AMINO ACID METABOLISM: ENZYME BIOGENESIS AND MUTATION

本申请中描述的研究继续侧重于 哺乳动物氨基酸三种酶的细胞和分子生物学 和有机酸代谢，其遗传缺陷导致 人类临床上重要的代谢性疾病：鸟氨酸 转氨甲基酶(OTC)、丙酰辅酶A羧基酶(PCC)和 甲基丙二酰辅酶A变位酶(MUT)。建议的实验是设计的1) 为了进一步描述细胞质前体通过何种途径 这些线粒体基质酶被靶向、移位、加工 并组装起来，特别强调细胞质因子的作用， 易位复合体的结构，以及参与 线粒体蛋白组装中的热休克蛋白或伴侣蛋白：2) 纯化、鉴定、克隆和测序这两个线粒体 负责处理非处方药前体的蛋白酶；3)澄清 生物素化在alphaPCC运输和加工中的作用；以及4) 应用发展到更好的信息、技术和试剂 对这些遗传性缺陷的分子基础的理解 为改善这些疾病的诊断方法而努力 活体内和子宫内。对于计划中的许多研究，将军 实验设计将涉及在体外重建不同的 大鼠肝脏线粒体蛋白的进口和加工方面。 所采用的具体技术将包括：分离完整的， 功能性大鼠肝线粒体及其部分：蛋白质 用常规技术和亲和技术进行纯化；抗体 产生和免疫沉淀；信使核糖核酸和基因组DNA的制备 来自患者材料；南部、北部和西部印迹：和 聚合酶链式反应扩增、克隆、操纵和 CDNA和基因组DNA的测序。这些研究将提供新的 关于胞内蛋白质分选基本过程的信息 和运输，并应提高对这一系统作用的理解 及其在正常动态平衡和病理生理状态下的成分 人类遗传病。

The research described in this application continues to focus on the cellular and molecular biology of three enzymes of mammalian amino acid and organic acid metabolism whose inherited deficiencies lead to clinically significant metabolic disease in man: ornithine transcarbamylase (OTC), propionyl CoA carboxylase (PCC), and methylmalonyl CoA mutase (MUT). The experiments proposed are designed 1) to further delineate the pathway by which the cytoplasmic precursors of these mitochondrial matrix enzymes are targeted, translocated, processed and assembled, with specific emphasis on the role of cytoplasmic factors, the structure of the translocation complex, and the participation of heat-shook proteins or chaperonins in mitochondrial protein assembly: 2) to purify, characterize, clone, and sequence the two mitochondrial protease responsible for processing the OTC precursor; 3) to clarify the role of biotinylation in the transport and processing of alphaPCC; and 4) to apply the information, techniques, and reagents developed to a better understanding of the molecular bases of inherited deficiencies of these enzymes and to the improvement of diagnostic methods for these disease in vivo and in utero. For many of the studies planned, the general experimental design will involve the reconstitution in vitro of various aspects of rat liver mitochondrial protein import and processing. Specific techniques employed will include: isolation of intact, functional rat liver mitochondria and fractions thereof: protein purification by both conventional and affinity techniques; antibody generation and immunoprecipitation; preparation of mRNA and genomic DNA from patient material; Southern Northern, and Western blotting: and polymerase chain reaction amplification, cloning, manipulation and sequencing of cDNA and genomic DNA. These studies will provide new information on the fundamental processes of intracellular protein sorting and transport and should improve understanding of the role of this system and its components in both normal homeostasis and the pathophysiology off human genetic disease.