probably not due to steric factors because the native enzyme is deadenylylated by snake venom phosphodiesterase under identical conditions. The inability of micrococcal nuclease to deadenosylate native glutamine synthetase may be due to the formation of an inactive complex because the native protein inhibited the nuclease activity on the denatured protein.

Regulation and biochemical characterization of the glutamine synthetase of Azotobacter vinelandii

We have investigated the regulation of the activity and synthesis of the glutamine synthetase ( lglutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH + 4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a ‘relaxed’, divalent-cation-free form which was catalytically inactive. The ‘taut’, active form could be restored with 1–5 mM Mg 2+, Mn 2+, Ca 2+ or CO 2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg 2+ and CO O2+ each supported biosynthetic catalysis, but with different substrate K m and V max values. l-Alanine, glycine and l-aspartate were the most potent of several inhibitors of the biosynthetic and the γ-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co 2+ -supported biosynthetic activity of A. vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1–10 mM aspartate.

Glutamine synthetase in preparations of RNA polymerase of Escherichia coli: a novel purification procedure.

A novel procedure of operational ease and reproducibility for the partial purification of DNA-dependent RNA polymerase [EC 2.7.7.6] from Escherichia coli is reported. It utilizes liquid phase partitions with polyethylene glycol and Dextran, ammonium sulfate fractionations and chromatography on a QAE-Sephadex A-50 column. A copurified protein in the partially purified preparation was isolated and identifed as glutamine synthetase [L-glutamate : ammonia ligase (ADP) EC 6.3.1.2].

Succinate dehydrogenase, monoamine oxidase and glutamine synthetase in developing human foetal brain regions.

In human foetal brain ontogeny the cerebral activity of succinate oxidoreductase (EC 1.3.99.1), i.e. succinate dehydrogenase (SDH), is higher than the cerebellar activity. With rise in foetal body weight the activity in all the brain regions gradually declines. SDH in all the brain regions shows two high-activity periods, one at 20-35 g and another at 110-220 g body weight. The enzyme exhibits a craniocaudal pattern of development. At all times of gestation, L-glutamate:ammonia ligase (EC 6.3.1.2), i.e. glutamine synthetase, activity in the spinal cord and medulla is higher than in the other three regions. At 190 g body weight glutamine synthetase shows an activity peak in all brain regions. Monoamine:oxygen oxidoreductase (EC 1.4.3.4). i.e. monoamine oxidase (MAO), is present much before the onset of electrical activity. It develops caudocranially and exhibits a biphasic pattern of development in all the regions. It increases considerably in the medulla and the spinal core towards late gestational periods.

Purification, physical characterization, and NH2-terminal sequence of glutamine synthetase from the cyanobacterium Anabaena 7120.

A procedure for the complete purification of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from the cyanobacterium (blue-green alga) Anabaena 7120 is described. The enzyme has structural characteristics in common with glutamine synthetases from other sources: a subunit molecular weight of approximately 50,000 and a native structure, determined by low dose exposure electron microscopy, consisting of a two-layered regular hexagon made up of 12 subunits. Sequence analysis suggests that the subunits are identical. There was no indication that the enzyme from cyanobacteria is adenylylated, a feature shared with the glutamine synthetase from Bacillus subtilis but not with the enzyme from Escherichia coli. NH2-terminal sequence analysis and the predicted conformation of the NH2-terminal regions showed definite homology among the enzymes from all three sources. The limited analysis suggested a stronger structural homology between the enzyme from Anabaena 7120 and that from E. coli.

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l‐glutamate: ammonia ligase, ADP‐forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn2+‐ and Mg2+‐dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg2+ and β‐mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg2+ and ATP. One‐tenth mM phenylmercuric acetate inhibited the Mg2+‐dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The Km values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH2OH, respectively. The constant for NH+4 was an order of magnitude higher in the presence of Mn2+ than Mg2+.When the amino acids were externally added to the reaction mixtures, the measurement of Pi exhibited a higher degree of enzyme inhibition than the measurement of γ‐glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg2+‐ and Mn2+‐dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l‐form) inhibited the enzyme 30% in the presence of either Mg2+ or Mn2+. Aspartate (Mg2+‐dependent) and histidine (Mn2+‐dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg2+‐dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.

