1887

Abstract

Summary: The purification, location, properties and regulation of glutamine synthetase (GS) from the facultative methylotroph X were investigated. The enzyme was purified to homogeneity by differential centrifugation, Blue Sepharose CL-6B chromatography and Sephadex G-25 gel filtration. The specific activity of the purified enzyme was 44.7 μmol min (mg protein). GS was cytoplasmic in location with no direct association with DNA, membranes or the enzyme glutamine: 2-oxoglutarate aminotransferase (GOGAT). The molecular mass of the native enzyme was 638 kDa; it was composed of 12 subunits of molecular mass 53 kDa. Double reciprocal plots showed that the enzyme followed Michaelis-Menten kinetics. The apparent values for hydroxamate and glutamine in the γ-glutamyl transferase assay were 7.0 and 5.0 mM respectively; those of ammonia, glutamate and ATP in the biosynthetic assay were 0.032, 31.1 and 0.37 mM respectively. GS activity was controlled by covalent modification, the presence of specific divalent cations and feedback inhibition by several end-products of glutamine metabolism.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-137-6-1345
1991-06-01
2021-05-15
Loading full text...

Full text loading...

/deliver/fulltext/micro/137/6/mic-137-6-1345.html?itemId=/content/journal/micro/10.1099/00221287-137-6-1345&mimeType=html&fmt=ahah

