Advertisement

Planta

, Volume 157, Issue 3, pp 245–253 | Cite as

Purification and properties of two forms of glutamine synthetase from the plant fraction of Phaseolus root nodules

  • Julie V. Cullimore
  • M. Lara
  • P. J. Lea
  • B. J. Miflin
Article

Abstract

Two forms of glutamine synthetase (GS) have been purified to apparent homogeneity from the plant fraction of Phaseolus vulgaris root nodules. One of these forms appears identical to the form of the enzyme found in roots but the other is probably specifically associated with the nodule. Free-living Rhizobium phaseoli also contain two forms of GS both of which have different molecular weights from the plant enzymes. Bacteroids contain solely the higher-molecular-weight form of rhizobial GS. There are only minor differences between the plant enzymes in Km or S0.5 values for the synthetase-reaction substrates and both forms have identical molecular weights of the holoenzyme (380,000 daltons) and its sub-units (41,000 daltons). They can be separated by ion-exchange chromatography on diethylaminoethyl-Sephacel and by native polyacrylamide-gel electrophoresis. The only other distinguishing feature observed is that the ratio of transferase: synthetase activity of the root form is threefold greater than that of the nodule-specific GS.

Key words

Glutamine synthetase Legume Rhizobium symbiosis Nitrogen assimilation Phaseolus (glutamine synthetase) Rhizobium Root nodule 

