1991 Fiscal Year Final Research Report Summary
Purification and cDNA cloning of galactosylceramidase 1
| Project/Area Number |
02454246
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurology
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| Research Institution | Kyushu University |
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Principal Investigator |
KOBAYASHI Takuro Kyushu University, Faculty of Medicine, Lecturer, 医学部, 講師 (40158902)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Takeshi Kyusuhu University, Faculty of Medicine, Associate, 医学部, 助手 (50230462)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Krabbe disease / Galactosylceramidase I |
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| 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.
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