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. 1977 Nov;132(2):596–603. doi: 10.1128/jb.132.2.596-603.1977

Nitrogen and Ammonia Assimilation in the Cyanobacteria: Purification of Glutamine Synthetase from Anabaena sp. Strain CA

Gary Stacey 1, F Robert Tabita 1, Chase Van Baalen 2
PMCID: PMC221901  PMID: 21167

Abstract

Glutamine synthetase was purified from the cyanobacterium Anabaena sp. strain CA, a newly isolated marine organism. This organism grows rapidly under nitrogen-fixing conditions and therefore is ideally suited for studies concerning cyanobacterial nitrogen metabolism. Studies were conducted to optimize the production of glutamine synthetase by Anabaena CA. The highest specific activities were obtained from cells grown in the presence of atmospheric N2 or KNO3 (13 mM); when NH4Cl was used as the nitrogen source, the specific activity was reduced by approximately 40%. Furthermore, through the use of a whole-cell γ-glutamylhydroxamate transferase assay, it was found that the maximum number of enzyme units is obtained in the late logarithmic stage of growth. Glutamine synthetase purification requires only three steps and results in a preparation that is electrophoretically homogeneous. The transferase specific activity (units per milligram of protein) of the purified enzyme is 78, whereas the biosynthetic specific activity is 2.2. The molecular weight of the native protein was found to be approximately 590,000, and the subunit molecular weight was determined to be about 50,000. Thus, this cyanobacterial enzyme closely resembles the enzyme obtained from other procaryotic sources, at least with regard to size. The purification of glutamine synthetase from Anabaena CA should stimulate a more detailed study of this enzyme and its role in cyanobacterial nitrogen metabolism.

