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

作为一个持续的项目，丙酮酸脱氢酶的所有亚单位 (PDH)多酶复合体被纯化到接近均一的水平。 牛肾和牛心脏。纯化的蛋白质随后被 用于PDH-激酶和PDH-磷酸酶的进一步纯化和 产生针对每个亚基的抗体。使用这些抗体 和寡聚脱氧核苷酸，我们还克隆和测定了 PDH E1`、E1~和E3全长cDNA的核苷酸结构 亚单位。我们还展示了这些PDH的差异表达 以细胞和组织特有的方式表达基因。尤其是一个 不同分子的PDH E1`亚型的免疫学差异 在大鼠睾丸中发现了肿块。仅检测到此PDH E1`异构体 由抗肽抗体和单特异性免疫沉淀 抗PDH E1~亚基的抗体，所有ALL都有PDH复合体的共同抗体 肾组织中PDH E1‘亚单位阳性，但未检出。 这种特殊的PDH E1‘亚型似乎存在于睾丸和 可能支持PDH同工酶在乙酸乙酯中的潜在作用的大脑， 因此，在酗酒者中以及在酒精中发现丁二醇的产生 动物模型。亚基的坐标、转录激活 脂肪细胞分化过程中PDH复合体的表达 演示了。PDH特异的蛋白激酶和磷酸酶也是 纯化至均一，其N-端氨基酸序列 下定决心。另一种具有PDH-激酶活性(M/r 68,000)的蛋白质是 同样提纯：多肽序列分析显示该蛋白 与钙电蛋白相同。根据部分氨基酸序列， 合成了几种寡聚脱氧核苷酸，并将其用于克隆 编码PDH特异性激酶和PDH-磷酸酶的基因，其 结构和监管还没有确定下来。

As a continuing project, all the subunits of the pyruvate dehydrogenase (PDH) multienzyme complex were purified to near homogeneity from bovine kidney and heart. The purified proteins were subsequently used for further purification of PDH-kinase and PDH-phosphatase and generation of antibodies against each subunit. Using these antibodies and oligodeoxynucleotides, we have also cloned and determined the nucleotide structures of full-length cDNAs for PDH E1`, E1~, and E3 subunits. We also demonstrated differential expression of these PDH genes in cell- and tissue-specific manners. Particularly an immunologically distinct isoform of PDH E1` with different molecular mass was identified in rat testis. This PDH E1` isoform is only detected by anti-peptide antibodies and immunoprecipitated by monospecific antibodies against PDH E1~ subunit, common to PDH complex in all tissues examined but not by those against PDH E1` subunit of kidney. This specific PDH E1` isotype appeared to be present in testis and brain which may support the potential role of PDH isozyme in acetoin, thus butanediol production found in alcoholic individuals as well as in animal models. The coordinate, transcriptional activation of the subunits of the PDH complex during adipocyte differentiation was also demonstrated. PDH-specific protein kinase and phosphatase were also purified to homogeneity and their N-terminal amino acid sequences were determined. Another protein with PDH-kinase activity (M/r 68,000) was also purified: the peptide sequence analysis revealed that this protein is identical with calelectrin. 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.