SYNTHESIS/ASSEMBLY-BRANCHED CHAIN KETOACID DEHYDROGENASE

总的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]。 已知遗传的人类突变特异性地影响 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了解，并提供改进的 为受影响的人提供治疗。 近期目标包括：1） 表达BCKD蛋白的人细胞中BCKD蛋白的免疫化学表征 催化活性功能缺陷，2）生化 的胞质合成和线粒体加工的表征 BCKD的前体蛋白; 3）cDNA探针的构建 指导这些蛋白质合成的基因和mRNA。 多克隆 和特异性识别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患者的交叉反应物质[CRM]。 CRM-将使用细胞 通过交叉免疫电泳方法定量BCKD蛋白。 蛋白质双向凝胶分离的Western Blot分析 线粒体蛋白也将用于研究突变蛋白。 在 从不同来源，特别是细胞中分离的RNA的体外翻译 诱导过度产生线粒体蛋白，将用于 合成这些蛋白质。 处理研究将分析条件 所需的摄取体外翻译的蛋白质的新鲜分离， 呼吸线粒体。 cDNA探针的构建涉及 使用具有人肝cDNA文库的Lambdagt 11表达载体， 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的选择和扩增中的应用 选择质粒载体中的克隆。

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.