Purification and some properties of glutamate dehydrogenase and glutamine synthetase from Free

Abstract

The purification and some properties of NADP-dependent glutamate dehydrogenase (GDH) and glutamine synthetase (GS) from the facultatively anaerobic Gram-negative bacterium were investigated. The enzymes were purified to homogeneity using a procedure which involved affinity chromatography on Blue Sepharose CL-6B as the major purification step. The recoveries in the purification of GDH and GS were 28% and 64%, respectively. The specific activity of purified GDH was 183 nkat (mg protein)(deaminating reaction). GDH was composed of subunits of molecular mass 47 kDa and the native enzyme was either a tetramer or hexamer. The apparent values for L-glutamate, NADP, 2-oxoglutarate, NADPH and ammonia were 1.5 m, 5.9 μ, 0.47m, 12.5 μ and 14 m, respectively. The specific activity of purified GS was 1125 nkat (mg protein)(transferase reaction). The molecular mass of native GS was 570 kDa; it was composed of 12 subunits of molecular mass 50.1 kDa. The apparent values for -glutamine and hydroxylamine in the transferase reaction were 2.1 and 2.4 mM, respectively; those of ammonia, -glutamate and ATP in the biosynthetic reaction were 0.03, 1 and 0.17 m, respectively. After the adenylylation of GS, the for -glutamine and L-glutamate increased and reached the values of 8.0 and 27 m, respectively. The effects of the changes in GS activity on the ammonia metabolism of are discussed.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-8-1587
1992-08-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/8/mic-138-8-1587.html?itemId=/content/journal/micro/10.1099/00221287-138-8-1587&mimeType=html&fmt=ahah

References

  1. Bellion E., Tan F. 1984; NADP-dependent glutamate dehydrogenase from a facultative methylotroph Pseudomonas sp. strain AMI. Journal of Bacteriology 157:435–439
    [Google Scholar]
  2. 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 Bacteriology 129:1001–1009
    [Google Scholar]
  3. Blanco F., Arana A., Llama M., Serra J. L. 1989; Purification and properties of glutamine synthetase from non-H2-fixing cyanobacterium Phormidium laminosum . Journal of Bacteriology 171:1158–1165
    [Google Scholar]
  4. Bradford M. M. 1976; A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  5. Deuel T. F., Stadtman E. R. 1970; Some kinetic properties of Bacillus subtilis glutamine synthetase. Journal of Biological Chemistry 245:5206–5213
    [Google Scholar]
  6. Duchars M. G., Attwood M. M. 1991; Purification, localization, properties and regulation of glutamine synthetase from Hyphomicrobium X. Journal of General Microbiology 137:1345–1354
    [Google Scholar]
  7. Ferguson S. J. 1987; Denitrification: a question of the control of organization of electron and ion transport. Trends in Biochemical Sciences 12:354–357
    [Google Scholar]
  8. Hemmila I. A., Mantsala P. I. 1978; Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium . Biochemical Journal 173:45–52
    [Google Scholar]
  9. Hussain H., Davidson V. L. 1987; Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans . Journal of Bacteriology 169:1712–1717
    [Google Scholar]
  10. Janssen D. B., Op den Camp H. J. M., Leenan P. J. M., van der Drift C. 1980; The enzymes of assimilation of ammonia in Pseudomonas aeruginosa . Archives of Microbiology 124:197–203
    [Google Scholar]
  11. Kanamori K., Weiss R. L., Roberts J. D. 1987; Ammonia assimilation in Bacillus polymyxa . Journal of Biological Chemistry 262:11038–11045
    [Google Scholar]
  12. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  13. LeJohn H. B., Suzuki J., Wright J. 1968; Glutamate dehydrogenase from Thiobacillus novellus . Journal of Biological Chemistry 243:118–128
    [Google Scholar]
  14. Lepo J. E., Stacey G., Wyss O., Tabita F. R. 1979; The purification of glutamine synthetase from Azotobacter and other procaryotes by blue sepharose chromatography. Biochimica et Biophysica Acta 568:428–436
    [Google Scholar]
  15. Magasanik B. 1988; Reversible phosphorylation of an enhancer binding protein regulates the transcription of bacterial nitrogen utilisation genes. Trends in Biochemical Sciences 13:475–479
    [Google Scholar]
  16. Mikeš V. 1991; Regulation of ammonia assimilation in Paracoccus denitrificans in state of nitrogen starvation. Biologia Bratislava 46:305–314
    [Google Scholar]
  17. Mikeš V., Chvalova H., Matlova L. 1991; Assimilation of ammonia in Paracoccus denitrificans . Folia Microbiologica 36:35–41
    [Google Scholar]
  18. Op den Camp H. J. M., Liem K. D., Meesters P., Hermans J. H. M., van der Drift C. 1989; Purification and characterization of NADP-dependent glutamate dehydrogenase from Bacillus fastidiosus . Antonie van Leeuwenhoek 55:303–311
    [Google Scholar]
  19. Robertson R., Jervis L. 1985; Multiple purification of ammonia assimilation enzymes of E. coli . Biochemical Society Transactions 13:377–378
    [Google Scholar]
  20. Sakamoto O. N., Kotre A. M., Savageau M. 1977; Glutamate dehydrogenase from E. coli: purification and properties. Journal of Bacteriology 124:775–783
    [Google Scholar]
  21. Schinkinger M. F., Redl B., Stoffler G. 1991; Purification and properties of thermostable glutamate dehydrogenase from the Archaebacterium Sulfolobus solfataricus . Biochimica et Biophysica Acta 1073:142–148
    [Google Scholar]
  22. Senior P. J. 1975; Regulation of nitrogen metabolism in E. coli and Klebsiella aerogenes. Studies of continuous-culture technique. Journal of Bacteriology 123:407–418
    [Google Scholar]
  23. Smith E. L., Austen B. M., Blumenthal K. M., Nyc J. F. 1975; Glutamate dehydrogenases. In The Enzymes11 pp. 293–367 Edited by Boyer P. D. New York: Academic Press;
    [Google Scholar]
  24. Sokolov A. P., Trotsenko Y. A. 1987; Purification and properties of NADP-dependent glutamate dehydrogenase from the obligate methylotrophic bacterium Methylophilus methanolovorus . Biokhimiya 52:1417–1421
    [Google Scholar]
  25. Soliman A., Nordlung S. 1989; Purification and partial characterization of glutamine synthetase from photosynthetic bacterium Rhodospirillum rubrum . Biochimica et Biophysica Acta 994:138–141
    [Google Scholar]
  26. Stadtman E. R., Ginsburg A. 1974; Glutamine synthetase of E. coli. Structure and control. In The Enzymes 10 pp. 755–807 Edited by Boyer P. D. New York: Academic Press;
    [Google Scholar]
  27. Valentine R. C., Shapiro B. M., Stadtman E. R. 1968; Regulation of glutamine synthetase XII. Electron microscopy of enzyme from E. coli . Biochemistry 7:2143–2152
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-138-8-1587
Loading
/content/journal/micro/10.1099/00221287-138-8-1587
Loading

Data & Media loading...

Most cited Most Cited RSS feed