| Project/Area Number |
02670423
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pediatrics
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| Research Institution | Tohoku University |
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Principal Investigator |
MIYABAHASHI Shigeaki Tohoku University, School of Medicine, Assistant Professor, 医学部, 講師 (20174203)
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| Co-Investigator(Kenkyū-buntansha) |
HANAMIZU Hiroshi Tohoku University, Hospital, Department of Pediatrics, Medical Staff, 医学部附属病院, 医員
ENDO Hitoshi Jichi Medical School, Department of Biochemistry, Instructor, 助手 (50221817)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
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| Keywords | Pyruvate dehydrogenase deficiency / Genetic analysis / Insertion / Atrandom inactivation of X chromosome / cDNA / SV40 / Cloned cells / ASO hybridization / ピルビン酸脱水素酵素欠損症 / 直接塩基決定法 / 一塩基変更 / 蛋白ブロット |
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| Research Abstract |
The biochemical, immunological and genetical characteristics of pyruvate dehydrogenase(PDH)were investigated in the fibroblasts from five patients with PDH deficiency. The PDH activities of the patients ranged from 2.64 to 13.1 pmol/min/mg priten and were 6 - 30% of the mean of controls(43.5 +- 13.2 pmol/min/mg proteins, mean +- SE). PDH in fibroblasts homogenate from the patients was found to have normal Km for pyruvate and normal pH optimum curves. Thermostability test at 39ﾟC revealed two different properties of the enzyme which was markedly unstable in three patients but normal in two. These results might be indicated the existence of at least two variants of PDH deficiency. Furthermore immunological analysis using antibody to purified PDHC from bovine heart was investigated. Immunoblot stains revealed four heterogeneities in El components of these patients. Elalpha component of one patient appeared to have a slightly higher molecular weight than that of control. On genetical analy
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sis of this patient, the four-nucleotide deletion was confirmed by three independent methods(ASO hybridization, Sl mapping and PCR amplification of another tissue). E. 1a component of other two-patients showed decreased cross-reacting materials. The components of both Elalpha and Elbeta in one female patient were undetectable. In this patient, the four-nucleotide insertion was clarified in Elalpha gene but no mutation in Elbeta gene. Immunoblot stains from other two patients showed decreased of Elbeta component. However, immunoblot in one patient did not exhibit these defects.
Structures of PDH Ela and Elbeta polypeptides concerned with complex association for PDHC were not well-known. In fibroblasts, mainly 4-bp inserted mRNA was expressed and normal Elbeta mRNA was also expressed. From protein blot analysis, PDH Elalpha and Elbeta polypeptides were undetectable. In this fact, it was speculated that the substitution of eight amino acids from C-terminus of Elalpha polypeptide caused by a frame shift influences the complex assembly and decreases the stability of not only Elalpha subunit, but also Elbeta subunit. Less
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