Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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Plant Physiology 99:938-944 (1992)
© 1992 American Society of Plant Biologists

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Metabolism and Enzymology

Purification and Characterization of Glutamine Synthetase and NADP-Glutamate Dehydrogenase from the Ectomycorrhizal Fungus Laccaria laccata 1

Annick Brun, Michel Chalot, Bernard Botton and Francis Martin

Laboratoire de Physiologie Végétale et Forestière, Université de Nancy I, B.P. 239, 54506 Vandoeuvre-les-Nancy Cedex, France, Laboratoire de Microbiologie Forestière, Centre de Recherches Forestières de Nancy, Institut National de la Recherche Agronomique, Champenoux, 54280 Seichamps, France

Glutamine synthetase (GS) and NADP-dependent glutamate dehydrogenase (NADP-GDH) play a key role in nitrogen assimilation in the ectomycorrhizal fungus Laccaria laccata (Scop. ex Fr. Cke) strain S 238. The two enzymes were purified to apparent electrophoretic homogeneity by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl and affinity chromatography, and DEAE-5PW fast protein liquid chromatography. This purification scheme resulted in a 23 and 62% recovery of the initial activity for GS and NADP-GDH, respectively. Purified GS had a specific activity of 713 nanomoles per second per milligram protein and a pH optimum of 7.2. Michaelis constants (millimolar) for the substrates were NH4+ (0.024), glutamate (3.2), glutamine (30), ATP (0.18), and ADP (0.002). The molecular weight (Mr) of native GS was approximately 380,000; it was composed of eight identical subunits of Mr 42,000. Purified NADP-GDH had a specific activity of 4130 nanomoles per second per milligram protein and a pH optimum of 7.2 (amination reaction). Michaelis constants (millimolar) for the substrates were NH4+ (5), 2-oxoglutarate (1), glutamate (26), NADPH (0.01), and NADP (0.03). Native NADP-GDH was a hexamer with a Mr of about 298,000 composed of identical subunits with Mr 47,000. Polyclonal antibodies were produced against purified GS and NADP-GDH. Immunoprecipitation tests and immunoblot analysis showed the high reactivity and specificity of the immune sera against the purified enzymes.

1 This work was financially supported by a research grant from the Institut National de la Recherche Agronomique (INRA, Action incitative programmée Recherches des mécanismes de régulation du métabolisme des associations mycorhiziennes) to B.B., a scholarship from the INRA and the Centre National Interprofessionnel de l'Horticulture to M.C., and a scholarship from the Ministère de la Recherche et de la Technologie to A.B.






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