Skip to main content
SpringerLink
Log in

Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (NADP-GDH) of Agaricus bisporus, a key enzyme in ammonia assimilation, was purified to apparent electrophoretic homogeneity with 27% recovery of the initial activity. The molecular weight of the native enzyme was 330 kDa. The enzyme is probably a hexamer, composed of identical subunits of 48 kDa. The isoelectric point of the enzyme was found at pH 4.8. The N-terminus appeared to be blocked. The enzyme was specific for NADP(H). The Km-values were 2.1, 3.2, 0.074, 27.0, and 0.117mM for ammonia, 2-oxoglutarate, NADPH, L-glutamate, and NADP respectively. The pH optima for the amination and deamination reactions were found to be 7.6 and 9.0, respectively. The temperature optimum was 33°C. The effect of several metabolites on the enzyme's activity was tested. Pyruvate, oxaloacetate, ADP, and ATP showed some inhibitory effect. Divalent cations slightly stimulated the aminating reaction. Antibodies raised against the purified enzyme were able to precipitate NADP-GDH activity from a cell-free extract in an anticatalytic immunoprecipitation test. Analysis of a Western blot showed the antibodies to be specific for NADP-GDH.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Literature Cited

  1. Ahmad I, Hellebust JA (1991) Enzymology of nitrogen assimilation in mycorrhiza. Methods Microbiol 23:181–202

    Google Scholar 

  2. Al-Gharawi A, Moore D (1977) Factors affecting the amount and the activity of the glutamate dehydrogenases of Coprinus cinereus. Biochim Biophys Acta 496:95–102

    Google Scholar 

  3. Arst HM, Parbtani AAM, Cove DJ (1975) A mutant of Aspergillus nidulans defective in NAD-linked glutamate dehydrogenase. Mol Gen Genet 138:165–171

    Google Scholar 

  4. Baars JJP, Op den Camp HJM, Hermans JMH, Van der Drift C, Van Griensven LJLD, Vogels GD (1994) Nitrogen assimilating enzymes in the white button mushroom Agaricus bisporus. Microbiology 140:1161–1168

    Google Scholar 

  5. Benachenhou-Lahfa N, Forterre P, Labedan B (1993) Evolution of glutamate dehydrogenase genes: evidence for two paralogous protein families and unusual branching patterns of the archaebacteria in the universal tree of life. J Mol Evol 36:335–346

    Google Scholar 

  6. Blumenthal KM, Smith EL (1973) Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. J Biol Chem 248:6002–6008

    Google Scholar 

  7. Botton B, Msatef Y (1983) Purification and properties of NADP-dependent glutamate dehydrogenase from Sphaerostilbe repens. Physiol Plant 59:438–444

    Google Scholar 

  8. Brun A, Chalot M, Botton B, Martin F (1992) Purification and characterization of glutamine synthetase and NADP-glutamate dehydrogenase from the ectomycorrhizal fungus Laccaria laccata. Plant Physiol 99:938–944

    Google Scholar 

  9. Dunbar BS, Schwoebel ED (1990) Preparation of polyclonal antibodies. Methods Enzymol 182:663–679

    Google Scholar 

  10. Fincham JRS (1962) Genetically determined multiple forms of glutamic dehydrogenase in Neurospora crassa. J Mol Biol 4:257–274

    Google Scholar 

  11. Goldin BR, Frieden C (1971) L-glutamate dehydrogenases. Curr Top Cell Regul 4:77–117

    Google Scholar 

  12. Hammond JBW, Nichols R (1975) Changes in respiration and soluble carbohydrates during the post-harvest storage of mushrooms (Agaricus bisporus). J Sci Food Agric 26:835–842

    Google Scholar 

  13. Hondmann DHA, Visser J (1990) Screening method for large numbers of dye-adsorbents for enzyme purification. J Chromatogr 510:155–164

    Google Scholar 

  14. Hudson RC, Daniel RM (1993) L-glutamate dehydrogenases: distribution, properties and mechanism. Comp Biochem Physiol 106B:767–792

    Google Scholar 

  15. Kessler RE, Thivierge BH (1983) Effects of substitution on polyglycerol phosphate-specific antibody binding to lopoteichoic acids. Infect Immun 41:549–555

    Google Scholar 

  16. Kinghorn JR, Pateman JA (1973) NAD and NADP L-glutamate dehydrogenase activity and ammonium regulation in Aspergillus nidulans. J Gen Microbiol 78:39–46

