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HATTORI, Takefumi

ITAYA, Masayuki

OHTA, Akira

SHIMADA, Mikio

Kyoto University Research Information Repository

Title<Preliminary>An Enzymatic Study on Isocitrate Metabolism inthe Ectomycorrhizal Fungus Laccaria amethysteaAuthor(s)ITAYA, Masayuki; HATTORI, Takefumi; OHTA, Akira;SHIMADA, MikioCitationWood research : bulletin of the Wood Research Institute KyotoUniversity (2001), 88: 46-47Issue Date2001-09-30URL http://hdl.handle.net/2433/53140RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityPreliminaryAn Enzymatic Study on Isocitrate Metabolism in theEctomycorrhizal Fungus Laccaria amethystea*lMasayuki hAYA*2, Takefumi HATTORI*2,Akira OHTA*3 and Mikio SHIMADA*2(Received May 31, 2001)Keywords: ectomycorrhiza1 fungus, Laccaria amethystea, isocitrate metabolism, isocitrate lyase, isocitrate dehydrogenaseIntroductionEctomycorrhizal fungi are supplied with carbohydratesas a sole carbon source from their host plants I). Themetabolism of carbohydrates in these fungi associated withwoody plants has been of widespread interest, but theirorganic acid metabolism has not been fully investigated.This study focussed on isocitrate metabolism in theectomycorrhizal fungi because isocitrate is the keyintermediate at a diverging point of the glyoxylate cycleand TCA cycle. Although the regulatory mechanisms forthe isocitrate flux to the glyoxylate cycle by isocitrate lyase(ICL) and TCA cycle by isocitrate dehydrogenase (ICDH)have been studied for Escherichia coli2 ), no enzymaticexperiment has been reported on the comparison of theactivity of ICDH with that of ICL in ectomycorrhizalfungi.Here we report that ICDH activity was always greaterthan that of ICL in the mycelial extracts from theectomycorrhizal fungus Laccaria amethystea grown in thepure culture and the symbiotic culture associated withPinus densiflora seedlings. The results are discussed inrelation to a possible isocitrate flux in the organic acidmetabolism in the fungus.Materials and MethodsFungal cultureL. amethystea was grown stationary in the 100 ml ofglucose (1 %) and yeast extract (0.1 %) medium at 21°Cunder the dark condition.Symbiotic cultivation by the sandwich methodThe symbiotic culture of P. densiflora seedlings and L.amethystea was conducted by the modified methods ofChilvers et al. 3 ).En;::,yme assayThe crude enzyme solution was prepared by extractionof the mycelia with 0.1 M potassium phosphate buffer (pH7.0) which contains 0.5 mM phenylmethylsulfonylfluoride. The activities of NAD-ICDH (ECl.l.1.41),NADP-ICDH (ECl.l.1.42) and ICL (EC4.1.3.1) weredetermined spectrophotometrically4,5).Results and discussionTable 1 shows the growth ofL. amethystea and the specificactivities ofNADP-ICDH and ICL in the cell-free extractsfrom the mycelia grown in the free-living culture. Thefungus did not grow better in the sucrose media than in theglucose and the fructose media. However, the activities ofNADP-ICDH were always greater than that of ICL in allcases. This finding is contrasted with the recent report onwood rotting fungi, in which the activity of ICL is greaterthan that of ICDH in vegetative mycelia of Fomitopsispalustris6 ). Thus, in relation to the isocitrate metabolism,the induction of ICL by ethanol was investigated for L.amethystea. Because it has been known that the glyoxylatecycle enzymes appear when microorganisms were grownon the non-sugar substrate such as ethanol and acetate7).However, the mycelia of L. amethystea could not sufficientlygrow in the ethanol media and ICL activity was notinduced by the addition of ethanol (data not shown).Thus, it has been found that L. amethystea can little utilizeethanol as a carbon source. Furthermore, the resultssuggest that the enzymic system for the isocitratemetabolism in this fungus is probably different from that ofTable 1. The growth and the activities of ICDH and ICL of L. amethystea in the free-living culturewith different carbon source.(mg D.W)Specific activity (nkat/mg protein)Culture days Carbon source GrowthICDH ICL21 Glucose 128.5 1.528 0.12321 Fructose 153.8 1.548 0.21821 Sucrose 53 0.754 0.047The values presented are averages of two independent replicates.*1 A part of this work was presented at the 45th Annual meeting of the Mycological Society of Japan in Tokyo, May, 2001.*2 Laboratory of Biochemical Control, Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan.*3 Shiga Forest Research Center, 978-95 Kitasakura, Yasu-cho, Yasu-gun, Shiga 520-2321, Japan.- 46-ITAYA et al.: An Enzymatic Study on Isocitrate Metabolism in the Ectomycorrhizal Fungus Laccaria amethysteaother microorganisms7), including the wood rotting fungusF. palustris6 ).During 5 weeks for symbiotic cultivation, ectomy-corrhizal synthesis was microscopically observed on theroots of P. densiflora associated with L. amethystea. Incontrast to the poor growth of fungus on the sucrose mediain the free-living cultivation, the mycelia could grow wellnear the roots of P. densiflora when sucrose was used as acarbon source. The NADP-ICDH activity was detectedbut ICL was not in the extracts from mycelia grown nearthe roots. However, the details of the results will bereported elsewhere.These results suggest that NADP-ICDH probably playamore important role than ICL in the isocitrate metabolismof L. amethystea. ICDH is one of the key enzymes in TCAcycle and regulates the flux of carbon to nitrogenmetabolism through synthesis of 2-oxoglutarate, which isnecessary for glutamate biosynthesis!). Therefore furtherresearch is needed to elucidate the metabolic pathway for2-oxoglutarate whether it is metabolized in TCA cycle orthrough y-aminobutyrate (GABA) shunt, which ISreported for other basidiomycetes8).ReferencesI) S.E. SMITH and D.J. READ: Mycorrhizal Symbiosis, 2ndEdition, Academic Press (1997).2) K. WALSH and D.E. KOSHLAND, JR. : ]. BioI. Chem., 259,9646-9654 (1984).3) G.A. CHILVERS et al.: New Phytol., 103, 3~17-402 (1986).4) G.H. DIXION and H.L. KONBERG: Biochem.].. 72, 3P (1959).5) S. GALVEZ and P. GADAL: Plant Sci., 105, 1-14 (1995).6) M. Erman et al.: J. Wood Sci., in press. (~~001).7) P. VANNI et al.: Com. Biocheme. Physiol., 95B(3), 431-458(1990).8) D. MOORE and J.O. EWAZE: ]. General Microbiol., 97,313-322 (1976).- 47-

<Preliminary>An Enzymatic Study on Isocitrate Metabolism in the Ectomycorrhizal Fungus Laccaria amethystea

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<Preliminary>An Enzymatic Study on Isocitrate Metabolism in the Ectomycorrhizal Fungus Laccaria amethystea

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