Abbreviations

DEAE-Sephacel

diethylaminoethyl-sephacel

GS

glutamine synthetase

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Awonaike, K.O., Lea, P.J., Miflin, B.J. (1981) The location of the enzymes of ammonia assimilation in root nodules of Phaseolus vulgaris L. Plant Sci. Lett. 23, 189–195Google Scholar
  2. Barratt, D.H.P. (1980) Method for the detection of glutamine synthetase activity on starch gels. Plant Sci. Lett. 18, 249–254Google Scholar
  3. Brown, C.M., Dilworth, M.J. (1975) Ammonia assimilation by Rhizobium cultures and bacteriods. J. Gen. Microbiol. 86, 39–48Google Scholar
  4. Cullimore, J.V., Sims, A.P. (1980) An association between photorespiration and protein catabolism: studies with Chlamydomonas. Planta 150, 392–396Google Scholar
  5. Cullimore, J.V., Sims, A.P. (1981) Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii. Phytochemistry 20, 933–940Google Scholar
  6. Cullimore, J.V., Lea, P.J., Miflin, B.J. (1982) Multiple forms of glutamine synthetase in the plant fraction of Phaseolus root nodules. Isr. J. Bot. 31, 155–162Google Scholar
  7. Darrow, R.A., Knotts, R.R. (1977) Two forms of glutamine synthetase in free-living root-nodule bacteria. Biochem. Biophys. Res. Commun. 78, 554–559Google Scholar
  8. Davis, B.J. (1964) Dise electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404–436Google Scholar
  9. Dunn, S.D., Klucas, R.V. (1973) Studies on possible routes of ammonia assimilation in soybean root nodule bacteroids. Can. J. Microbiol. 19, 1493–1499Google Scholar
  10. Dye, M. (1979) Functions and maintenance of a Rhizobium collection. In: Recent advances in biological nitrogen fixation, pp. 435–471, Subba Rao, N.S., ed. Oxford & IBH Publ. Co., New DelhiGoogle Scholar
  11. Eaglesham, A.R.J., Minchin, F.R., Summerfield, R.J., Dart, P.J., Huxley, P.A., Day, J.M. (1977) Nitrogen nutrition of cowpea (Vigna unguiculata). III. Distribution of nitrogen within effectively nodulated plants. Exp. Agric. 13, 369–380Google Scholar
  12. Forde, B.G., Kreis, M., Bahramian, M.B., Matthews, J.A., Miflin, B.J. (1981) Molecular cloning and analysis of cDNA sequences derived from poly A+ RNA from barley endosperm: identification of B hordein related clones. Nucleic Acids Res. 9, 6689–6707Google Scholar
  13. Fuchs, R.L., Keister, D.L. (1980a) Identification of two glutamine synthetases in Agrobacterium. J. Bacteriol. 141, 996–998Google Scholar
  14. Fuchs, R.L., Keister, D.L. (1980b) Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp. J. Bacteriol. 144, 641–648Google Scholar
  15. Guiz, C., Hirel, B., Shedlofsky, G., Gadal, P. (1979) Occurrence and influence of light on the relative proportions of two glutamine synthetases in rice leaves. Plant Sci. Lett. 15, 271–277Google Scholar
  16. Hirel, B., Gadal, P. (1980) Glutamine synthetase in rice. A comparative study of the enzymes from roots and leaves. Plant Physiol 66, 619–623Google Scholar
  17. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685Google Scholar
  18. Lara, M., Cullimore, J.V., Lea, P.J., Miflin, B.J., Johnston, A.W.B., Lamb, J.W. (1983) Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L. Planta 157, 254–258Google Scholar
  19. Legocki, R.P., Verma, D.P.S. (1980) Identification of “nodule specific” host proteins (nodulins) involved in the development of Rhizobium-legume symbiosis. Cell 20, 153–164Google Scholar
  20. Lowry, H.O., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275Google Scholar
  21. Mann, A.F., Fentem, P.A., Stewart, G.R. (1979) Identification of two forms of glutamine synthetase in barley (Hordeum vulgare). Biochem. Biophys. Res. Commun. 88, 515–521Google Scholar
  22. McParland, R.H., Guevara, J.G., Becker, R.R., Evans, H.J. (1976) The purification and properties of the glutamine synthetase from the cytosol of soya-bean root nodules. Biochem. J. 153, 597–606Google Scholar
  23. Miflin, B.J., Lea, P.J. (1977) Amino acid metabolism. Annu. Rev. Plant Physiol. 28, 299–329Google Scholar
  24. O'Neal, D., Joy, K.W. (1973) Glutamine synthetase of pea leaves. I. Purification, stabilization and pH optima. Arch. Biochem. Biophys. 159, 113–122Google Scholar
  25. Planqué, K., Kennedy, I.R., de Vries, G.E., Quispel, A., van Brussel, A.A.N. (1977) Location of nitrogenase and ammonia-assimilatory enzymes in bacteroids of Rhizobium leguminosarum and Rhizobium lupini. J. Gen. Microbiol. 102, 94–104Google Scholar
  26. Rao, R.V., Darrow, R.A., Keister, D.L. (1978) Effect of oxygen tension on nitrogenase and on glutamine synthetase I and II in Rhizobium japonicum 61A76. Biochem. Biophys. Res. Commun. 81, 224–231Google Scholar
  27. Robetson, J.G., Farnden, K.J.F., Warburton, M.P., Banks, J.M. (1975) Induction of glutamine synthetase during nodule development in lupin. Aust. J. Plant. Physiol. 2, 265–272Google Scholar
  28. Robertson, J.G., Farnden, K.J.F. (1980) Ultrastructure and metabolism of the developing legume root nodule. In: The biochemistry of plants, vol. 5, pp. 65–113. Miflin, B.J., ed. Academic Press, New York LondonGoogle Scholar
  29. Shapiro, B.M., Stadtman, E.R. (1970) The regulation of glutamine synthesis in microorganisms. Annu. Rev. Microbiol. 24, 501–524Google Scholar
  30. Siegel, L.M., Monty, K.J. (1966) Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductase. Biochim. Biophys Acta. 112, 346–362Google Scholar
  31. Sims, A.P., Toone, J., Box, V. (1974) The regulation of glutamine synthesis in the food yeast Candida utilis: the purification and subunit structure of glutamine synthetase and aspects of enzyme deactivation. J. Gen. Microbiol. 80, 485–499Google Scholar
  32. Stasiewicz, S., Dunham, V.L. (1979) Isolation and characterization of two forms of glutamine synthetase from soybean hypocotyl. Biochem. Biophys. Res. Commun. 87, 627–634Google Scholar
  33. Stewart, G.R., Mann, A.F., Fentem, D.A. (1980) Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase and glutamate synthase. In: The biochemistry of plants, vol. 5, pp. 271–327, Miflin, B.J., ed. Academic Press, New York LondonGoogle Scholar
  34. Streeter, J.G. (1979) Asparaginase and asparagine transaminase in soybean leaves and root nodules. Plant Physiol. 60, 235–239Google Scholar
  35. Verma, D.P.S. (1980) Expression of host genes during symbiotic nitrogen fixation. In: Genome organisation and expression in plants, pp. 439–452, Leaver, C., ed. Plenum Press, New York LondonGoogle Scholar
  36. Wallsgrove, R.M., Keys, A.J., Bird, I.F., Cornelius, M.J., Lea, P.J., Miflin, B.J. (1980) The location of glutamine synthetase in leaf cells and its role in the reassimilation of ammonia released via photorespiration. J. Exp. Bot. 31, 1005–1017Google Scholar
  37. Wong, D.P., Evans, H.J. (1971) Poly-β-hydroxybutyrate utilisation by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity. Plant Physiol. 47, 750–755Google Scholar
  38. Woolfolk, C.A., Shapiro, B., Stadtman, E.R. (1966) Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch. Biochem. Biophys. 116, 177–192Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • Julie V. Cullimore
    • 1
  • M. Lara
    • 1
  • P. J. Lea
    • 1
  • B. J. Miflin
    • 1
  1. 1.Biochemistry DepartmentRothamsted Experimental StationHarpendenUK

Personalised recommendations