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. 1988 Oct;170(10):4897–4902. doi: 10.1128/jb.170.10.4897-4902.1988

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum.

M Martinez-Bilbao 1, A Martinez 1, I Urkijo 1, M J Llama 1, J L Serra 1
PMCID: PMC211535  PMID: 3139639

Abstract

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-fixing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30 degrees C it showed a specific activity of 32.9 mumol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (Km = 1.25 mM) and NADPH (Km = 64 microM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [S]0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60 degrees C (Ea = 5.1 kcal/mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is 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. Blumenthal K. M., Smith E. L. Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. I. Isolation, subunits, amino acid composition, sulfhydryl groups, and identification of a lysine residue reactive with pyridoxal phosphate and N-ethylmaleimide. J Biol Chem. 1973 Sep 10;248(17):6002–6008. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Camardella L., Di Prisco G., Garofano F., Guerrini A. M. Purification and properties of NADP-dependent glutamate dehydrogenase from yeast nuclear fractions. Biochim Biophys Acta. 1976 Apr 8;429(2):324–330. doi: 10.1016/0005-2744(76)90280-1. [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. 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]
  6. Gayler K. R., Morgan W. R. An NADP-dependent Glutamate Dehydrogenase in Chloroplasts from the Marine Green Alga Caulerpa simpliciuscula. Plant Physiol. 1976 Sep;58(3):283–287. doi: 10.1104/pp.58.3.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hemmings B. A., Sims A. P. The regulation of glutamate metabolism in Candida utilis. Evidence for two interconvertible forms of NAD-dependent glutamate dehydrogenase. Eur J Biochem. 1977 Oct 17;80(1):143–151. doi: 10.1111/j.1432-1033.1977.tb11866.x. [DOI] [PubMed] [Google Scholar]
  8. Hernández G., Sánchez-Pescador R., Palacios R., Mora J. Nitrogen source regulates glutamate dehydrogenase NADP synthesis in Neurospora crassa. J Bacteriol. 1983 Apr;154(1):524–528. doi: 10.1128/jb.154.1.524-528.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hooper A. B., Hansen J., Bell R. Characterization of glutamate dehydrogenase from the ammonia-oxidizing chemoautotroph Nitrosomonas europaea. J Biol Chem. 1967 Jan 25;242(2):288–296. [PubMed] [Google Scholar]
  10. Hornby D. P., Engel P. C. Characterization of Peptostreptococcus asaccharolyticus glutamate dehydrogenase purified by dye-ligand chromatography. J Gen Microbiol. 1984 Sep;130(9):2385–2394. doi: 10.1099/00221287-130-9-2385. [DOI] [PubMed] [Google Scholar]
  11. Israel D. W., Gronostajski R. M., Yeung A. T., Schmidt R. R. Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella. J Bacteriol. 1977 May;130(2):793–804. doi: 10.1128/jb.130.2.793-804.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kane J. F., Wakim J., Fischer R. S. Regulation of glutamate dehydrogenase in Bacillus subtilis. J Bacteriol. 1981 Dec;148(3):1002–1005. doi: 10.1128/jb.148.3.1002-1005.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kindt R., Pahlich E., Rasched I. Glutamate dehydrogenase from peas: isolation, quaternary structure, and influence of cations on activity. Eur J Biochem. 1980 Dec;112(3):533–540. doi: 10.1111/j.1432-1033.1980.tb06116.x. [DOI] [PubMed] [Google Scholar]
