Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Apr;170(4):1940–1944. doi: 10.1128/jb.170.4.1940-1944.1988

Purification and properties of glutamate synthase from Nocardia mediterranei.

B G Mei 1, R S Jiao 1
PMCID: PMC211054  PMID: 3350793

Abstract

Glutamate synthase was purified about 250-fold from Nocardia mediterranei U32 and characterized. The native enzyme has a molecular weight of 195,000 +/- 5,000 and is composed of two nonidentical subunits with molecular weights of 145,000 +/- 5,000 and 55,000 +/- 3,000. This enzyme is a complex of iron-sulfur flavoproteins with absorption maxima at 278, 375, 410, and 440 nm. It contains 1.1 mol of flavin adenine dinucleotide, 1.0 mol of flavin mononucleotide, 7.5 mol of nonheme iron, and 7.2 mol of acid-labile sulfur per 200,000 g of protein. Km values for L-glutamine, alpha-ketoglutarate, and NADPH were 77, 53, and 110 microM, respectively. The activity of this glutamate synthase is inhibited by its products (i.e., glutamate and NADP), several amino acids, and tricarboxylic acid cycle intermediates.

Full text

PDF
1940

Images in this article

1942Image
on p.1942
1942Image
on p.1942

Selected References

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

  1. Adachi K., Suzuki I. Purification and properties of glutamate synthase from Thiobacillus thioparus. J Bacteriol. 1977 Mar;129(3):1173–1182. doi: 10.1128/jb.129.3.1173-1182.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beinert H. Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal Biochem. 1983 Jun;131(2):373–378. doi: 10.1016/0003-2697(83)90186-0. [DOI] [PubMed] [Google Scholar]
  3. Boland M. J., Benny A. G. Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupin nodules. Eur J Biochem. 1977 Oct 3;79(2):355–362. doi: 10.1111/j.1432-1033.1977.tb11816.x. [DOI] [PubMed] [Google Scholar]
  4. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  5. DOEG K. A., ZIEGLER D. M. Simplified methods for the estimation of iron in mitochondria and submitochondrial fractions. Arch Biochem Biophys. 1962 Apr;97:37–40. doi: 10.1016/0003-9861(62)90041-3. [DOI] [PubMed] [Google Scholar]
  6. Hemmilä I. A., Mäntsälä P. I. Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium. Biochem J. 1978 Jul 1;173(1):45–52. doi: 10.1042/bj1730045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jiao R. S., Liu C. J., Jin Z. K., Zhang X. C., Ni L. Y., Lu Z. M. The route of incorporation of nitrogen atom into rifamycin during its biosynthesis. Sci Sin B. 1984 Apr;27(4):380–390. [PubMed] [Google Scholar]
  8. 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]
  9. Light D. R., Walsh C., Marletta M. A. Analytical and preparative high-performance liquid chromatography separation of flavin and flavin analog coenzymes. Anal Biochem. 1980 Nov 15;109(1):87–93. doi: 10.1016/0003-2697(80)90014-7. [DOI] [PubMed] [Google Scholar]
  10. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  11. Masters D. S., Jr, Meister A. Inhibition of homocysteine sulfonamide of glutamate synthase purified from Saccharomyces cerevisiae. J Biol Chem. 1982 Aug 10;257(15):8711–8715. [PubMed] [Google Scholar]
  12. Meers J. L., Tempest D. W., Brown C. M. 'Glutamine(amide):2-oxoglutarate amino transferase oxido-reductase (NADP); an enzyme involved in the synthesis of glutamate by some bacteria. J Gen Microbiol. 1970 Dec;64(2):187–194. doi: 10.1099/00221287-64-2-187. [DOI] [PubMed] [Google Scholar]
  13. Miller R. E., Stadtman E. R. Glutamate synthase from Escherichia coli. An iron-sulfide flavoprotein. J Biol Chem. 1972 Nov 25;247(22):7407–7419. [PubMed] [Google Scholar]
  14. Mäntsälä P., Zalkin H. Glutamate synthase. Properties of the glutamine-dependent activity. J Biol Chem. 1976 Jun 10;251(11):3294–3299. [PubMed] [Google Scholar]
  15. 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]
  16. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  17. Ratti S., Curti B., Zanetti G., Galli E. Purification and characterization of glutamate synthase from Azospirillum brasilense. J Bacteriol. 1985 Aug;163(2):724–729. doi: 10.1128/jb.163.2.724-729.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Robertson J. G., Warburton M. P., Farnden K. J. Induction of glutamate synthase during nodule development in lupin. FEBS Lett. 1975 Jul 15;55(1):33–37. doi: 10.1016/0014-5793(75)80950-1. [DOI] [PubMed] [Google Scholar]
  19. Schreier H. J., Bernlohr R. W. Purification and properties of glutamate synthase from Bacillus licheniformis. J Bacteriol. 1984 Nov;160(2):591–599. doi: 10.1128/jb.160.2.591-599.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Trotta P. P., Platzer K. E., Haschemeyer R. H., Meister A. Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4607–4611. doi: 10.1073/pnas.71.11.4607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tyler B. Regulation of the assimilation of nitrogen compounds. Annu Rev Biochem. 1978;47:1127–1162. doi: 10.1146/annurev.bi.47.070178.005403. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES