| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1991: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Pyruvate dehydrogenase(PDH)complex is a mitochondrial multiple enzyme complex and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDH complex consists of six components, PDH, lipoate acetyltransferase, lipoamide dehydrogenase, PDH phosphatase, PDH kinase and protein-X. The activity of PDH complex is regulated by dephosphorylation(activation)and phosphorylation(inactivation), catalyzed by PDH phosphatase and PDH kinase, respectively. The defect of PDH complex and the defect in the activation of PDH complex are common causes of the disorders leading to congenital actic acidemia.
The PDH phosphatase activities in cultured skin fibroblasts from three patients with lactic acidemia due to the defect of the activation of PDH complex were determined by our newly developed assay method. The enzyme activities were significantly reduced in the three patients. The markedly reduced amounts of alpha and beta subunits of PDH which was a substrate for PDH phosphatase, were revealed i
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n the fibroblasts from two of the three patients by the immunoblot technique. In contrast, the fibroblasts from one of the three patients had approximately similar amounts of the alpha and beta subunits to control. These results suggest that the defect of the activation of PDH complex might be primarily due to the abnormalities in PDH and PDH phosphatase in the two patients and the other patient, respectively.
A mutation, 4-bp insertion, in the gene for alpha subunit of PDH was found in a female patient with PDH deficiency due to the rapid degradation of alpha and beta subunit proteins of PDH. This 4-bp insertion caused frameshift that altered amino acid sequence and created the premature stop codon. This female patient was a heterozygote of the normal and this mutant alleles. However, most of cultured skin fibroblasts from this patient expressed the mutant allele. These results suggested that in this female patient, X chromosome containing the normal allele was predominantly inactivated so that she showed lactic acidemia and neurological abnormalities in spite of a heterozygote of the normal and the mutant alleles and that the mutant alpha subunit protein failed to form a stable structure of PDH and both alpaha and beta subunit proteins were degraded rapidly. Less
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