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. 1977 Mar;129(3):1545–1555. doi: 10.1128/jb.129.3.1545-1555.1977

Formation of glutamine from [13n]ammonia, [13n]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrica.

J Thomas, J C Meeks, C P Wolk, P W Shaffer, S M Austin
PMCID: PMC235134  PMID: 14927

Abstract

A method is described for the isolation of metabolically active heterocysts from Anabaena cylindrica. These isolated heterocysts accounted for up to 34% of the acetylene-reducing activity of whole filaments and had a specific activity of up to 1,560 nmol of C2H4 formed per mg of heterocyst chlorphyll per min. Activity of glutamine synthetase was coupled to activity of nitrogenase in isolated heterocysts as shown by acetylene-inhibitable formation of [13N]NH3 and of amidelabeled [13N]glutamine form [13N]N2. A method is also described for the production of 6-mCi amounts of [13N]NH3. Isolated heterocysts formed [13N]glutamine from [13N]NH3 and glutamate, and [14C]glutamine from NH3 and [14C]glutamate, in the presence of magnesium adenosine 5'-triphosphate. Methionine sulfoximine strongly inhibited these syntheses. Glutamate synthase is, after nitrogenase and glutamine synthetase, the third sequential enzyme involved in the assimilation of N2 by intact filaments. However, the kinetics of solubilization of the activity of glutamate synthase during cavitation of suspensions of A. cylindrica indicated that very little, if any, of the activity of that enzyme was located in heterocysts. Concordantly, isolated heterocysts failed to form substantial amounts of radioactive glutamate from either [13N]glutamine or alph-[14C]ketoglutarate in the presence of other substrates and cofactors of the glutamate synthase reaction. However, they formed [14C]glutamate rapidly from alpha-[14C]ketoglutarate by aminotransferase reactions, with various amino acids as the nitrogen donor. The implication of these findings with regard to the identities of the substances moving between heterocysts and vegetative cells are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Fay P. Cell differentiation and pigment composition in Anabaena cylindrica. Arch Mikrobiol. 1969;67(1):62–70. doi: 10.1007/BF00413682. [DOI] [PubMed] [Google Scholar]
  3. Fay P., Stewart W. D., Walsby A. E., Fogg G. E. Is the heterocyst the site of nitrogen fixation in blue-green algae? Nature. 1968 Nov 23;220(5169):810–812. doi: 10.1038/220810b0. [DOI] [PubMed] [Google Scholar]
  4. Fay P., Walsby A. E. Metabolic activities of isolated heterocysts of the blue-green alga Anabaena cylindrica. Nature. 1966 Jan 1;209(5018):94–95. doi: 10.1038/209094a0. [DOI] [PubMed] [Google Scholar]
  5. Fleming H., Haselkorn R. Differentiation in Nostoc muscorum: nitrogenase is synthesized in heterocysts. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2727–2731. doi: 10.1073/pnas.70.10.2727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Haystead A., Robinson R., Stewart W. D. Nitrogenase activity in extracts of heterocystous and non-heterocystous blue-green algae. Arch Mikrobiol. 1970;74(3):235–243. doi: 10.1007/BF00408884. [DOI] [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. Lea P. J., Miflin B. J. The occurrence of glutamate synthase in algae. Biochem Biophys Res Commun. 1975 Jan 2;64(3):856–862. doi: 10.1016/0006-291x(75)90126-6. [DOI] [PubMed] [Google Scholar]
  9. Lex M., Carr N. G. The metabolism of glucose by heterocysts and vegetative cells of Anabaena cylindrica. Arch Microbiol. 1974;101(2):161–167. doi: 10.1007/BF00455936. [DOI] [PubMed] [Google Scholar]
