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1993 Fiscal Year Final Research Report Summary

Crystallization and X-ray crystallographic analysis of Complexes III and I in mitochondrial respiratory system

Research Project

Project/Area Number 03454565
Research Category

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

Allocation TypeSingle-year Grants
Research Field 生物物性学
Research InstitutionHimeji Institute of Technology

Principal Investigator

YOSHIKAWA Shinya  Himeji Inst. of Tech. Prof., 理学部, 教授 (40068119)

Co-Investigator(Kenkyū-buntansha) TSUKIHARA Tomitake  Tokushima Univ. Prof., 工学部, 教授 (00032277)
SHINZAWA-ITOH Kyoko  Himeji Inst. of Tech. Asis. Prof., 理学部, 助手 (70206316)
TSUKIHARA Tomitake  Tokushima Univ. Prof. (00032277)
TSUKIHARA Tomitake  Tokushima Univ. Prof. (00032277)
TSUKIHARA Tomitake  Tokushima Univ. Prof. (00032277)
Project Period (FY) 1990 – 1993
KeywordsNADH-Ubiquinone reductase / Ubiquinol-cytochrome c reductase / Membrane protein / Electron transfer system / respiratory chain / Protein crystallizatioin / Crystallographic analysis
Research Abstract

Based on the experience about crystallization of beef heart cytochrome c oxidase, purification procedure of beef heart Complex III, reported by Rieske, was improved to increase the yeald and the reproducibility. Complete replacement of the residual cholate in the purified sample with a nonionic detergent, dodecyl sucrose, increased the stability of the sample significantly. The preparation dissolved in Tris-HCl buffer was crystallizable in a monoclinic form with polyethyleneglycol as the precipitant, giving an x-ray diffraction pattern with 7.5 A resolution. However, the crystals were fragil and easily to be twinned. The preparation in K-phosphate buffer provided hexagonal crystals which diffracted x-rays upto 6.5 A resolution. An addition of An^<2+> increased the reproducibility of crystallization of the hexagonal crystals. On the other hand, the preparation purified with crystallization gave tetragonal crystals with a parfection similar to the hexagonal one.
Purification procedure by Rieske for Complex I was also reexamined to establish the reproducibility and the yeald(0.9 grams from 1.2 kg of beef heart muscle). Survey of the crystallization conditions with "crystal screen"(Hampton Research Inc.) provided spherlite crystals which showed birefringe under polarized light.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] Yoshikawa,S.: "Strategies for crystallization of membrane protein complexes." J.Cryst.Growth. 122. 298-302 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kubota,T.: "Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase." J.Biochem.111. 91-98 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kubota,T.: "Crystallization and preliminary X-ray crystallographic studies of bovine heart mitochondrial cytochrome bc_1 complex." J.Mol.Biol.221. 379-382 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kawamoto,M.: "New crystal forms and preliminary X-ray diffraction studies of mitochondrial cytochrome bc_1 complex from bovine heart." J.Mol.Biol.(submitted).

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yoshikawa, S., Shinzawa-Itoh, K., Ueda, H., Tsukihara, T., Fukumoto, Y., Kubota, T., Kawamoto, M., Fukuyama, K.and Matsubara, H.: "Strategies for crystallization of membrane protein complexes." J.Cryst. Growth. 122. 298-302 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kubota, T., Yoshikawa, S.and Matsubara, H.: "Kinetic mechanism of beef heart ubiquinol : cytochrome c oxido-reductase." J.Biochem.111. 91-98 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kubota, T., Kawamoto, M., Shinzawa-Itoh, K., Yoshikawa, S., Matsubara, H.: "Crystallization and preliminary X-ray crystallographic studies of bovine heart mitochodrial cytochrome bc_1 complex." J.Mol. Biol.221. 379-382 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kawamoto, M., Kubota, T., Matsunaga, T., Fukuyama, K., Matsubara, H., Shinzawa-Itoh, K., and Yoshikawa, S.: "New crystal form and preliminary X-ray diffraction studies of mitochondrial cytochrome bc_1 comples from bovine heart." submitted to J.Mol. Biol.

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

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Published: 1995-03-27  

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