SYNTHESIS/ASSEMBLY-BRANCHED CHAIN KETOACID DEHYDROGENASE

总的目标是定义指导合成的基因 线粒体酶复合体，支链α-酮酸脱氢酶 [BCKD]。遗传的人类突变是已知的，它特别影响 BCKD的功能称为枫糖浆尿病[MSUD]。这些研究 将增加我们对缺陷的了解，并提供改进的 对受影响个人的治疗。近期目标包括：1) 人细胞表达BCKD蛋白的免疫化学特性 催化活性功能缺陷，2)生化 人血清白蛋白胞质合成和线粒体加工特征 BCKD的前体蛋白，以及3)构建用于研究BCKD的c DNA探针 控制这些蛋白质合成的基因和mRNAs。多克隆 以及特异性识别BCKD蛋白的单抗 用于筛选来自于成纤维细胞的线粒体蛋白 MSUD患者的交叉反应材料[CRM]。将使用CRM-CELES 采用交叉免疫电泳法对BCKD蛋白进行定量。 蛋白质二维凝胶分离的Western Blot分析 线粒体蛋白也将用于研究突变蛋白。在……里面 不同来源的RNA的体外翻译，特别是细胞 诱导产生过量的线粒体蛋白，将用于 这些蛋白质的合成。处理研究将分析条件 是新分离的细胞摄取体外翻译蛋白所必需的， 呼吸能力强的线粒体。构建cDNA探针涉及到 将Lambdagt11表达载体与人肝cDNA文库结合使用 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筛选和克隆扩增中的应用 在质粒载体中筛选克隆。

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.