AMINO ACID METABOLISM: ENZYME BIOGENESIS & MUTATION

该项目保留了其科学重点的细胞和分子 催化氨基酸和有机物反应的四种酶的生物学 酸代谢，其遗传缺陷导致临床 显著代谢紊乱：鸟氨酸转氨甲酰酶（OTC）; 甲基丙二酰辅酶A羧化酶（MUT）;和 胱硫醚β-合酶（CS）。 拟议的调查旨在 具体地说：1）进一步表征生物起源的途径， 组中的三种线粒体基质酶（OTC，PCC，MUT），其 亚基是核编码的，细胞质合成的， 在离职后重新安置和处理，特别强调 负责线粒体识别、蛋白水解 裂解和组装成功能酶; 2）阐明 翻译后，有限的蛋白水解的生理调节， 仅胞质酶组（CS）; 3）使用克隆的cDNA定义 对于PCC、MUT和CS，它们的编码和侧翼序列的核苷酸序列 区域，它们各自的核酸内切酶图谱，以及它们的基因组 组织，并从衍生的完整氨基酸序列预测 这些多肽、其前导肽的结构、蛋白酶 切割位点和辅因子结合位点; 4）测试该理论， 这些酶的遗传性或后天缺乏有时会导致 从异常分选或加工;和5）延长产前检测 遗传性缺乏这些酶的分子水平使用核 酸性探针和Southern印迹程序以及从活检中获得的DNA 绒毛膜绒毛 将采用多种实验方法 包括：无细胞合成多肽， 肝RNA或由其衍生的高度富集的单个mRNA; 将线粒体蛋白前体与完整线粒体孵育，或 其组分;从线粒体膜分离用于 线粒体蛋白质前体，并从线粒体基质的 内切蛋白酶催化其裂解;随后合成cDNA 通过它们在适当的质粒载体中的增殖和通过 核酸杂交或序列匹配方案。 这些研究 应该增加新的信息， 这些模型酶系统，以及它们在遗传或 获得性疾病状态。

This project retains its scientific focus on the cellular and molecular biology of four enzymes which catalyze reactions of amino acid and organic acid metabolism and whose inherited deficiency leads to clinically significant metabolic disorders: ornithine transcarbamylase (OTC); propionyl CoA carboxylase (PCC); methylmalonyl CoA mutase (MUT); and cystathionine Beta-synthase (CS). The proposed investigations are aimed specifically at: 1) characterizing further the pathway of biogenesis of the three mitochondrial matrix enzymes in the group (OTC, PCC, MUT) whose subunits are nuclear-encoded, cytoplasmically synthesized, and posttranslationally translocated and processed, with particular emphasis on the systems responsible for mitochondrial recognition, proteolytic cleavage, and assembly into functional enzymes; 2) clarifying the role of posttranslational, limited proteolysis in the physiologic regulation of the only cytosolic enzyme of the group (CS); 3) defining, using cloned cDNAs for PCC, MUT, and CS, the nucleotide sequence of their coding and flanking regions, their respective endonuclease maps, and their genomic organization, and predicting from derived complete amino acid sequence of these polypeptides, the structure of their leader peptides, protease cleavage sites, and cofactor binding sites; 4) testing the thesis that inherited or acquired deficiency of these enzymes in man sometimes results from aberrant sorting or processing; and 5) extending prenatal detection of inherited deficiency of these enzymes to the molecular level using nuclei acid probes and Southern blotting procedures and DNA obtained from biopsies of chorionic villi. Numerous experimental approaches will be employed including: cell-free synthesis of polypeptides programmed with total hepatic RNA or highly enriched individual mRNAs derived therefrom; incubation of mitochondrial protein precursors with intact mitochondria or fractions thereof; isolation from mitochondrial membranes of receptors for mitochondrial protein precursors, and from mitochondrial matrix of the endoprotease(s) which catalyze their cleavage; synthesis of cDNAs followed by their propagation in appropriate plasmid vectors and identification by nuclei acid hybridization or sequence-matching protocols. These studies should add new information concerning both the fundamental regulation of these model enzymatic systems, and their modulation in inherited or acquired disease states.