References

  1. Baginski E. S., Foa P. P., Zak B. 1967; Microdetermination of inorganic phosphate, phospholipids and total phosphate in biological materials. Clinical Chemistry 13:326–330
    [Google Scholar]
  2. Bellion E., Bolbot J. R. 1983; Nitrogen assimilation in facultative methylotrophic bacteria. Current Microbiology 9:37–44
    [Google Scholar]
  3. Bender R. A., Streicher S. L. 1979; Glutamine synthetase. Regulation, adenylylation state and strain specificity analysed by polyacrylamide gel electrophoresis. Journal of Bacteriology 137:1000–1007
    [Google Scholar]
  4. Bender R. A., Janssen K. A., Resnick A. D., Blumenberg M., Foor F., Magasanik B. 1977; Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. Journal of Bacteri-ology 129:1001–1009
    [Google Scholar]
  5. Bhatnagar L., Zeikus J. G., Aubert J.-P. 1986; Purification and characterization of glutamine synthetase from the archaebacterium Methanobacterium ivanovi. Journal of Bacteriology 165:638–643
    [Google Scholar]
  6. Blackmore M. A., Quayle J. R. 1970; Microbial oxidation of oxalate by a route not involving glyoxylate carboligase. Biochemical Journal 118:53–59
    [Google Scholar]
  7. Bodusing S. J., Brantt P. W., Robb F. T., Woods D. R. 1985; Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain. Archives of Microbiology 140:369–374
    [Google Scholar]
  8. Brooke A. G., Duchars M. G., Attwood M. M. 1987; Nitrogen assimilation in the facultative methylotroph Hyphomicrobium X. FEMS Microbiology Letters 41:41–45
    [Google Scholar]
  9. Brown C. M. 1980; Ammonia assimilation and utilization in bacteria and fungi. Microorganisms and Nitrogen Sources511–538 Payne J. W. New York: Academic Press;
    [Google Scholar]
  10. Brown M. S., Segal A., Stadtman E. R. 1971; Modulation of glutamine synthetase adenylylation and deadenylylation is mediated by metabolic transformation of the Pn-regulatory protein. Proceedings of the National Academy of Sciences of the United States of America 682949–2943
    [Google Scholar]
  11. Caballero P., Romero F., Roldan J. M. 1987; Purification and characterisation of glutamine synthetase from Rhodomicrobium vannielii. FEMS Microbiology Letters 48:15–17
    [Google Scholar]
  12. Dalton H. 1979; Utilization of inorganic nitrogen by microbial cells. International Reviews of Biochemistry 21:227–266
    [Google Scholar]
  13. Deuel T. F., Stadtman E. R. 1970; Some kinetic properties of Bacillus subtilis glutamine synthetase. Journal of Biological Chemistry 245:5206–5213
    [Google Scholar]
  14. Duchars M. G, Attwood M. M. 1989; The influence of C:N ratio in the growth medium on the cellular composition and regulation of enzyme activity in Hyphomicrobium X. Journal of General Microbiology 135:787–793
    [Google Scholar]
  15. Ely B., Amarasinghe A. B. C., Bender R. A. 1978; Ammonia assimilation and glutamate formation in Caulobacter crescentus. Journal of Bacteriology 133:225–230
    [Google Scholar]
  16. Florencio F. J., Romos J. L. 1985; Purification and characterization of glutamine synthetase from the unicellular cyanobacterium Anacystis nidulans. Biochimica et Biophysica Acta 838:39–48
    [Google Scholar]
  17. Ginsburg A. 1969; Conformational changes in glutamine synthetase from Escherichia coli. II. Some characteristics of the equilibrium binding of feedback inhibitors to the enzyme. Biochemistry 8:1726–1740
    [Google Scholar]
  18. Ginsburg A., Stadtman E. R. 1973; Regulation of glutamine synthetase in Escherichia coli. The Enzymes of Glutamine Metabolism9–44 Prusiner S., Stadtman E. R. New York: Academic Press;
    [Google Scholar]
  19. Ginsburg A., Yeh J., Henning S. B., Denten M. D. 1970; Some effects of adenylylation on the biosynthetic properties of the glutamine synthetase from Escherichia coli. Biochemistry 9:633–649
    [Google Scholar]
  20. Harder W., Attwood M. M. 1978; Biology, physiology and biochemistry of hyphomicrobia. Advances in Microbial Physiology 17:303–359
    [Google Scholar]
  21. Harder W., Attwood M. M., Quayle J. R. 1973; Methanol assimilation by Hyphomicrobium sp. Journal of General Microbiology 78:155–163
    [Google Scholar]
  22. Hubbard J. S., Stadtman E. R. 1967a; Regulation of glutamine synthetase. V. Partial purification and properties of glutamine synthetase of Bacillus licheniformis. Journal of Bacteriology 94:1007–1015
    [Google Scholar]
  23. Hubbard J. S., Stadtman E. R. 1976; Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase. Journal of Bacteriology 94:1016–1024
    [Google Scholar]
  24. Johnson P. A., Quayle J. R. 1964; Microbial growth on C, compounds. 6. Oxidation of methanol, formaldehyde and formate by methanol-grown Pseudomonas AMI. Biochemical Journal 93:281–290
    [Google Scholar]
  25. Kingdom H. S., Shapiro B. M., Stadtman E. R. 1967; Regulation of glutamine synthetase. VIII. ATPiglutamine synthetase: an enzyme that catalyses alterations in the regulatory properties of glutamine synthetase. Proceedings of the National Academy of Sciences of the United States of America 581703–1714
    [Google Scholar]
  26. Kingdom H. S., Hubbard J., Stadtman E. R. 1968; Regulation of glutamine synthetase. XI. The nature and implications of a lag phase in the Escherichia coli glutamine synthetase reaction. Biochemistry 7:2136–2142
    [Google Scholar]
