o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

Messenger RNA for glutamine synthetase

Molecular and Cellular Biochemistry - Of the various eucaryotic tissues, where glutamine synthetase (GS) mRNA and its regulation have been investigated, the induction of GS by glucocorticoids in...     

Distribution of glutamine synthetase and an inverse relationship between glutamine synthetase expression and intramuscular glutamine concentration in the horse

Glutamine plays important roles in the interorgan transport of nitrogen, carbon and energy but little is known about glutamine metabolism in the horse. In this study we determined the tissue distribution of glutamine synthetase expression in three Standardbred mares. Expression of glutamine synthetase was highest in kidney and mammary gland, and relatively high in liver and adipose tissue. Expression was lower in gluteus muscle, thymus, colon and lung, and much lower in small intestine, pancreas and uterus. The pattern of glutamine synthetase expression in the horse is similar to that of other herbivores and it is likely that skeletal muscle, liver, adipose tissue and lungs are the major sites of net glutamine synthesis in this species. Expression did not differ between adipose tissue depots but did vary between different muscles. Expression was highest in gluteus and semimembranous muscles and much lower in diaphragm and heart muscles. The concentration of intramuscular free glutamine was inversely correlated with expression of glutamine synthetase (r=-0.81, p=0.0017). The concentration of free glutamine was much higher in heart muscle (21.6+/-0.9 micromol/g wet wt) than in gluteus muscle (4.19+0.33 micromol/g wet wt), which may indicate novel functions and/or regulatory mechanisms for glutamine in the equine heart.     

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.     

Zinc-induced paracrystalline aggregation of glutamine synthetase

The unique capacity of glutamine synthetase to form highly insoluble paracrystalline aggregates in the presence of Zn²⁺ and Mg²⁺ mixtures is the basis of a new simple procedure for the isolation of the enzyme from crude extracts of Escherichia coli. Under optimal conditions (pH 5.85, 25 °C, 1.5 mm ZnSO₄ and 50 MgCl₂ over 95% of the enzyme is precipitated from crude extracts; differential extraction of the precipitate with dilute buffer (pH 7.0) containing 2.5 mm MgCl₂ leads to high yields of almost pure glutamine synthetase. Polyacrylamide gel electrophoresis of the purified enzyme shows it to consist of one major protein and two minor protein components, all of which exhibit glutamine synthetase activity. The major component appears to be identical with the enzyme previously isolated by the older more tedious procedure of Woolfolk et al. (1966). The γ-glutamyl transferase activity of enzyme isolated by the new procedure is the same as that isolated by the older method, but its biosynthetic activity is 25–35% lower. In all other respects examined (i.e., divalent ion specificity, pH optimum, apparent K_m values for substrates, susceptibility to feedback inhibition and physical properties) enzymes prepared by the old and the new procedures are indistinguishable. From studies with pure glutamine synthetase isolated by either procedure, it has been established that paracrystalline aggregation does not occur until 9–10 equivs of Zn²⁺ are bound per mole of enzyme. The high specificity of Zn²⁺ in inducing enzyme aggregation, suggests that its binding provokes a unique conformational state of the enzyme. This is supported by the fact that addition of Zn²⁺ to relaxed (divalent cation free) enzyme elicits a change in the ultraviolet spectrum of the enzyme that is qualitatively different from that caused by either Mg²⁺ or Mn²⁺. Moreover, in contrast to Mg²⁺, the binding of Zn²⁺ decreases the fluorescence associated with the binding of 2-p-toludinyl-naphthalene-6-sulfonic acid to the enzyme, suggesting that Zn²⁺ binding is accompanied by a decrease in the number of exposed hydrophobic regions on the enzyme.     

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.     

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.     

Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase.

The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).     

Inactivation of bacterial glutamine synthetase by ADP-ribosylation

Glutamine synthetase from Escherichia coli was inactivated by chemical modification with arginine-specific reagents (Colanduoni, J. A., and Villafranca, J. J. (1985) Biochem. Biophys. Res. Commun. 126, 412-418). E. coli glutamine synthetase was also a substrate for an erythrocyte NAD:arginine ADP-ribosyltransferase. Transfer of one ADP-ribosyl group/subunit of glutamine synthetase caused loss of both biosynthetic and gamma-glutamyltransferase activity. The ADP-ribose moiety was enzymatically removed by an erythrocyte ADP-ribosylarginine hydrolase, resulting in return of function. The site of ADP-ribosylation was arginine 172, determined by isolation of the ADP-ribosylated tryptic peptide. Arginine 172 lies in a central loop that extends into the core formed by the 12 subunits of the native enzyme. The central loop is important in anchoring subunits together to yield the spatial orientation required for catalytic activity. ADP-ribosylation may thus inactivate glutamine synthetase by disrupting the normal subunit alignment. Enzyme-catalyzed ADP-ribosylation may provide a simple, specific technique to probe the role of arginine residues in the structure and function of proteins.     

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.     

Regulation of glutamine synthetase I fromRhizobium meliloti

Glutamine synthetase I fromRhizobium meliloti was found to be inhibited by adenosine 5-monophosphate, alanine, glycine, carbamyl phosphate, cytidine 5-triphosphate, tryptophan, histidine, and glucosamine-6-phosphate. Each inhibitor was independent in its action and the effect was cumulative when more than one inhibitor was added.