ENZYME DEFECTS IN DISORDERS OF PROTOPORPHYRIN METABOLISM

原卟啉代谢紊乱中的酶缺陷

• 批准号: 3227897
• 负责人: JOSEPH R BLOOMER
• 金额: $ 22.11万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3227897
• 关键词: antioxidants; autosomal dominant trait; cell membrane; complementary DNA; congenital hepatic porphyria; cow; enzyme induction /repression; enzyme linked immunosorbent assay; enzyme mechanism; fibroblasts; heme; human tissue; hybrid cells; iron disorder; laboratory rat; liposomes; liver disorder; liver metabolism; molecular pathology; orphan disease /drug; porphyrin biosynthesis; protoporphyria; tissue /cell culture; zinc

This research focuses on understanding the basis for the biochemical abnormalities in the porphyrias, and defining the pathogenesis of liver disease in some of these disorders. Three areas will be examined: (1) Particular attention is paid to protoporphyria, a disorder in which there is a defect in ferrochelatase, the terminal enzyme of the heme biosynthesis pathway. The disease is inherited in humans as an autosomal dominant trait. The disease has also been found in cattle, where it is inherited in a homozygous fashion. A kinetic ELISA assay has been developed which will be used to quantify the amount of ferrochelatase protein in human and bovine tissues. The possibility exists that protoporphyria in humans results from more than one type of mutation which may be differentiated by the amount of immunoreactive protein present. Peptide analysis of ferrochelatase will be performed in order to compare the normal and protoporphyria enzymes. Initial studies will be done with the bovine enzymes, since this is facilitated by the homozygous defect in ferrochelatase in bovine protoporphyria. Emphasis will be on identifying the peptide sequences which contain the active site of the enzyme. Molecular studies of the ferrochelatase defect will also be initiated. Bovine cDNA libraries obtained from both normal and protoporphyria tissues will be screened, and a clone containing the cDNA coding for ferrochelatase will be isolated The cDNA will be sequenced in order to infer the complete peptide sequences for normal and protoporphyria enzymes. Comparison of the primary sequences will determine whether bovine protoporphyria is due to a point mutation in the structural gene for ferrochelatase. The cDNA for normal bovine ferrochelatase will also be used to probe for human ferrochelatase cDNA and initiate studies of the molecular defect in the human condition. (2) The activity of ferrochelatase in the mitochondrial is likely to be influenced by its lipid environment. This will be examined using cultured cells which are incubated with media containing specific fatty acids. The lipid effect will also be examined by incorporating purified ferrochelatase into liposomes of defined composition. The results may suggest a dietary means of modulating ferrochelatase activity in vivo (3) A mechanism for hepatocellular damage in porphyria cutanea tarda is postulated in which the oxidation of uroporphyrinogen to uroporphyrin in the presence of molecular oxygen and iron causes the formation of activated oxygen species which damage cellular membranes. This will be tested using a model system composed of liposomes, uroporphyrinogen, and various metals. The interaction of antioxidants with this system will also be studied. The results of this of this study may provide a rationale for using antioxidant therapy in porphyria cutanea tarda in humans.

这项研究重点是理解 卟啉的生化异常，并定义 其中一些疾病中肝病的发病机理。 三 将检查区域：（1）特别注意 原质畸形，一种缺陷的疾病 铁螯合酶，血红素生物合成的末端酶 路径。 该疾病是人类遗传为常染色体 主要特征。 该疾病也在牛中发现 它是以纯合的方式继承的。 动力学ELISA分析有 已开发用于量化的数量 人和牛组织中的铁链蛋白酶蛋白。 这 存在人类原质质的可能性更多 比一种可以通过数量区分的突变类型的突变 存在免疫反应性蛋白。 肽分析 将执行铁螯合酶，以比较正常情况和 原晶酶。 最初的研究将与 牛酶，因为这是由纯合缺陷促进的 在牛原畸化中的铁螯酶中。 重点将在 识别包含包含活性位点的肽序列 酶。 铁铬酶缺陷的分子研究将 也可以启动。 牛cDNA文库从两个正常 将筛选和原核组织，并包含一个克隆 将分离为铁铬酶编码的cDNA将分离出cDNA将 进行测序以推断出的完整肽序列 正常和原态酶。 比较 序列将确定牛原生质是否是由于 铁螯合酶的结构基因中的点突变。 这 正常牛铁胆管酶的cDNA也将用于探测 用于人铁胆管酶cDNA并开始研究分子 在人类状况下的缺陷。 （2）铁铬酶的活性 在线粒体中可能会受到其脂质的影响 环境。 这将使用培养的单元进行检查 与含有特定脂肪酸的培养基孵育。 脂质 效果也将通过合并纯化来检查 铁螯合酶进入定义成分的脂质体。 结果 可能建议一种调节铁铬酶活性的饮食手段 体内（3）卟啉症肝细胞损伤的机制 假设Cutanea Tarda在其中氧化 在分子存在的情况下 氧和铁导致活化氧的形成 哪些损害细胞膜。 这将使用模型进行测试 由脂质体，泌尿吡啶原和各种金属组成的系统。 抗氧化剂与该系统的相互作用也将是 研究。 这项研究的结果可能会提供理由 用于在人类中使用卟啉症的porphyria cutanea tarda。