The enzyme glutamine synthetase I of Drosophila melanogaster is associated with a modified RNA

Glutamine synthetase I (L-glutamate:ammonia ligase, ADP forming; EC 6.3.1.2) was purified from Drosophila melanogaster larvae. The complete enzyme has an apparent molecular weight of 380,000. The subunit of the active enzyme has an apparent molecular weight of 43,000 after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Routine preparations yield enzymes which have at least another polypeptide component of apparent molecular weight of 64,000. Several factors suggest that the 64,000-dalton polypeptide might be a transformation product of the 43,000-dalton subunit which occurs in association with enzyme inactivation. Distinct from its protein subunit, from pure glutamine synthetase I a material can be extracted which can be labeled with 32P-labeled gamma-ATP using polynucleotide kinase. After alkaline hydrolysis the majority of the radioactivity is recovered as 5'2' and 5'3' ribonucleotide diphosphates, and after venom phosphodiesterase digestion as 5' ribonucleotide. We therefore conclude that the native glutamine synthetase I enzyme contains, or at least is reproducibly associated with, an RNA component. Several characteristics of the labeled material indicate that the RNA is small in size and is bound to polymer molecules different from RNA.     

6-Phosphogluconate dehydrogenase from Drosophila melanogaster. I. Purification and properties of the a isozyme

6-Phosphogluconate dehyrogenase is evident at all developmental stages of Drosophila melanogaster. The activity level is highest in early third instar larvae and declines to a lower, but relatively constant, level at all later stages of development. The enzyme is localized in the cytosolic portion of the cell. The A-isozymic form of 6-phosphogluconate dehydrogenase was purified to homogeneity and has a molecular weight of 105,000. The enzyme is a dimer consisting of subunits with molecular weights of 55,000 and 53,000. For the oxidative decarboxylation of 6-phosphogluconate the K_m for substrate is 81 µm while that for NADP⁺ is 22.3 µm. The optimum pH for activity is 7.8 while the optimum temperature is 37 C.This work was supported by National Research Council of Canada Grant A5860 and by the University of Calgary Research Policy and Grants Committee.     

Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster: Purification and properties

Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster has been purified 1400-fold over a crude 230,000g supernatant fraction. The optimum activity of the enzyme occurs at magnesium concentrations above 10 mm at 37 °C and pH 7.5. At a 50 mm Mg²⁺ concentration, NH₄⁺ stimulates the ATP-PP₁ exchange reaction as much as 2-fold. Ammonium chloride causes an increase in the V with no change in the K_m with phenylalanine as substrate. Homologous (Drosophila) tRNA, in the presence of NH₄⁺, further stimulates the ATP-PP_i, exchange reaction but inhibits the reaction in the absence of NH₄⁺.In the presence of its substrates the enzyme is inactivated by NEM to varying degrees depending upon the substrate or combinations of substrates used. In the presence of phenylalanine the enzyme is partially protected but both ATP and tRNA make the enzyme more susceptible to inactivation. NEM together with ATP and tRNA or all three substrates results in near-total inactivation.     

Purification and structural properties of adenylylated and deadenylylated glutamine synthetase from Rhodopseudomonas sphaeroides

Glutamine synthetase (GS) of Rhodopseudomonas sphaeroides is regulated by adenylylation and deadenylylation. The extent of adenylylation/deadenylylation of the enzyme in cell free extracts was influenced by inorganic phosphate (P _i), -ketoglutarate, ATP and other nucleotides. While P _i and -ketoglutarate stimulated deadenylylation, ATP and other nucleotides enhanced adenylylation of the GS. By using proper combinations of the effectors and incubation conditions, any desired adenylylation state of GS could be adjusted in vitro. The enzyme was purified to electrophoretic homogenity by three steps including affinity chromatography on 5-AMP-Sepharose. Adenylylated and deadenylylated enzyme showed different UV-spectra and isoelectric points. The native enzyme had a molecular weight of 600,000, deadenylylated subunits of 50,000±1,000. Electron microscopic investigations revealed a dodecameric arrangement of subunits in two hexameric planes.     

Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp

Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae.     

Purification and characterization of GTP cyclohydrolase I from Drosophila melanogaster

The enzyme GTP cyclohydrolase I, which catalyzes the first step in the pteridine biosynthetic pathway, has been purified by at least 4400-fold from Drosophila melanogaster. The active complex has an apparent molecular mass of 575,000 daltons, as estimated from gel filtration. This high molecular mass complex appears to be composed of a number of 39,000-dalton subunits. A polyspecific antiserum generated against the active complex has been used to identify this polypeptide as being severely affected by mutations in Punch, the structural gene for GTP cyclohydrolase. Enzyme activity is inhibited by divalent cations and high ionic strength buffers. No cofactors have been demonstrated to be required for enzyme activity. The enzyme displays positive cooperativity in phosphate buffer, a Hill number of 2.1, but only slight cooperativity in Tris buffer, a Hill number of 1.2.     

Genetic determinants of glutamine synthetase inDrosophila melanogaster: A gene for glutamine synthetase I resides in the 21B3-6 region

Recombinational and deletion mapping of electrophoretic variants of the glutamine synthetase I isozyme (GSI) in Drosophila melanogaster locates the gene in the 21B region on the second chromosome. We have conducted a genetic analysis of the region extending cytologically from 21A to 21B4-6. Recessive lethal mutations were generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU) mutagenesis and by hybrid dysgenesis (HD). These lethals fall into seven functional groups, which were partially ordered by complementation with cytologically defined deficiencies of this region generated by hybrid dysgenesis. Two of the EMS- and two of the ENU-induced lethals fulfill biochemical criteria expected for null alleles of the GSI gene.     

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

Glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) was isolated from chicken liver, brain and neural retina. 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. Slight differences were detectable among them in relation to the amino acid compositions and regulation of their activities by the several effectors tested.     

Expression of a functional Drosophila melanogaster CMP-sialic acid synthetase. Differential localization of the Drosophila and human enzymes

CMP-N-acetylneuraminic acid is a critical metabolite in the generation of glycoconjugates that play a role in development and other physiological processes. Whereas pathways for its generation are firmly established in vertebrates, the presence and function of the relevant synthetic enzyme in insects and other protostomes is unknown. In this study, we characterize the first functional CMP-sialic acid synthase (DmCSAS) from any protostome lineage expressed from a D. melanogaster cDNA clone. Homologous genes were subsequently identified in other insect species. The gene is developmentally regulated, with expression first appearing at 12-24 h of embryogenesis, low expression through larval and pupal stages, and greatly enriched expression in the adult head, suggesting a possible role in the central nervous system. Activity of the enzyme was verified by an increase in in vitro and in vivo CMP-N-acetylneuraminic acid levels when expressed in a heterologous host. Unlike all known vertebrate CMP-sialic acid synthetase (CSAS) proteins that localize to the nucleus, the D. melanogaster CSAS protein was targeted to the Golgi compartment when expressed in both heterologous mammalian and insect cell lines. Replacement of the N-terminal leader sequence of DmCSAS with the human CSAS N-terminal sequence resulted in the redirection of the chimeric CSAS protein to the nucleus but with a concomitant loss of enzymatic activity. The localization of CSAS orthologs to different intracellular organelles represents, to our knowledge, the first example of differential protein targeting of orthologs in eukaryotes and reveals how the sialylation pathway diverged during the evolution of protostomes and deuterostomes.     

Purification and characterisation of glutamine synthetase fromNocardia corallina

Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold fromNocardia corallina. The apparentM _r of the GS subunit was approximately 56,000. Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000. The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine. The pH profiles assayed by the -glutamyl transferase method were similar for NH₄ ⁺-treated and untreated cell extracts and an isoactivity point was not obtained from these curves. GS activity was repressed by (NH₄)₂SO₄ and glutamate. Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity. The GS ofN. corallina cross-reacted with antisera prepared against GS from a Gram-negativeThiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positiveClostridium acetobutylicum strain.