Nitrate Reduction in the Marine Brown Alga Giffordia mitchellae (Harv.) Ham.

Summary Nitrate reductase (NADH-nitrate oxidoreductase, E.C. 1.6.6.2) and glutamine synthetase (L-glutamate: ammonia ligase (ADP), EC 6.3.1.2) have been preliminarily characterized for the first time in a Brown Alga, Giffordia mitchellae (Harv.) Ham. Nitrate reductase activity could be estimated in an «in vivo» assay system only. For determination of glutamine synthetase activity solely the transferase reaction (L-γ-glutamylhydroxamate formation) gave reliable results. In nitrogen-starved algal cultures nitrate reductase is inducible by nitrate, a lag phase of approx. 5 hours preceding induction. Ammonium ions rapidly inactivate nitrate reductase, inactivation is overcome by addition of excess nitrate to ammonium-containing cultures. Nitrate reductase activity in Giffordia is markedly reduced under the influence of glutamine administered to the cultures. Glutamine synthetase activity is not affected by glutamine in the medium. However NO3--and NH4+-ions exert a significant influence on the activity level of this enzyme in nitrogen-depleted cultures. Nitrate leads to an temporary enhancement of the activity level, whereas ammonium reduces glutamine synthetase activity. Nitrate reductase activity exhibits pronounced diurnal fluctuations. This rhythmicity, occurring under continuous light and constant temperature conditions, is initiated by addition of nitrate to nitrogen-depleted algae.

Glutamine synthetase from the pumpkin leaf cytosol

Glutamine synthetase ( l-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from pumpkin leaf cytosol is an oligomer that consists of eight identical monomers. The enzyme has a partial specific volume of 0.720 ml/g. The pH optimum in the presence of Mg 2+ is 7.2. It is shown by means of electron microscopy that the enzyme consists of elongated monomers, which are arranged with point 42 symmetry at the vertices of two squares. These squares are twisted about the 4-fold axis at 40° relative to each other.

Purification and properties of CTP synthetase from Ehrlich ascites tumor cells

CTP synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) was purified 200-fold from Ehrlich ascites tumor cells with about 50% purity as judged by polyacrylamide gel electrophoresis. The molecular weight was estimated to be 118 000 by gel filtration. The optimal pH was 8.6. 2-Mercaptoethanol was required for optimal activity and stabilization of the enzyme. Magnesium was essential for the reaction and other divalent cations were ineffective. Ammonia could replace glutamine as the amino donor. When other substrates were at saturating concentrations, Michaelis-Menten kinetics were observed yielding K m values for UTP, ATP and glutamine of 0.18, 0.8 and 0.13 mM, respectively. With ATP at subsaturating concentration, the double-reciprocal plot for UTP saturation was sinusoidal and the Hill plot showed an n value of 1.3. The double-reciprocal plot for ATP saturation, when UTP was at subsaturating concentration, departed from Michaelis-Menten kinetics with an n value of 1.4. These data suggest the existence of cooperative interactions between enzyme and substrates at subsaturating concentrations of ATP or UTP. GTP was not essential, but it acted as an activator on the glutamine reaction; optimal activation was observed at 1 mM GTP. The affinity for glutamine was not affected by GTP.

The involvement of proteolysis in conformational stability of the carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa

The pyrimidine-3 gene of Neurospora crassa codes for a bifunctional enzyme catalysing the first two steps of the pyrimidine biosynthetic pathway. Difficulties have been experienced in purification due to the lability of the enzyme. The enzyme loses carbamoyl-phosphate synthetase (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) activity and undergoes a change in apparent molecular weight from the native 650 000 to 100 000 of the only detectable fragment. Attempts have been made therefore to stabilize the enzyme so as to minimise these effects. Elastinal, a protease inhibitor, reduces the effects, as do certain ultra-violet-sensitive mutant strains which lack a minor protease. The nature of the loss of carbamoyl-phosphate synthetase suggests an instability in the tertiary structure of the enzyme which can be reduced by the use of glycerol. Glycerol also exhibits a protease-inhibiting effect in this system. Although a range of protease inhibitors and use of uvs mutants can reduce the rate of decay of carbamoyl-phosphate synthetase activity, only glycerol can stabilize the native molecular weight. Our results support the hypothesis that the loss of carbamoyl-phosphate synthetase activity and change in molecular weight of the enzyme is a three-step sequence of proteolysis, conformational shift and cleavage of a further non-covalent bond.

