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1991 Fiscal Year Final Research Report Summary

Purification and cDNA cloning of galactosylceramidase 1

Research Project

Project/Area Number 02454246
Research Category

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

Allocation TypeSingle-year Grants
Research Field Neurology
Research InstitutionKyushu University

Principal Investigator

KOBAYASHI Takuro  Kyushu University, Faculty of Medicine, Lecturer, 医学部, 講師 (40158902)

Co-Investigator(Kenkyū-buntansha) YAMADA Takeshi  Kyusuhu University, Faculty of Medicine, Associate, 医学部, 助手 (50230462)
Project Period (FY) 1990 – 1991
KeywordsKrabbe disease / Galactosylceramidase I
Research Abstract

Krabbe disease (GLD) is one of the geneti leukodystrophies, in which galactosylc eramidase I is deficient. For 10 years, we have studied the pathogenesis of this particular disorder and found that the hydrolysis of galactosylceramide is catalyzed by 2 acid hydrolases, namely galactosylceramidase I and 11, and only the former enzyme is deficient in GLD. This finding could answer the question why galactosylsphingosine but not galactosylceramide accumulates in the tissue of GLD patient. Because the accumulated galactosylsphingosine is cytotoxic, it is suggested that myelin-forming cells, oflgodendroglia and Schwann cells, are dead and demyelination occurs.
The aim of this project is to characterize the molecular properties of the defici ent enzyme in GLD. The purification of the enzyme has been very difficult, and no in vestigators could succeed to purify it. In 1990, we have purified the enzyme up to I 0000 folds from the crude sample of human placenta, using several chromatographic tec hniques. The final product contained 2 bands of 58kDa and 2OkDa, as cheeked with SDS -PAGE. After blotting the 2 bands to an appropriatemembrane and the corresponding proteins were digested by endopeptidase. But the amino acid sequence of the 58kDa band revealed the homology to IgG, as checked by compute research. We could not detect amino acid in the 2OkDa band protein. Therefore, in 1991, we changed the starting material to porcine kidney which contained relatively high specific acitivit of the enzyme. After purification to about 20000 folds, the final product contained a main band of 54 kDa and several faint bands. The amino acid sequence of the main protein was that which has never been reported. We are now trying to clone the CDNA of the protein, and the results will soon be able to be obtained.

  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] 光尾 邦彦: "若年型Sandhoff病の一例" 臨床神経. 30. 179-183 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mitsuo,K.: "Juvenile Sandhoff disease:a Japanese patient carrying a mutation identical to that found earlier in a Canadian patient" J.Neurol.Sci.98. 277-286 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Toda K.: "Lysosulfatide(sulfogalactosylsphingosine)accumulation in tissues from patients with meta-chromatic leukodystrophy" J.Neurochem. 55. 1585-1591 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yoshimura T.: "Accumulation of galactosylsphingosine(psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse" Neurochem.Res.15. 963-967 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kobayashi T.: "A sensitive assay of lysoganglioside using high-performance liquid chromatography" Biochim.Biophys.Acta. 1081. 159-166 (1991)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mitsuo, K.: "A case of jivenile Sandhoff disease" Clin. Neurol.30. 179-183 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Mitsuo, K.: "Juvenile Sandhoff disease : a Japanese patient carrying a mutation identical to that found earlier in a Canadian patient." J. Neurol. Sci.98. 277-286 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Toda, K.: "Lysosulfatide (sulfogalactosylsphingosine) accumulation in tissues from patients with metachromatic leukodystrophy." J. Neurochem.55. 1585-1591 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yoshimura, T.: "Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse." Neurochem. Res.15. 963-967 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kobayashi, T.: "A sensitive assay of lysoganglioside using high-performance liquid chromatography." Biochem. Biophys. Acta. 1081. 159-166 (1991)

    • 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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