  27. Kleinschmidt J. A., Kleiner D. 1978; The glutamine synthetase from Azotobacter vinelandii. purification, characterisation, regulation and localisation. European Journal of Biochemistry 89:51–60
    [Google Scholar]
  28. Kumar S., Nicholas D. J. D. 1984; Purification, properties and regulation of glutamine synthetase from Nitrobacter agilis. Journal of General Microbiology 130:959–966
    [Google Scholar]
  29. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature London: 227680–685
    [Google Scholar]
  30. Laemmli U. K., Favre K. 1973; Maturation of the head of bacteriophage T4. 1. DNA packaging events. Journal of Molecular Biology 80:575–599
    [Google Scholar]
  31. Loginova N. V., Govorukhina N. I., Trotsenko Y. A. 1982; Enzymes of the assimilation of ammonium in bacteria with various pathways of C1 metabolism. Mikrobiologiya 51:38–42 (English translation)
    [Google Scholar]
  32. Mangum J. H., Magni G., Stadtman E. R. 1973; Regulation of glutamine synthetase adenylylation and deadenylylation by the enzymatic uridylylation and deadenylylation of the PII regulatory protein. Archives of Biochemistry and Biophysics 158:514–525
    [Google Scholar]
  33. Meers J. L., Tempest D. W., Brown C. W. 1970; Glutamine (amide) : 2-oxoglutarate aminotransferase oxido-reductase (NADP): an enzyme involved in the synthesis of glutamate by some bacteria. Journal of General Microbiology 64:187–194
    [Google Scholar]
  34. Murrell J. C., Dalton H. 1983; Purification and properties of glutamine synthetase from Methylococcus capsulatus (Bath). Journal of General Microbiology 129:1187–1196
    [Google Scholar]
  35. Orr J., Haselkorn R. 1981; Kinetics and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120. Journal of Biological Chemistry 256:13099–13104
    [Google Scholar]
  36. Segal A., Stadtman E. R. 1972a; Effects of cobaltous ion on various catalytic parameters and on heterologous subunit interactions of Escherichia coli glutamine synthetase. Archives of Biochemistry and Biophysics 152:356–366
    [Google Scholar]
  37. Segal A., Stadtman E. R. 1972b; Variation of the conformational status of Escherichia coli glutamine synthetase by interactions with different divalent cations. Archives of Biochemistry and Biophysics 152:367–377
    [Google Scholar]
  38. Segal A., Brown M. S., Stadtman E. R. 1974; Metabolite regulation of the state of adenylylation of glutamine synthetase. Archives of Biochemistry and Biophysics 161:319–327
    [Google Scholar]
  39. Shapiro B. M., Stadtman E. R. 1967; Regulation of glutamine synthetase. IX. Reactivity of the sulfhydryl groups of the enzyme from Escherichia coli. Journal of Biological Chemistry 242:5069–5079
    [Google Scholar]
  40. Shapiro B. M., Stadtman E. R. 1970; The regulation of glutamine synthesis in microorganisms. Annual Review of Microbiology 24:501–524
    [Google Scholar]
  41. Shishkina V. N., Trotsenko Y. A. 1979; Pathways of ammonia assimilation in obligate methane utilizers. FEMS Microbiology Letters 5:187–191
    [Google Scholar]
  42. Stadtman E. R., Ginsburg A., Ciardi E., Yeh S., Hennig B., Shapiro B. M. 1970; Multiple molecular forms of glutamine synthetase produced by enzyme catalysed adenylylation and deadenylylation reactions. Advances in Enzyme Regulation 8:99–118
    [Google Scholar]
  43. Streicher S. L., Tyler B. 1980; Purification of glutamine synthetase from a variety of bacteria. Journal of Bacteriology 142:69–78
    [Google Scholar]
  44. Tempest D. W., Meers J. L., Brown C. M. 1973; Glutamate synthase (GOGAT) : a key enzyme in the assimilation of ammonia by prokaryotic organisms. The Enzymes of Glutamine Metabolism167–182 Prusiner S., Stadtman E. R. New York: Academic Press;
    [Google Scholar]
  45. Tyler B. 1978; Regulation of the assimilation of nitrogenous compounds. Annual Review of Biochemistry 47:1127–1162
    [Google Scholar]
  46. Valentine R. C., Shapiro B. M., Stadtman E. R. 1968; Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry 7:2143–2152
    [Google Scholar]
  47. Wedler F. C., Hoffman F. M. 1974; Glutamine synthetase of Bacillus stearothermophilus. I. Purification and basic properties. Biochemistry 13:3207–3214
    [Google Scholar]
  48. Wedler F. C., Carfi J., Ashour A. E. 1976; Glutamine synthetase of Bacillus stearothermophilus. Regulation, site interactions and functional information. Biochemistry 15:1749–1755
    [Google Scholar]
  49. Woolfolk C. A., Stadtman E. R. 1967; Regulation of glutamine synthetase. III. Cumulative feedback inhibition of glutamine synthetase from Escherichia coli. Archives of Biochemistry and Biophysics 118:736–755
    [Google Scholar]
  50. Woolfolk C. A., Shapiro B. M., Stadtman E. R. 1964; Cumulative feedback inhibition in the multiple end product regulation of glutamine synthetase in Escherichia coli. Biochemical and Biophysical Research Communications 17:313–319
    [Google Scholar]
  51. Woolfolk C. A., Shapiro B. M., Stadtman E. R. 1966; Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Archives of Biochemistry and Biophysics 116:177–192
    [Google Scholar]
  52. Wulff K., Mecke D., Holzer H. 1967; Mechanism of the inactivation of glutamine synthetase from E. coli. Biochemical and Biophysical Research Communications 28:740–745
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-137-6-1345
Loading
/content/journal/micro/10.1099/00221287-137-6-1345
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error