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Selected References

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  1. Deuel T. F., Ginsburg A., Yeh J., Shelton E., Stadtman E. R. Bacillus subtilis glutamine synthetase. Purification and physical characterization. J Biol Chem. 1970 Oct 25;245(20):5195–5205. [PubMed] [Google Scholar]
  2. Dharmawardene M. W., Haystead A., Stewart W. D. Glutamine synthetase of the nitrogen-fixing alga Anabaena cylindrica. Arch Mikrobiol. 1973 Apr 26;90(4):281–295. doi: 10.1007/BF00408924. [DOI] [PubMed] [Google Scholar]
  3. Holzer H., Mecke D., Wulff K., Liess K., Heilmeyer L., Jr Metabolite-induced enzymatic inactivation of glutamine synthetase in E. coli. Adv Enzyme Regul. 1967;5:211–225. doi: 10.1016/0065-2571(67)90017-9. [DOI] [PubMed] [Google Scholar]
  4. Hubbard J. S., Stadtman E. R. Regulation of glutamine synthetase. V. Partial purification and properties of glutamine synthetase from Bacillus licheniformis. J Bacteriol. 1967 Oct;94(4):1007–1015. doi: 10.1128/jb.94.4.1007-1015.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kingdon H. S., Stadtman E. R. Two E. coli glutamine synthetases with different sensitivities to feedback effectors. Biochem Biophys Res Commun. 1967 May 25;27(4):470–473. doi: 10.1016/s0006-291x(67)80008-1. [DOI] [PubMed] [Google Scholar]
  6. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  7. Lea P. J., Miflin B. J. Glutamate synthase in blue-green algae. Biochem Soc Trans. 1975;3(3):381–384. doi: 10.1042/bst0030381. [DOI] [PubMed] [Google Scholar]
  8. McParland R. H., Guevara J. G., Becker R. R., Evans H. J. The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules. Biochem J. 1976 Mar 1;153(3):597–606. doi: 10.1042/bj1530597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Miller R. E., Shelton E., Stadtman E. R. Zinc-induced paracrystalline aggregation of glutamine synthetase. Arch Biochem Biophys. 1974 Jul;163(1):155–171. doi: 10.1016/0003-9861(74)90465-2. [DOI] [PubMed] [Google Scholar]
  10. Orr M. D., Blakley R. L., Panagou D. Discontinuous buffer systems for analytical and preparative electrophoresis of enzymes on polyacrylamide gel. Anal Biochem. 1972 Jan;45(1):68–85. doi: 10.1016/0003-2697(72)90008-5. [DOI] [PubMed] [Google Scholar]
  11. PROVASOLI L., MCLAUGHLIN J. J., DROOP M. R. The development of artificial media for marine algae. Arch Mikrobiol. 1957;25(4):392–428. doi: 10.1007/BF00446694. [DOI] [PubMed] [Google Scholar]
  12. Palacios R. Neurospora crassa glutamine synthetase. Purification by affinity chromatography and characterization of subunit structure. J Biol Chem. 1976 Aug 10;251(15):4787–4791. [PubMed] [Google Scholar]
  13. Pearce J., Leach C. K., Carr N. G. The incomplete tricarboxylic acid cycle in the blue-green alga Anabaena variabilis. J Gen Microbiol. 1969 Mar;55(3):371–378. doi: 10.1099/00221287-55-3-371. [DOI] [PubMed] [Google Scholar]
  14. Prival M. J., Brenchley J. E., Magasanik B. Glutamine synthetase and the regulation of histidase formation in Klebsiella aerogenes. J Biol Chem. 1973 Jun 25;248(12):4334–4344. [PubMed] [Google Scholar]
  15. Shapiro B. M., Stadtman E. R. The regulation of glutamine synthesis in microorganisms. Annu Rev Microbiol. 1970;24:501–524. doi: 10.1146/annurev.mi.24.100170.002441. [DOI] [PubMed] [Google Scholar]
  16. Stadtman E. R., Ginsburg A., Ciardi J. E., Yeh J., Hennig S. B., Shapiro B. M. Multiple molecular forms of glutamine synthetase produced by enzyme catalyzed adenylation and deadenylylation reactions. Adv Enzyme Regul. 1970;8:99–118. doi: 10.1016/0065-2571(70)90011-7. [DOI] [PubMed] [Google Scholar]
  17. Stadtman E. R., Shapiro B. M., Kingdon H. S., Woolfolk C. A., Hubbard J. S. Cellular regulation of glutamine synthetase activity in Escherichia coli. Adv Enzyme Regul. 1968;6:257–289. doi: 10.1016/0065-2571(68)90017-4. [DOI] [PubMed] [Google Scholar]
  18. Tabita F. R., Stevens S. E., Jr, Gibson J. L. Carbon dioxide assimilation in blue-green algae: initial studies on the structure of ribulose 1,5-bisphosphate carboxylase. J Bacteriol. 1976 Feb;125(2):531–539. doi: 10.1128/jb.125.2.531-539.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tabita R., Lundgren D. G. Glucose-6-phosphate dehydrogenase from the chemolithotroph Thiobacillus ferrooxidans. J Bacteriol. 1971 Oct;108(1):343–352. doi: 10.1128/jb.108.1.343-352.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thomas J., Wolk C. P., Shaffer P. W., Austin S. M., Galonsky A. The initial organic products of fixation of 13N-labeled nitrogen gas by the blue-green alga Anabaena cylindrica. Biochem Biophys Res Commun. 1975 Nov 17;67(2):501–507. doi: 10.1016/0006-291x(75)90840-2. [DOI] [PubMed] [Google Scholar]
  21. Tronick S. R., Ciardi J. E., Stadtman E. R. Comparative biochemical and immunological studies of bacterial glutamine synthetases. J Bacteriol. 1973 Sep;115(3):858–868. doi: 10.1128/jb.115.3.858-868.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  23. Wedler F. C., Hoffmann F. M. Glutamine synthetase of Bacillus stearothermophilus. I. Purification and basic properties. Biochemistry. 1974 Jul 30;13(16):3207–3214. doi: 10.1021/bi00713a002. [DOI] [PubMed] [Google Scholar]
  24. Wolk C. P., Thomas J., Shaffer P. W., Austin S. M., Galonsky A. Pathway of nitrogen metabolism after fixation of 13N-labeled nitrogen gas by the cyanobacterium, Anabaena cylindrica. J Biol Chem. 1976 Aug 25;251(16):5027–5034. [PubMed] [Google Scholar]
  25. Woolfolk C. A., Shapiro B., Stadtman E. R. Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch Biochem Biophys. 1966 Sep 26;116(1):177–192. doi: 10.1016/0003-9861(66)90026-9. [DOI] [PubMed] [Google Scholar]

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