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， 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）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 多酶复合物将PDH复合物的所有亚基纯化， 来自牛肾脏和心脏的接近同质性。纯化的蛋白质 随后用于抗体生成。然后我们克隆了 鉴定了PDH E1 a、E1 b和E3亚基的全长cDNA， 基因分别位于X、3和7号染色体上。的核苷酸 人脑PDH E1 a克隆序列与肝PDH E1 a克隆序列完全一致 表明2，3-丁二醇生产的差异 在脑和肝中，可能不是由于PDH E1 a的结构差异， 而是由于代谢调节剂或组织特异性 由PDH特异性激酶不同地调节的PDH的调节， 磷酸酶。然而，我们的克隆与其他cDNA克隆不同 分离自包皮和胎儿肝脏。两种不同类型的cDNA克隆 的PDH E1 b亚基也被鉴定和完全表征。的 我们克隆的序列是完全相同的，但它们不同于 从几个地区的包皮中分离出来。正确的序列 我们的克隆通过聚合酶链反应和DNA测序的方法进行了验证 测序本文还研究了PDH复合物的差异调节。 从乳酸酸中毒患者中培养的人成纤维细胞。两种细胞 Leigh综合征患者100例，PDH复合物未见异常。 观察到，而在TCA循环中观察到PDH复合物中的主要问题 缺陷细胞系。这些细胞中的缺陷被确定为与 PDH蛋白质的翻译和加工问题。PDH特异性蛋白 还纯化了激酶和磷酸酶， 序列已确定。根据部分氨基酸序列， 合成了几种寡脱氧核苷酸并用于克隆基因 编码PDH特异性激酶以及PDH-磷酸酶，其结构 和调节的特征还没有被描述出来。

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.