Synthesis, intracellular transport, and processing of the precursors for mitochondrial ornithine transcarbamylase and carbamoyl-phosphate synthetase I in isolated hepatocytes.

The synthesis and intracellular transport of the mitochondrial matrix enzymes ornithine transcarbamylase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) and carbamoyl-phosphate synthetase (ammonia) I [carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] were studied in isolated rat hepatocytes. In pulse experiments at 37 degrees C, the larger precursors of the two enzymes appeared in the cytosol of the liver cells, where radioactivity levels of the precursors reached a plateau in 10-20 min after the pulse. The pulse-labeled mature enzymes appeared in the particulate fraction (containing mitochondria) after a time lag and increased almost linearly with time up to 40 min. The specific radioactivities of the precursors in the cytosol were much higher than those of the mature enzymes in the particulate fraction. In pulse--chase experiments, the labeled precursors disappeared from the cytosol with estimated half-lives of about 1-2 min. These results indicate that ornithine transcarbamylase and carbamoyl-phosphate synthetase I are initially synthesized as larger precursors and exist in a cytosolic pool from which they are transported into mitochondria and processed there to the mature enzymes concomitantly with or immediately after transport. Although the rates of synthesis, transport, and processing were decreased about 3-fold at 25 degrees C (as compared to incubation at 37 degrees C), the pool size of the precursors in the cytosol were somewhat larger at this temperature.

Gut bacteria recycle uric acid nitrogen in termites: A strategy for nutrient conservation

Reticulitermes flavipes termites synthesize uric acid via purine-nucleoside phosphorylase (purine-nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) and xanthine dehydrogenase (xanthine:NAD(+) oxidoreductase, EC 1.2.1.37), but their tissues lack uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) or any other enzyme that degrades uric acid. Nevertheless, uricolysis occurs in termites, but as an anaerobic process mediated by hindgut bacteria. (14)C-Tracer experiments showed that termites transport uric acid from the site of synthesis and storage (fat body tissue) to the site of degradation (hindgut microbiota) via Malpighian tubules. Moveover, [1,3-(15)N]uric acid dissimilated by gut bacteria in vivo leads to assimilation of (15)N into termite tissues. NH(3), a product of uricolysis, is a potential N source for termites, either directly via glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] activity of fat body tissue or indirectly through microbe assimilation. Symbiotic recycling of uric acid N appears to be important to N conservation in these oligonitrotrophic insects.

A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated.

The coding sequence for Anabaena 7120 glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.1] are shown to be contained within a 7.5-kilobase-pair (kbp) HindIII fragment that has been cloned by plaque hybridization. The hybridization probe for the cyanobacterial gene was a recombinant plasmid containing the glnA gene from Escherichia coli K-12. Evidence that the cloned Anabaena fragment contains the glnA gene includes complementation of a glnA deletion mutant of E. coli and immunological identity of the enzyme produced by the cloned Anabaena fragment in E. coli with glutamine synthetase purified from Anabaena 7120. Heteroduplex analysis reveals 0.65 kbp of homology between the 7.5-kbp Anabaena 7120 fragment and an 11-kbp E. coli fragment that codes for E. coli glutamine synthetase. Studies of Anabaena glnA gene activity in E. coli suggest that the cyanobacterial gene is not repressible and that the Anabaena 7120 glutamine synthetase is not adenylylated in E. coli.

Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome.

