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1991 Fiscal Year Final Research Report Summary

Research for Improvement in Efficiency of Energy Metabolism of Soybean Rhizobium.

Research Project

Project/Area Number 02660068
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 土壌・肥料
Research InstitutionKagawa University

Principal Investigator

TAJIMA Shigeyuki  KAGAWA Univ., Dept. Agriculture, Associate Professor, 農学部, 助教授 (50116894)

Project Period (FY) 1990 – 1991
Keywordsnitrogen fixation / nodules / malic enzyme / Bradyrhizobiom / gene cloning / emergy metabolism / nod-35 / soybean
Research Abstract

Role of malic enzyme in Bradyrhizobium japonicum energy metabolism were surveyed, and the data suggested the enzyme was a key enzyme to improve the energy metabolism.
An NADP-malic enzyme, a possible key enzyme for the energy metabolism of Bradyrhizobium japonicum A1017, was purified to homogeneity by ammonium sulfate precipitation, DEAE-cellulose column chromatography, preparative isoelectric-focusing, Sephacryl S-200 column, 2', 5'-ADP-Sepharose 4B column and NAD-agarose column chromatographies.
The purified enzyme showed its optimun pH at 7.4, and the pI value was 6.4. The reaction was dependent on NADP, with the Km values of 4.9 muM and 61 muM against NADP and malate respectively. The molecular weight of the enzyme subunit was calculated to be 59, 000 on SDS-PAGE, and the result of Sephacryl S-200 gel chromatography suggested that the native enzyme was present as dimer. Among metabolites present in the nodule cell extract, malonate, succinate, oxalacetate, ATP, ADP and NADPH in the enzyme reaction system showed strong regulatory effect on the activity under physiological concentrations.
From the purified enzyme, the N-terminal amino acid sequence was analysed. From the data 4 DNA probes were synthesized. A genomic DNA library was also prepared after selecting ca. 2 kbp DNA fragment by sucrose density gradient centrifugation. Following ligating and packaging of the DNA to lamda phage, the plaque hybridization was performed and selected the positive clone.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] Tajima,S.and H.Kouchi: "Metabolism of C^4-dicarboxylates and amino acids in soybean nodule bacteroids,and supply to nitrogenae" Nitrogen Fixation:Achievementa and objectives. 309-314 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tajima,S.et al.: "Purification and characteristics of NADP-malic enzyme in Bradyrhizobium japonicum" Nitrogen Fixation:Achievementa and objectives. 590 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tajima,S.et al.: "Soybean cotyledons contain a uricase that cross-reacts with antibodies raised against the nodule uricase(nod-35)." Plant Cell Physiol.32(8). 1307-1311 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tajima,S.et al.: "Purification and characteristics of NADP-malic enzyme in Bradyrhizobium japonicum A1017." Biosci.Biotec.Biochem.

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Tajima, S. and H. Kouchi: "Metabolism of C_4-dicarboxylates and amino acids in soybean nodule bacteroids, and their role in energy supply to nitrogenase" Nitrogen Fixation : Achievementa and objectives. 309-314 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tajima, S. et al.: "Purification and characteristics of NADP-malic enzyme in Bradyrhizobium Japonicum A1017" Nitrogen Fixation : Achievementa and objectives. 590 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tajima, S. et al.: "soybean cotyledons contain a uricase that cross-reacts with antibodies raised against the nodule uricase (nod-35)." Plant Cell Physiol. 32 (8). 1307-1311 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Tajima, S. et al.: "Purification and characteristics of NADP-malic enzyme in Dradyrhizobium japonicum A1017." Biosci, Biotec. Biochem.

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-03-16  

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