  14. Loyola-Vargas V. M., de Jimenez E. S. Differential role of glutamate dehydrogenase in nitrogen metabolism of maize tissues. Plant Physiol. 1984 Oct;76(2):536–540. doi: 10.1104/pp.76.2.536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maulik P., Ghosh S. NADPH/NADH-dependent cold-labile glutamate dehydrogenase in Azospirillum brasilense. Purification and properties. Eur J Biochem. 1986 Mar 17;155(3):595–602. doi: 10.1111/j.1432-1033.1986.tb09530.x. [DOI] [PubMed] [Google Scholar]
  16. Meeks J. C., Wolk C. P., Lockau W., Schilling N., Shaffer P. W., Chien W. S. Pathways of assimilation of [13N]N2 and 13NH4+ by cyanobacteria with and without heterocysts. J Bacteriol. 1978 Apr;134(1):125–130. doi: 10.1128/jb.134.1.125-130.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Meeks J. C., Wolk C. P., Thomas J., Lockau W., Shaffer P. W., Austin S. M., Chien W. S., Galonsky A. The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica. J Biol Chem. 1977 Nov 10;252(21):7894–7900. [PubMed] [Google Scholar]
  18. Molin W. T., Cunningham T. P., Bascomb N. F., White L. H., Schmidt R. R. Light requirement for induction and continuous accumulation of an ammonium-inducible NADP-specific glutamate dehydrogenase in chlorella. Plant Physiol. 1981 Jun;67(6):1250–1254. doi: 10.1104/pp.67.6.1250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Neet K. E., Ainslie G. R., Jr Hysteretic enzymes. Methods Enzymol. 1980;64:192–226. doi: 10.1016/s0076-6879(80)64010-5. [DOI] [PubMed] [Google Scholar]
  20. Neumann P., Markau K., Sund H. Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition. Eur J Biochem. 1976 Jun 1;65(2):465–472. doi: 10.1111/j.1432-1033.1976.tb10362.x. [DOI] [PubMed] [Google Scholar]
  21. Prunkard D. E., Bascomb N. F., Robinson R. W., Schmidt R. R. Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana. Plant Physiol. 1986 Jun;81(2):349–355. doi: 10.1104/pp.81.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sarada K. V., Rao N. A., Venkitasubramanian T. A. Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46. Biochim Biophys Acta. 1980 Oct;615(2):299–308. doi: 10.1016/0005-2744(80)90498-2. [DOI] [PubMed] [Google Scholar]
  23. Smits R. A., Pieper F. R., Van der Drift C. Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation. Biochim Biophys Acta. 1984 Sep 7;801(1):32–39. doi: 10.1016/0304-4165(84)90209-5. [DOI] [PubMed] [Google Scholar]
  24. Stewart W. D. Some aspects of structure and function in N2-fixing cyanobacteria. Annu Rev Microbiol. 1980;34:497–536. doi: 10.1146/annurev.mi.34.100180.002433. [DOI] [PubMed] [Google Scholar]
  25. Talley D. J., White L. H., Schmidt R. R. Evidence for NADH- and NADPH-specific isozymes of glutamate dehydrogenase and the continuous inducibility of the NADPH-specific isozyme throughout the cell cycle of the eucaryote Chlorella. J Biol Chem. 1972 Dec 25;247(24):7927–7935. [PubMed] [Google Scholar]
  26. Vierula P. J., Kapoor M. A study of derepression of NAD-specific glutamate dehydrogenase of Neurospora crassa. J Gen Microbiol. 1986 Apr;132(4):907–915. doi: 10.1099/00221287-132-4-907. [DOI] [PubMed] [Google Scholar]
  27. Yamaya T., Oaks A., Matsumoto H. Characteristics of glutamate dehydrogenase in mitochondria prepared from corn shoots. Plant Physiol. 1984 Dec;76(4):1009–1013. doi: 10.1104/pp.76.4.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yeung A. T., Turner K. J., Bascomb N. F., Schmidt R. R. Purification of an ammonium-inducible glutamate dehydrogenase and the use of its antigen affinity column-purified antibody in specific immunoprecipitation and immunoadsorption procedures. Anal Biochem. 1981 Jan 1;110(1):216–228. doi: 10.1016/0003-2697(81)90138-x. [DOI] [PubMed] [Google Scholar]

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