REGULATION OF THIAMINE-DEPENDENT ENZYMES INVOLVED IN GLUCOSE METABOLISM

参与葡萄糖代谢的硫胺依赖性酶的调节

• 批准号: 3745202
• 负责人: B J SONG
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3745202
• 关键词: Krebs' cycle; SDS polyacrylamide gel electrophoresis; animal tissue; conformation; enzyme activity; enzyme complex; enzyme induction /repression; enzyme inhibitors; enzyme structure; fluorescent dye /probe; glucose metabolism; isocitrate dehydrogenase; mitochondria; molecular cloning; oxoglutarate dehydrogenase; phosphorylation; posttranslational modifications; protein purification; protein sequence; pyruvate dehydrogenase; site directed mutagenesis; transaldolase /transketolase

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.

作为一个正在进行的项目，生物化学和分子特征 对参与糖代谢的硫胺依赖酶进行了研究。 这些酶是线粒体丙酮酸脱氢酶(PDH)复合体和 酮戊二酸脱氢酶(`-KGDH)复合体和胞浆 转酮醇酶。这些来自牛和大鼠组织的酶被提纯到 SDS-聚丙烯酰胺凝胶电泳法的表观均一性及其应用 用于生化鉴定。一种疏水性荧光探针，双- ANS能有效地抑制PDH复合体和`-KGDH复合体的活性。 这些酶的构象变化是关于 添加变构调节剂，如三磷酸腺苷或二磷酸腺苷。此外，PDH 用PDH-E2亲和柱层析纯化磷酸酶。这 纯化的酶更敏感地被几种抗精神病药物抑制 以非竞争性方式开发钙调蛋白拮抗剂 药效顺序：氟奋乃静大于氯丙嗪 硫代咪嗪大于奋乃静大于三氟丙嗪 比异丙嗪更有效。然而，PDH复合体的活性很低或 受影响最小。近全长胞浆转酮醇酶基因的克隆 并测定了其核苷酸序列。肝脏特异性激活 用克隆的cdna探针和多克隆技术鉴定转酮醇酶 抗体。根据纯化产物的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+特异的异柠檬酸脱氢酶也被鉴定出来， 特色化的。