    Google Scholar 

  17. Kusnan MB, Klug K, Fock HP (1989) Ammonia assimilation by Aspergillus nidulans: [15N] ammonia study. J Gen Microbiol 135:729–738

    Google Scholar 

  18. LeJohn HB (1971) Enzyme regulation, lysine pathways and cell wall structures as indicators of major lines of evolution in fungi. Nature 231:164–168

    Google Scholar 

  19. Lilley KS, Engel PC (1992) The essential active-site lysines of clostridial glutamate dehydrogenase. Eur J Biochem 207:533–540

    Google Scholar 

  20. Martin F, Msatef Y, Botton B (1983) Nitrogen assimilation in mycorrhizas. I. Purification and properties of the nicotinamide dinucleotide phosphate-specific glutamate dehydrogenase of the ectomycorrhizal fungus Cenococcum graniforme. New Phytol 93:415–422

    Google Scholar 

  21. Martin F, Stewart GR, Genetet I, Mourot B (1988) The involvement of glutamate dehydrogenase and glutamine synthetase in ammonia assimilation by the rapidly growing ectomycorrhizal ascomycete, Cenococcum geophilum Fr. New Phytol 110:541–550

    Google Scholar 

  22. Miller SM, Magasanik B (1990) Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae. J Bacteriol 172:4927–4935

    Google Scholar 

  23. Ploug M, Jensen AL, Barkholt V (1989) Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from Tricinesodiumdodecylsulfate-polyacrylamide gel electrophoresis. Anal Biochem 811:33–39

    Google Scholar 

  24. Schwartz T, Kusnan MB, Fock HP (1991) The involvement of glutamate dehydrogenase and glutamine synthetase/glutamate synthase in ammonia assimilation by the basidiomycete fungus Stropharia semiglobata. J Gen Microbiol 137:2253–2258

    Google Scholar 

  25. Smith EL, Austen BM, Blumenthal KM, Nyc JF (1975) Glutamate dehydrogenases. In: Boyer PD (ed) The enzymes, vol 11. New York: Academic Press, pp 293–367

    Google Scholar 

  26. Son D, Jo J, Sugiyama T (1991) Purification and characterization of alanine aminotransferase from Panicum miliaceum leaves. Arch Biochem Biophys 289:262–266

    Google Scholar 

  27. Stewart GR, Mann AF, Fentem PA (1980) Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase and glutamate synthase. In: Miflin BJ (ed) The biochemistry of plants, a comprehensive treatise, vol 5. New York: Academic Press, pp 271–327

    Google Scholar 

  28. Tijssen P (1985) Practice and theory of enzyme immunoassays. In: Burdon RH, van Knippenberg PH (eds) Laboratory techniques in biochemistry and molecular biology, vol 15. Amsterdam: Elsevier

    Google Scholar 

  29. Valinger Z, Engel PP, Metzler DE (1993) Is pyridoxal 5′-phosphate an affinity label for phosphate-binding sites in proteins?: the case of bovine glutamate dehydrogenase. Biochem J 294:835–839

    Google Scholar 

  30. Vallejos CE (1983) Enzyme activity staining. In: Tanksley SD, Orton TJ (eds) Isozymes in plant genetics and breeding, part A. Amsterdam: Elsevier, pp 469–516

    Google Scholar 

  31. Watson DH, Harvey MJ, Dean PDG (1978) The selective retardation of NADP+-dependent dehydrogenases by immobilized Procion Red HE-3B. Biochem J 173:591–596

    Google Scholar 

  32. Wootton JC (1983) Re-assessment of ammonium-ion affinities of NADP-specific glutamate dehydrogenases. Biochem J 209: 527–531

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED, Nijmegen, The Netherlands

    J. J. P. Baars, H. J. M. Op den Camp, A. H. A. M. van Hoek, C. van der Drift & G. D. Vogels

  2. Mushroom Experimental Station, Postbus 6042, NL-5960 AA, Horst, The Netherlands

    L. J. L. D. Van Griensven

  3. Section of Molecular Genetics of Industrial Microorganisms, Agricultural University of Wageningen, Dreyenlaan 2, NL-6703 HA, Wageningen, The Netherlands

    J. Visser

Authors
  1. J. J. P. Baars
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. J. M. Op den Camp
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. H. A. M. van Hoek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. van der Drift
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. L. J. L. D. Van Griensven
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J. Visser
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G. D. Vogels
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baars, J.J.P., Op den Camp, H.J.M., van Hoek, A.H.A.M. et al. Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus . Current Microbiology 30, 211–217 (1995). https://doi.org/10.1007/BF00293635

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00293635

Keywords

Search

Navigation