Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Chalot, Michel; Brun, Annick; Debaud, Jean Claude; Botton, Bernard

doi:10.1034/j.1399-3054.1991.830118.x

Cited by 8 publications

(12 citation statements)

References 16 publications

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“…However, its GS activities under these conditions are ver\' low. Moreover, with each nitrogen source, H. crustuliniforme exhibits highest activities of both GS and NADPH-GDH in the post-exponential period, whereas the reverse is true for amtnoniumand glutamate-grown H. cylindrosporum {Chalot et al, 1991). These results indicate a divergence in both the expression and regulation of GS and NADPH-GDH in these two closely related species of ectomycorrhiza!…”

Section: Discussionmentioning

confidence: 64%

“…Laccaria bicolor shows high levels of both GS and NADPH-GDH when ammonium is replaced by nitrate as the nitrogen source, and when both nitrate and ammonium are present the activities of GS and NADPH-GDH are greater in rapidly growing hyphae than in stationary cultures (Ahmad et al, 1990), A somewhat similar induction of GS and NADPH-GDH activities has been show^n to occur in the basidiomycete Hebeloma cylindrosporum when ammonium is replaced by glutamate (Chalot et al, 1991). It appears, therefore, that both the external nitrogen source and the fungal growth status have a major influence on the participation of GS and NADPH-GDH pathways in ectomycorrhizal fungi.…”

mentioning

confidence: 69%

“…According to Chalot et al (1991), high activities of both GS and NADPH-GDH are present in rapidly growing mycelia of H. cylindrosporum. The present study shows that a highly active NADPH-GDH is also present in the mycelia of H. crustuliniforme growing exponentially with different nitrogen sources in the presence of glucose.…”

Section: Discussionmentioning

confidence: 99%

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Nitrogen metabolism in the ectomycorrhizal fungus Hebeloma crustuliniforme

et al. 1995

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SUMMARYWith glucose as the carbon source, the ectomycorrhizal basidiomycete Hebeloma crustuliniforme (Bull. ex. Amans) Quel. utilizes nitrate, ammonium and amino acids as sources of nitrogen. Inoculated as a suspension of fine hyphae in such liquid media, the fungus expands rapidly, exhibiting a distinct exponential phase with a doubling time of 1-2 d and a maximum content of mycelial protein during the first 7 d of growth. Its growth when amino acids serve as sole carbon and nitrogen source is, however, less rapid, and requires several days of induction under these conditions. In H. crustuliniforme, assimilation of ammonium appears to be carried out primarily via the NADPHglutamate dehydrogenase (GDH) pathway. Its NADPH-GDH levels in exponentially growing mycelia are several times greater than the apparent nitrogen assimilation rates of the mycelia. Its levels of glutamine synthetase activity in comparison are very small during the exponential period of growth. The greatest levels of NADH-GDH are found in glucose-grown cultures during the post-exponential period, and in glucose-free amino acidgrown cultures during the active period of growth. This suggests a catabolic role for the NADH-GDH. Aspartate aminotransferase and alanine aminotransferase in H. crustuliniforme are highly active during the exponential period of growth, and their concentrations are increased further in the presence of amino acids as sole nitrogen sources, suggesting a key role for these enzymes in interactions between pathways of amino acid metabolism and carbon metabolism in tbis fungus.

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Section: Discussionmentioning

confidence: 64%

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confidence: 69%

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Nitrogen metabolism in the ectomycorrhizal fungus Hebeloma crustuliniforme

et al. 1995

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“…In addition, glutamate and glutamine can support biomass production comparable to that on ammonium in different ectomycorrhizal fungi (Abuzinadah & Read, 1988 ;Chalot et al, 1991a;Finlay e t al., 1992). As pointed out by Abuzinadah & Read (1989), assimilation of amino acids derived from proteolytic activity can supply up to 10 YO of the total C gained by the host over a period of 50 d, highlighting the importance of amino acids as a potential C source.…”

Section: Sequential G D H / G S Activity In Spruce Ectomycorrhizasmentioning

confidence: 99%

Metabolism of [14C]glutamate and [14C]glutamine by the ectomycorrhizal fungus Paxillus involutus

et al. 1994

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To examine pathways of glutamate and glutamine metabolism in the ectomycorrhizal fungus Paxillus involutus, tracer kinetic experiments were performed using ~-[U-l~C]glutamate and ~-[U-~~C]glutamine and the enzyme inhibitors methionine sulfoximine (MSX), azaserine (AZA) and aminooxyacetate (AOA). When [14C]glutamate was supplied to fungal cultures, 25% of the radioactivity of the amino acid fraction was incorporated into glutamine after 5 min feeding, but MSX inhibited incorporation of 14C into glutamine by 85 %, suggesting the rapid operation of glutamine synthetase. Conversely, when P. involutus was fed with [14C]glutamine, 46% of the label was found in glutamate within 30 min of feeding and AZA inhibited glutamate formation by 90%. Taken together, these data indicate that glutamate synthase (GOGAT) is the major enzyme of glutamine degradation. In addition, the strong inhibition of glutamine utilization by AOA indicates that glutamine catabolism in P. involutus might involve a transamination process as an alternative pathway to GOGAT for glutamine degradation. The high l4CO, evolution shows that glutamate and glutamine are further actively consumed as respiratory substrates, being channelled through the tricarboxylic acid (TCA) cycle and oxidized as CO, . It appears that synthesis of amino acid precursors during TCA cycle operation is an essential step for aspartate and alanine synthesis through aminotransferase activities in P. involutus.

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“…Conversely, GS and, to a lesser extent, NADP-GDH have not been extensively characterized in ectomycorrhizal fungi. Only their activity in fungal mycelia and ectomycorrhizas has been studied (1,8,10,28), and their role in NH4' assimilation by ectomycorrhizal fungi and ectomycorrhizas investigated (9,11,19). Indeed, the activity and amount of fungal NADP-GDH polypeptide were strongly suppressed in beech associations (10,19).…”

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confidence: 99%

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

Brun¹,

Chalot²,

Botton³

et al. 1992

Plant Physiol.

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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 M, 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. on regulation, physical properties, and kinetic characteristics of GS and NADP-GDH is derived from studies on higher plants (20,21,26), yeasts (7,25

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Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Cited by 8 publications

References 16 publications

Nitrogen metabolism in the ectomycorrhizal fungus Hebeloma crustuliniforme

Nitrogen metabolism in the ectomycorrhizal fungus Hebeloma crustuliniforme

Metabolism of [14C]glutamate and [14C]glutamine by the ectomycorrhizal fungus Paxillus involutus

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

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