STRUCTURE & REGULATION OF PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX (III) GENES

来自我们实验室的数据表明，2，3-丁二醇是一种 在人类酗酒者中发现的不寻常的代谢物，可能是由 丙酮酸脱氢酶(PDH)的副反应位于脑和睾丸。 基于这一假设，我们已经开始研究PDH的基因 多酶复合体。PDH复合体的所有亚基都被纯化到 来自牛肾脏和心脏的近乎同质的。纯化的蛋白质是 随后用于产生抗体。然后我们克隆了 鉴定了PDH E1a、E1b和E3亚基的全长cDNA，其 基因分别位于X、3和7染色体上。核苷酸 人脑PDH E1a克隆序列与肝脏序列一致 克隆表明2，3-丁二醇产量的差异 可能不是由于PDH E1a的结构差异，而是 而是由于代谢调节因子或组织特异性的改变 PDH特异性激酶和PDH对PDH的不同调节作用 磷酸酶。然而，我们的克隆与其他cdna克隆不同。 从包皮和胎儿肝脏中分离出来。两种不同类型的cDNA克隆 对于PDH，也鉴定了E1b亚基，并对其进行了完整的鉴定。这个 我们克隆的序列完全相同，但它们不同于 在几个地区从包皮上分离出来的。正确的序列 用聚合酶链式反应和DNA方法对克隆进行了验证 测序。还研究了PDH复合体的差异性调节。 乳酸酸中毒患者培养的人成纤维细胞。在两个单元格中 Leigh综合征患者的PDH波群未见异常 在TCA循环中观察到PDH复合体中的主要问题 有缺陷的细胞系。这些细胞中的缺陷被确定为与 解决了PDH蛋白的翻译和加工问题。PDH特异性蛋白 还纯化了激酶和磷酸酶，其N-端氨基酸 确定了序列。根据部分氨基酸序列， 合成了几个寡核苷酸，并用于克隆这些基因 PDH特异性激酶和PDH-磷酸酶的编码，其结构 和监管还没有被描述出来。

The data from our laboratory indicated that 2,3-butanediol, one of the unusual metabolites found in human alcoholics, could be generated by the side reaction of pyruvate dehydrogenase (PDH) located in brain and testis. Based on this hypothesis, we have started to study the genes for the PDH multienzyme complex. All the subunits of the PDH complex were purified to near homogeneity from bovine kidney and heart. The purified proteins were subsequently used for antibody generation. We have then cloned and characterized full-length cDNAs for PDH E1a, E1b, and E3 subunits whose genes are located in chromosome X, 3, and 7, respectively. The nucleotide sequence of human brain PDH E1a clone was identical with that of liver clone indicating that the differences in the production of 2,3-butanediol in brain and liver may not be due to a structural difference in PDH E1a but rather due to alteration in metabolic regulators or tissue specific regulation of PDH differently modulated by PDH-specific kinase and phosphatase. Our clones, however, were different from other cDNA clones isolated from foreskin and fetal liver. Two different types of cDNA clones for PDH E1b subunits were also identified and completely characterized. The sequences of our clones were exactly identical, but they are different from that isolated from foreskin in several regions. The correct sequences of our clones were verified by a method of polymerase chain reaction and DNA sequencing. The differential regulation of PDH complex was also studied in cultured human fibroblasts from patients of lactic acidosis. In two cell lines of Leigh syndrome patients, no abnormality of PDH complex was observed while a major problem in PDH complex was observed in a TCA-cycle defective cell line. The defect in these cells was determined to be related to problems of PDH protein translation and processing. PDH-specific protein kinase and phosphatase were also purified and their N-terminal amino acid sequences were determined. Based on the partial amino acid sequences, several oligodeoxynucleotides were synthesized and used to clone the genes coding for PDH-specific kinase as well as PDH-phosphatase, whose structures and regulation were not characterized yet.