SYNTHESIS/ASSEMBLY-BRANCHED CHAIN KETOACID DEHYDROGENASE

合成/组装支链酮酸脱氢酶

• 批准号: 3237629
• 负责人: DEAN J DANNER
• 金额: $ 13.69万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-15 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3237629
• 关键词: complementary DNA; enzyme complex; fibroblasts; gel electrophoresis; gene expression; gene mutation; genetic library; genetic manipulation; human subject; human tissue; immunochemistry; immunoelectrophoresis; ketoacid; maple syrup urine disease; messenger RNA; mitochondria; monoclonal antibody; oxidoreductase; tissue /cell culture

The overall goal is to define the genes directing the synthesis of the mitochondrial enzyme complex, branched chain Alpha-ketoacid dehydrogenase [BCKD]. Inherited human mutations are known which specifically affect the function of BCKD known as Maple Syrup Urine Disease [MSUD]. These studies will increase our understanding of the defect and provide improved treatment for affected individuals. Immediate goals include: 1) Immunochemical characterization of BCKD proteins in human cells expressing defective function of the catalytic activity, 2) Biochemical characterization of the cytosolic synthesis and mitochondrial processing of the precursor proteins to BCKD, and 3) Construction of cDNA probes to study the genes and mRNAs directing the synthesis of these proteins. Polyclonal and monoclonal antibodies which specifically recognize the proteins of BCKD are used to screen mitochondrial proteins from fibroblasts derived from MSUD patients for cross-reactive material [CRM]. CRM- cells will be used to quantify BCKD proteins by crossed-immunoelectrophoretic methods. Western Blot analysis of two-dimensional gel separation of the mitochondrial proteins will also be used to study the mutant proteins. In vitro translation of RNA isolated from different sources, especially cells induced to overproduce mitochondrial proteins, will be used for the synthesis of these proteins. Processing studies will analysize conditions required for uptake of in vitro translated proteins by freshly isolated, respiratory competent mitochondria. Construction of cDNA probes involve using Lambdagt11 expression vectors with a human liver cDNA library to select clones producing the BCKD proteins. This involves to use of monospecific antibodies in selection of the clones and expansion of the selected clones in plasmid vectors.

总体目标是确定指导合成的基因 线粒体酶复合物，支链α-酮酸脱氢酶 [BCKD]。 已知遗传性人类突变特别影响 BCKD 的功能被称为枫糖浆尿病 [MSUD]。 这些研究 将增加我们对缺陷的理解并提供改进 为受影响的个人提供治疗。 近期目标包括：1) 人类细胞表达 BCKD 蛋白的免疫化学特征 催化活性功能缺陷，2) 生化 细胞质合成和线粒体加工的表征 BCKD 的前体蛋白，以及 3) 构建 cDNA 探针进行研究 指导这些蛋白质合成的基因和 mRNA。 多克隆 以及特异性识别 BCKD 蛋白的单克隆抗体 用于筛选来自成纤维细胞的线粒体蛋白 MSUD 患者存在交叉反应物质 [CRM]。 将使用 CRM- 单元 通过交叉免疫电泳方法定量 BCKD 蛋白。 二维凝胶分离的蛋白质印迹分析 线粒体蛋白也将用于研究突变蛋白。 在 从不同来源（尤其是细胞）分离的 RNA 的体外翻译 诱导过量产生线粒体蛋白，将用于 这些蛋白质的合成。 加工研究将分析条件 新鲜分离的体外翻译蛋白的摄取所需， 具有呼吸能力的线粒体。 cDNA探针的构建包括 使用 Lambdagt11 表达载体和人肝脏 cDNA 文库 选择产生 BCKD 蛋白的克隆。 这涉及到使用 单特异性抗体在克隆选择和扩增中的应用 质粒载体中选定的克隆。