AMINO ACID METABOLISM: ENZYME BIOGENESIS & MUTATION

这个项目保持了对细胞和分子的科学关注。 四种催化氨基酸与有机反应的酶的生物学 酸代谢及其遗传缺陷导致临床 显著代谢紊乱：鸟氨酸转氨甲基酶(OTC)； 丙酰辅酶A羧基酶(PCC)；甲基丙二酸辅酶A变位酶(MUT)；以及 胱硫醚β-合酶(CS)。拟议的调查旨在 具体地说：1)进一步刻画了细菌的生物发生途径 组中的三种线粒体基质酶(OTC、PCC、MUT) 亚基是核编码的，细胞质合成的，以及 翻译后的转移和加工，特别强调 负责线粒体识别、蛋白质分解的系统 切割，并组装成功能酶；2)阐明 翻译后，有限的蛋白分解在生理调节中的作用 仅限(CS)组的胞浆酶；3)使用克隆的cDNA定义 对于PCC、MUT和CS，其编码和侧翼的核苷酸序列 区域及其各自的内切酶图谱和它们的基因组 组织，并根据推导的完整氨基酸序列进行预测 这些多肽，它们的前导肽的结构，蛋白酶 裂解位点和辅因子结合位点；4)检验论文 人类遗传性或获得性这些酶的缺陷有时会导致 防止异常分拣或处理；以及5)扩大产前检测 利用细胞核将这些酶的遗传性缺陷提升到分子水平 酸性探针和Southern blotting程序以及从活检组织中获得的DNA 绒毛膜的。将采用许多实验方法。 包括：用全程序无细胞合成多肽 肝脏RNA或由其衍生的高度浓缩的个体RNA； 线粒体蛋白前体与完整线粒体的孵育 其组分；从受体的线粒体膜分离 线粒体蛋白前体，以及来自线粒体基质的 催化它们切割的内切酶(S)；随后是cDNA的合成 通过在适当的质粒载体中繁殖并通过 核酸杂交或序列匹配方案。这些研究 应该增加关于基本规则的新信息 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.