1887

Abstract

Glutamate synthase (GOGAT), glutamine synthetase (GS), NAD-dependent glutamate dehydrogenase (GDH) and NAD-dependent alanine dehydrogenase (AlaDH) activities were detected in cell extracts of PH24, a strain deficient in NADP-dependent GDH. GDH and GOGAT activities were low under most growth conditions and GOGAT was not detectable in extracts of cells grown with amino acids as carbon and nitrogen source. AlaDH and GS activities were more variable, the former being high in cells grown on -alanine as carbon and nitrogen source. GS was repressed during growth with high concentrations of NHC1 as nitrogen source but a corresponding increase in AlaDH activities suggests that this enzyme may replace NADP-dependent GDH as the main enzyme for ammonia assimilation under these conditions.

GOGAT was purified 40-fold using affinity chromatography on NADPH-Sepharose. The molecular weight of the partially purified enzyme was estimated to be 160000 and values for NADPH, 2-oxoglutarate and -glutamine were 22, 15 and 29 μ, respectively. Glutamine could be replaced by NHC1 as nitrogen donor ( 44 m) but the rate was only 10% to 15% that of the -glutamine-dependent reaction. The pH optimum for glutamine-dependent activity was 8·0 and the temperature optimum 75 C: the enzyme displayed a discontinuous Arrhenius plot over the range 30 °C to 75 °C. Azaserine, -methionine sulphone and Cibacron Blue 3GA were all inhibitors and the enzyme was rapidly inactivated in the presence of NADPH when -glutamine and 2-oxoglutarate were absent.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-128-8-1713
1982-08-01
2021-05-10
Loading full text...

Full text loading...

/deliver/fulltext/micro/128/8/mic-128-8-1713.html?itemId=/content/journal/micro/10.1099/00221287-128-8-1713&mimeType=html&fmt=ahah

References

  1. Amelunxen R. E., Murdock A. L. 1978 In Microbial Life in Extreme Environments pp. 217–278 Kushner D. J. Edited by London: Academic Press.;
    [Google Scholar]
  2. Andrews P. 1965; The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochemical Journal 96:595–606
    [Google Scholar]
  3. Barnett J. A., Ingram M. 1955; Techniques in the study of yeast assimilation reactions. Journal of Applied Bacteriology 18:131–143
    [Google Scholar]
  4. Buswell J. A., Twomey D. G. 1975; Utilization of phenol and cresols by Bacillus stearothermophilus strain PH24. Journal of General Microbiology 87:377–379
    [Google Scholar]
  5. Deshpande K. L., Kane J. F. 1980; Glutamate synthase from Bacillus subtilis: in vitro reconstitution of an active amido-transferase. Biochemical and Biophysical Research Communications 93:308–314
    [Google Scholar]
  6. Eisenthal R., Cornish-Bowden A. 1974; The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochemical Journal 139:715–720
    [Google Scholar]
  7. Elmerich C., Aubert J.-P. 1971; Synthesis of glutamate by a glutamine:2 oxogluterate amido-transferase (NADP-oxidoreductase) in Bacillus megaterium . Biochemical and Biophysical Research Communications 42:371–376
    [Google Scholar]
  8. Epstein I., Grossowicz N. 1975; Purification and properties of glutamate dehydrogenase from a thermophilic bacillus. Journal of Bacteriology 122:1257–1264
    [Google Scholar]
  9. Epstein I., Grossowicz N. 1976; Regulatory control and function of alanine dehydrogenase from a thermophilic bacillus. Biochimica et biophysica acta 445:549–557
    [Google Scholar]
  10. Hayes M. B., Wellner D. 1969; Microheterogeneity of l-amino acid oxidase. Journal of Biological Chemistry 244:6636–6644
    [Google Scholar]
  11. Hemmilä I. A., Mäntsälä P. I. 1978; Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium . Biochemical Journal 173:45–52
    [Google Scholar]
  12. Hong M. M., Shen S. C., Braunstein A. G. 1959; Distribution of l-alanine dehydrogenase and l-glutamate dehydrogenase in bacilli. Biochimica et biophysica acta 36:288–289
    [Google Scholar]
  13. Inman J. K., Dintzis H. M. 1969; The derivatization of cross-linked polyacrylamide beads. Controlled introduction of functional groups for the preparation of special purpose, biochemical adsorbents. Biochemistry 8:4074–4082
    [Google Scholar]
  14. Lumsden J., Coggins J. R. 1977; The subunit structure of the arom multienzyme complex of Neurospora crassa . Biochemical Journal 161:599–607
    [Google Scholar]
  15. Mäntsälä P., Zalkin H. 1976; Glutamate synthase: properties of the glutamine-dependent activity. Journal of Biological Chemistry 251:3294–3299
    [Google Scholar]
  16. Miflin B. J., Lea P. J. 1976; The pathway of nitrogen assimilation in plants. Phytochemistry 15:873–885
    [Google Scholar]
  17. Miller R. E., Stadtman E. R. 1972; Glutamate synthase from Escherichia coli . Journal of Biological Chemistry 247:7407–7419
    [Google Scholar]
  18. Nagatani H., Shimizu M., Valentine R. C. 1971; The mechanism of ammonia assimilation in nitrogen fixing bacteria. Archiv für Mikrobiologie 79:164–175
    [Google Scholar]
  19. Nishikawa A. H., Bailon P. 1975; Affinity purification methods. Improved procedure for cyanogen bromide reaction on agarose. Analytical Biochemistry 64:268–275
    [Google Scholar]
  20. Schmidt C. N. G. 1980; Affinity purification of glutamate synthase from Escherichia coli . Analytical Biochemistry 104:127–129
    [Google Scholar]
  21. Tempest D. W., Meers J. L., Brown C. M. 1970; Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route. Biochemical Journal 117:405–407
    [Google Scholar]
  22. Tempest D. W., Meers J. L., Brown C. M. 1973; Glutamate synthase (GOGAT); a key enzyme in the assimilation of ammonia by prokaryotic organisms. In The Enzymes of Glutamine Metabolism pp. 167–182 Prusiner S., Stadtman E. R. Edited by New York: Academic Press.;
    [Google Scholar]
  23. Wedler F. C., Hoffman F. M. 1974; Glutamine synthetase of Bacillus stearothermophilus. I. Purification and basic properties. Biochemistry 13:3207–3214
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-128-8-1713
Loading
/content/journal/micro/10.1099/00221287-128-8-1713
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