  10. Miflin B. J., Lea P. J. Glutamine and asparagine as nitrogen donors for reductant-dependent glutamate synthesis in pea roots. Biochem J. 1975 Aug;149(2):403–409. doi: 10.1042/bj1490403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nagatani H., Shimizu M., Valentine R. C. The mechanism of ammonia assimilation in nitrogen fixing Bacteria. Arch Mikrobiol. 1971;79(2):164–175. doi: 10.1007/BF00424923. [DOI] [PubMed] [Google Scholar]
  12. Peterson R. B., Burris R. H. Properties of heterocysts isolated with colloidal silica. Arch Microbiol. 1976 May 3;108(1):35–40. doi: 10.1007/BF00425090. [DOI] [PubMed] [Google Scholar]
  13. Rivera-Ortiz J. M., Burris R. H. Interactions among substrates and inhibitors of nitrogenase. J Bacteriol. 1975 Aug;123(2):537–545. doi: 10.1128/jb.123.2.537-545.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rowell P., Stewart W. D. Alanine dehydrogenase of the N2-fixing blue-green alga, Anabaena cylindrica. Arch Microbiol. 1975 Sep;107(2):115–124. doi: 10.1007/BF00446830. [DOI] [PubMed] [Google Scholar]
  15. Smith R. V., Evans M. C. Soluble nitrogenase from vegetative cells of the blue-green alga Anabaena cylindrica. Nature. 1970 Mar 28;225(5239):1253–1254. doi: 10.1038/2251253a0. [DOI] [PubMed] [Google Scholar]
  16. Stewart W. D., Haystead A., Pearson H. W. Nitrogenase activity in heterocysts of blue-green algae. Nature. 1969 Oct 18;224(5216):226–228. doi: 10.1038/224226a0. [DOI] [PubMed] [Google Scholar]
  17. Stewart W. D., Rowell P. Effects of L-methionine-DL-sulphoximine on the assimilation of newly fixed NH3, acetylene reduction and heterocyst production in Anabaena cylindrica. Biochem Biophys Res Commun. 1975 Aug 4;65(3):846–856. doi: 10.1016/s0006-291x(75)80463-3. [DOI] [PubMed] [Google Scholar]
  18. Tel-Or E., Stewart W. D. Photosynthetic electron transport, ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica. Biochim Biophys Acta. 1976 Feb 16;423(2):189–195. doi: 10.1016/0005-2728(76)90177-8. [DOI] [PubMed] [Google Scholar]
  19. Tempest D. W., Meers J. L., Brown C. M. Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route. Biochem J. 1970 Apr;117(2):405–407. doi: 10.1042/bj1170405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thomas J., David K. A. Site of nitrogenase activity in the blue-green alga Anabaena sp. L-31. Nat New Biol. 1972 Aug 16;238(85):219–221. doi: 10.1038/newbio238219a0. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Winkenbach F., Wolk C. P. Activities of enzymes of the oxidative and the reductive pentose phosphate pathways in heterocysts of a blue-green alga. Plant Physiol. 1973 Nov;52(5):480–483. doi: 10.1104/pp.52.5.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wolk C. P., Austin S. M., Bortins J., Galonsky A. Autoradiographic localization of 13N after fixation of 13N-labeled nitrogen gas by a heterocyst-forming blue-green alga. J Cell Biol. 1974 May;61(2):440–453. doi: 10.1083/jcb.61.2.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wolk C. P. Physiological basis of the pattern of vegetative growth of a blue-green alga. Proc Natl Acad Sci U S A. 1967 May;57(5):1246–1251. doi: 10.1073/pnas.57.5.1246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wolk C. P., Quine M. P. Formation of one-dimensional patterns by stochastic processes and by filamentous blue-green algae. Dev Biol. 1975 Oct;46(2):370–382. doi: 10.1016/0012-1606(75)90113-x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. van Gorkom H. J., Donze M. Localization of nitrogen fixation in Anabaena. Nature. 1971 Nov 26;234(5326):231–232. doi: 10.1038/234231b0. [DOI] [PubMed] [Google Scholar]

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