Molecular biological studies on congenital lactic acidemia due to pyruvate dehydrogenase deficiency

丙酮酸脱氢酶（PDH）复合物是一种线粒体多重酶复合物，催化丙酮酸氧化脱羧为乙酰辅酶a。PDH复合物由PDH、脂酸乙酰转移酶、脂酰胺脱氢酶、PDH磷酸酶、PDH激酶和protein-X六种组分组成。PDH复合物的活性受PDH磷酸酶和PDH激酶分别催化的去磷酸化（活化）和磷酸化（失活）调控。PDH复合物的缺陷和PDH复合物的激活缺陷是导致先天性乳酸血症的常见原因。采用新方法测定了3例乳酸性乳酸血症患者皮肤成纤维细胞中PDH磷酸酶活性。3例患者的酶活性明显降低。免疫印迹技术显示，3例患者中2例的成纤维细胞中PDH的α亚基和β亚基（PDH磷酸酶的底物）的数量明显减少。相比之下，来自三名患者中的一名的成纤维细胞具有大约相似数量的α和β亚基来控制。这些结果提示，PDH复合物激活缺陷可能主要是由于两例患者和另一例患者的PDH和PDH磷酸酶异常所致。由于PDH α和β亚基蛋白的快速降解，在一名女性患者中发现了PDH α亚基基因的4 bp插入突变。这个4 bp的插入引起移码，改变了氨基酸序列，产生了过早终止密码子。这名女性患者是正常等位基因和突变等位基因的杂合子。然而，该患者培养的大多数皮肤成纤维细胞表达突变等位基因。这些结果表明，在该女性患者中，含有正常等位基因的X染色体主要失活，尽管存在正常等位基因和突变等位基因的杂合子，但仍出现乳酸血症和神经系统异常，突变α亚基蛋白未能形成稳定的PDH结构，α亚基蛋白和β亚基蛋白均被迅速降解。少

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 … More 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