REGULATION OF THIAMINE-DEPENDENT ENZYMES INVOLVED IN GLUCOSE

作为一个正在进行的项目， 研究了参与葡萄糖代谢的硫胺素依赖性酶。 这些酶是线粒体丙酮酸脱氢酶（PDH）复合物， “-酮戊二酸脱氢酶（”-KGDH）复合物和胞质 转酮醇酶将来自牛和大鼠组织的这些酶纯化， SDS聚丙烯酰胺凝胶电泳上的表观均一性， 进行生化鉴定疏水荧光探针，双- ANS能有效地抑制PDH复合物和KGDH复合物的活性。 这些酶中的构象变化是在 添加别构调节剂如ATP或ADP。此外，PDH 使用PDH E2亲和柱层析纯化磷酸酶。这 纯化的酶被几种抗精神病药物更敏感地抑制 药物钙调素拮抗剂通过非竞争性的方式与以下 效力顺序：氟奋乃静大于氯丙嗪大于 硫利达嗪大于奋乃静大于三氟丙嗪 而不是精神病而PDH复合物的活性很低， 影响最小。克隆了近全长胞质转酮醇酶 并测定其核苷酸序列。肝脏特异性激活 用克隆的cDNA探针和多克隆的 抗体基于纯化的N-末端氨基酸序列， 蛋白质，线粒体PDH磷酸酶的cDNA克隆，NADP+特异性， 和NAD+特异性异柠檬酸脱氢酶也被鉴定， 表征了

As an ongoing project, the biochemical and molecular characteristics of thiamine-dependent enzymes involved in glucose metabolism were studied. These enzymes are mitochondrial pyruvate dehydrogenase (PDH) complex and `-ketoglutarate dehydrogenase (`-KGDH) complex, and cytosolic transketolase. These enzymes from bovine and rat tissues were purified to apparent homogeneity on SDS polyacrylamide gel electrophoresis and used for biochemical characterizations. A hydrophobic fluorescent probe, bis- ANS, potently inhibited the activity of PDH complex and `-KGDH complex. The conformational changes in these enzymes were demonstrative upon the addition of allosteric regulators such as ATP or ADP. In addition, PDH phosphatase was purified using PDH E2 affinity column chromatography. This purified enzyme was more sensitively inhibited by several antipsychotic drugs calmodulin antagonists via non-competitive manner with a following potency order: fluphenazine greater than chlorpromazine greater than thioridazine greater than perphenzine greater than triflupromazine greater than promazine. However, the activity of PDH complex was little or minimally affected. Near full-length cytosolic transketolase was cloned and its nucleotide sequence was determined. Liver-specific activation of transketolase was demonstrated using cloned cDNA probe and polyclonal anti-bodies. Based on the N-terminal amino acid sequences of the purified proteins, cDNA clones for mitochondrial PDH phosphatase, NADP+-specific, and NAD+-specific isocitrate dehydrogenases were also identified and characterized.