We have cloned and characterized a fragment of the Escherichia coli chromosome spanning glnA, the structural gene for glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The fragment also carriers glnG, whose product is necessary for regulation of glnA expression, and a previously unidentified gene whose function we have not discovered. Transcription of glnA and the newly identified gene occurs divergently from a region between the two genes. Transcription of glnA proceeds toward glnG, which is transcribed in the same direction. A region of DNA between glnA and glnG contains genetic information whose loss may result in the inability to reduce expression of glnA and other operons in response to ammonia (the GlnC phenotype).

Carbamoyl phosphate synthetase I of human liver. Purification, some properties and immunological cross-reactivity with the rat liver enzyme

The purification of mitochondrial carbamoyl phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) from small samples of human liver is described. The enzyme is composed of a single polypeptide of M r 160 000 ± 500 as shown by SDS-polyacrylamide gel electrophoresis in the presence of reducing agents. The synthetase migrates in polyacrylamide gradient gels in the absence of detergents at a rate corresponding to a M r of 165 000. Estimates of the molecular weight of the native enzyme by gel filtration and density gradient centrifugation yield a value of 178 000. These results indicate that the enzyme exists predominantly as monomers. Amino acid composition, isoelectric point, stability K m values and the ability to catalyze partial reactions have been measured and compared with known properties of carbamoyl phosphate synthetases from other sources. From the available data a high degree of evolutionary conservation of the ammonia-dependent synthetase is suggested. This is also supported by the demonstration of extensive immunological cross-reactivity between the human and rat enzymes.

Nitrogen regulatory locus "glnR" of enteric bacteria is composed of cistrons ntrB and ntrC: identification of their protein products.

The nitrogen regulatory locus "glnR" of Escherichia coli and Salmonella typhimurium is composed of two cistrons, which we propose to call ntrB and ntrC (nitrogen regulation B and C). Frameshift mutations in ntrB and ntrC were isolated on a lambda phage that carries the E. coli ntrB and ntrC genes and the closely linked glnA gene, the structural gene encoding glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]; mutations were selected as suppressors of glnF (which we propose to rename ntrA), a selection used previously to isolate glnR mutations. Phage DNA from one mutant (ntrB) failed to direct synthesis of a 36-kilodalton (kDal) protein whose synthesis was directed by DNA from the parent phage (ntrB+) in a coupled in vitro transcription/translation system. DNA from three other mutants (ntrC) failed to direct synthesis of a 54-kDal protein; DNA from two of these mutants instead directed synthesis of smaller proteins, 53 and 50 kDal, respectively. In all four cases, DNA from frameshift revertants directed synthesis of both the 36-kDal and 54-kDal proteins. These results suggested that ntrB and ntrC were separate genes which encoded 36-kDal and 54-kDal protein products, respectively. Frameshift mutations in ntrB and ntrC complemented each other with regard to regulation of glnA expression in vivo and growth on arginine as nitrogen source, another nitrogen-controlled phenotype; this confirmed that ntrB and ntrC are separate cistrons that encode diffusible products. The ntrB and ntrC genes were also defined in S. typhimurium. Studies of mutant strains provided information on the roles of the ntrB and ntrC products in activation and repression of glnA expression and raised the possibility that these products function as a protein complex in regulating expression of nitrogen-controlled genes.

Turnover of bacterial glutamine synthetase: oxidative inactivation precedes proteolysis.

We partially purified a preparation from Escherichia coli that proteolytically degrades the enzyme glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The degradation is at least a two-step process. First, the glutamine synthetase undergoes an oxidative modification. This modification leads to loss of catalytic activity and also renders the protein susceptible to proteolytic attack in the second step. The oxidative step displays characteristics of a mixed-function oxidation, requiring both molecular oxygen and a reduced nucleotide. This step can also be catalyzed by a purified, mammalian cytochrome P-450 system, as well as by a model system consisting of ascorbic acid and oxygen. Catalase blocks this oxidative modification step. Thus, the overall process of proteolytic degradation can be observed only if care is taken to remove catalase activity from the extracts. The inactivation reaction is dependent on the state of adenylylation of the glutamine synthetase, suggesting that this a physiologically important reaction. If so, then mixed-function oxidases are now implicated in the process of intracellular protein turnover.