| Project/Area Number |
61440090
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Kyoto Sangyo University (1989)
Kyoto University (1986-1988) |
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Principal Investigator |
YAMASHINA Ikuo KYOTO SANGYO UNIV., FAC. OF ENGINEERING, PROFESSOR, 工学部, 教授 (70025675)
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| Co-Investigator(Kenkyū-buntansha) |
SUGAHARA Kazuyuki KYOTO UNIV., FAC. OF PHARM. SCI., ASSISTANT, 薬学部, 助手 (60154449)
FUNAKOSHI Ikuo KYOTO SANGYO UNIV., FAC. OF ENGINEERING, PROFESSOR, 工学部, 教授 (10025702)
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| Project Period (FY) |
1986 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥29,700,000 (Direct Cost: ¥29,700,000)
Fiscal Year 1989: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 1988: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1987: ¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1986: ¥12,600,000 (Direct Cost: ¥12,600,000)
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| Keywords | Low's Syndrome / Nucleotide Pyrophosphatase (NPPase) / Phosphodiesterase I / Active Sulfate (PAPS) / Primary Structure of NPPase / Genomic DNA of NPPase / Elevation of Enzyme Activity in a Genetic Disease / Transcription and Translation Relevant to Elevation of Enzyme Activity / ロー症候群 / ヌクレオチドピロホスファターゼ / 先天性代謝異常 / ヌクレオチドピロホスファターゼ遺伝子 / ヒト繊維芽細胞cDNAライブラリー / ヒト線維芽細胞cDNAライブラリー / Lowe症候群 / 糖タンパク質 |
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| Research Abstract |
Lowe's synarome is a genetic disease with very serious symptoms. We have investigated the biochemical etiology of this disease. Initially we discovered the disorder of glycosaminoglycan sulfation which turned out to be due to enhanced degradation of active sulfate, PAPS. The enzyme which is involved in the PAPS degradation was then identified as nucleotide pyrophosphatase (NPPase) or phosphodiesterase I. The enzyme was purified from human placenta with several steps including the use of a monoclonal antibody to homogeneity. The amounts of the enzyme in fibroblasts from patients and normal individuals were assayed with the use of rabbit anti-NPPase antiserum, and it was found that the enzyme activities in the cells were proportional to the amount of the enzyme.
To obtain a clue to reveal the mechanism to cause the elevation of NPPase in the patients' cells, we have tried to clone the gene encoding NPPase. Nucleotide probes were synthesized based on the sequences of peptides obtained from the purified NPPase by enzymatic digestion. The probes were used to clone cDNA coding the NPPase polypeptide. The longest cDNA had a size of about 3 kb which corresponded to most of the polypeptide except for a small N-terminal region. This cDNA was sequenced. A primer extension method was applied to obtain cDNA corresponding to the N-terminal region plus leader peptide region, which led to elucication of whole amino acid sequence of NPPase. Using the longest cDNA, the amount of MRNA in fibroblasts were determined, and it was found that the amount of enzyme was proportional to the amount of mRNA. Although the mechanism of the elevation of NPPasw in patients' cells has not yet been revealed, the chromosomal location of NPPase and results of other genetic analyses will lead to understanding of biochemical etiology of Lowe's syndrome.
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