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Intracellular fate of nitrate reductase in higher plants

Research Project

Project/Area Number 01540558
Research Category

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

Allocation TypeSingle-year Grants
Research Field 植物生理学
Research InstitutionChiba University

Principal Investigator

NAKAGAWA Hiroki  Chiba University of Horticulture Professor, 園芸学部, 教授 (70009330)

Co-Investigator(Kenkyū-buntansha) ANDO Akikazu  Chiba University of Horticulture Associate professor, 園芸学部, 助教授 (80125898)
SATO Takahide  Chiba University of Horticulture Associate professor, 園芸学部, 助教授 (60125929)
Project Period (FY) 1989 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1991: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Keywordsnitrate reductase / protein degradation / nitrate reductase inactivator / 硝酸還元酵素(NR) / NR不活性化因子 / ホウレンソウ / 分解中間体(タンパク質の) / モノクロ-ナル抗体 / cDNA(硝酸還元酵素)
Research Abstract

Proteolytic fragments were obtained by limited proteolysis of 120 kDa nitrate reductase from Spinacia oleracea L. using trypsin and Staphylococcus aureus V8 protease. Determination of NH4-terminal sequences in 9 to 14 Edman degradation steps allowed the exact localization of the fragments within the amino-acid sequence of CDNA clone pSPNR117. This clone has a 2324 base insert, and the amino acid sequence deduced from its open reading frame, which contains 640 residues.
A sequence identity of 61.2-80.1% was found in the amino acid sequences deduced from the CDNA sequences as obtained by spinach and other higher plant nitrate reductases. However, the amino acid sequences surrounding the proteolytic cleavage sites of nitrate reductase had poor homology.
A nitrate reductase-inactivator protein has been purified 16, 000-fold from spinach leaves by PH 5 treatment, chromatography on SE53, Con A-Sepharose, and chromatofocusing. -The yield was 12%, the specific activity was 115 u/mg. Polyacrylamide gel electrophoresis of the final purified inactivator yield 2 major protein bands and both bands exhibited nitrate reductase-inactivator activity. Analysis of this inactivator protein by gel filtration and SDS-gel electrophoresis revealed protein stainable material only in a molecular weight range of 110, 000115, 000. SDS gel electrophoresis under reducing conditions yielded 2 protein bands corresponding to molecular weight of 51, 000 and 53, 00'0- The proteolytic mapping for the two separated subunits appeared similar and possibly identical.

Report

(4 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • 1989 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] N.Shiraishi,Y.Kubo,G.Takeba,S.Kiyota,K.Sakano and H.Nakagawa: "Sequence analysis of cloned cDNA and Proteolytic fragments for nitrate reductase from Spinacia oleracea" Plant Cell Physiology. 32. 1031-1038 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T.Yoshimura,N.Sekino,K.Okuo,T.Sato, N.Ogura and H.Nakagawa: "A nitrate reductase inactivator protein from spinach.Purification,molecular weight and subunit composition" Plant Cell Physiology. (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] N. Shiraishi, Y. Kubo, G. Takeba, S. Kiyota, K. Sakano and H. Nakagawa: "Sequence analysis of cloned cDNA and proteolytic fragments for nitrate reductase from Spinacia oleracea L" Plant Cell Physiol.32 (7). 1031-1038 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Yoshimura, N. Sekino, K. Okuo, T. Sato, N. Ogura and H. Nakagawa: "A nitrate reductase inactivator protein from spinach. Purification, molecular weight and subunit composition." Plant Cell Physiol.(1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Naomasa Shiraishi: "Sequence analysis of cloned cDNA and Proteolytic Fragments for Nitrote reductase from Spinacia oleracea L." Plant Cell Physiol. 32. 1031-1038 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Yukiko Sato: "Arginine and Lysine Residue as NADHーbinding sites in NADHーnitrate reductase from spinach." Phytochemistry. (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] Tomoaki Yoshimura: "A nitrate reductase inactiuator protein from spimach.Purification,molecular weight and subunit composition" Plant Cell Physiol. (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] Kuni Sueyoshi,Nagao Ogura,Hirok Nakagawa: "Identification of Possible Intermediates in in vivo degradation of spinach nitrate reductase" Agr.Biol.Chem.53. 151-156 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] W.Bruggemann,PーR,Moog,H.Nakagawa,P.J.C.Kuiper: "Plasma membraneーbound NADHーFe^<3+>ーEDTA reductase and iron deficiency in tomato.Is thae a turbo reductase?" Physiol.Planfrum. 79. 339-346 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] P.Askerlund,P.Laurent,H.Nakagawa,J.C.Kader: "NADHーferricyanide reductase of Leat plasma membranes Partial pvrification and immunological relation to potato tuber microsomal NADHーferricyanide reductase and spinach leaf NADHーMitrate recluctase" Plant Physiol. (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] H.Nakagawa,Y.Kubo,N.Shiraishi: "Functional domains in higher plant nitrate reducase" Program and abstructs of 3rd Internctional symposium “NO^ー_3 assimiration molecular and genetic aspects". 78-80 (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] N.Shiraishi,H.Nakagawa,他4名: "Seqvence analysis of cloned cDNA and Proteolytic fragments for nitrate reductase from spinacis oleracea." Plant Cell Physiol.

    • Related Report
      1990 Annual Research Report
  • [Publications] Kuni Sueyoshi,Nagao Ogura,Hiroki Nakagawa: "Identification of Possible Intermediates in in vivo Degradation of Spinach Nitrate reductase" Agr.Biol.Chem.53. 151-156 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Naomasa Shiraishi,Hiroki Nakagawa 他4名: "Sequence analysis of cloned cDNA and proteolytic fragments for nitrate reductase from spinacia oleracea L" Biochin.Biophys.Acta.

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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