ENZYME DEFECTS IN DISORDERS OF PROTOPORPHYRIN METABOLISM

原卟啉代谢紊乱中的酶缺陷

• 批准号: 2137865
• 负责人: JOSEPH R BLOOMER
• 金额: $ 22.64万
• 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 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2137865
• 关键词: chickens; cow; disease /disorder model; enzyme activity; enzyme induction /repression; enzyme mechanism; enzyme structure; hemoprotein structure; human subject; inborn metabolism disorder; laboratory rabbit; lyase; mass spectrometry; molecular pathology; nuclear magnetic resonance spectroscopy; nucleic acid sequence; orphan disease /drug; polymerase chain reaction; porphyrin metabolism; protein structure function; protoporphyria

The ongoing effort of this research project is to define the pathogenesis of the biochemical and clinical manifestations in the porphyrias. The focus is on protoporphyria, in which a deficiency of ferrochelatase (FC) activity causes an excess accumulation and excretion of protoporphyrin. The FIRST AIM is to identify the FC gene mutations which have occurred in human subjects with protoporphyria, correlating genotype with phenotype. The hypothesis is that CRIM-positive mutations will cause the greatest reduction in FC activity because the functional state of FC in mitochondria is that of a dimer. A large number of patients with a broad range of elevated protoporphyrin levels is available to examine this hypothesis. Lymphoblastoid cell lines will be established on the patients and used to measure FC activity and the level of immunoreactive FC protein. FC cDNA will be amplified by a specific reverse transcriptase-PCR method, and the PCR products will be sequenced to define the gene mutation. FC mRNA will also be quantified. Mutant FC cDNA clones will be examined in an expression vector in order, to confirm that the FC protein has lost enzyme activity. This analysis should define CRIM-positive and CRIM-negative mutations, in most situations identifying the FC gene defects which have occurred, and allow correlation of genotype with phenotype. The SECOND AIM is to identify the FC gene mutation in bovine protoporphyria. This is a naturally occurring animal model of protoporphyria which is expressed only in the homozygous state. Using a radiolabeled fragment of human FC cDNA, positive clones have been identified in a bovine placenta FC cDNA library and are being purified for sequencing. The expected homology of bovine FC with human FC should determine if bovine FC cDNA has been cloned. When the sequence for bovine FC cDNA has been determined, the FC gene defect in bovine protoporphyria will be examined in cells from homozygous animals. Once it has been defined, a rapid genetic test will be developed so that heterozygous animals can be identified. Most cattle with protoporphyria in the United States should have the same FC gene mutation as a result of common ancestry. The THIRD AIM is to identify a putative porphyrin-peptide conjugate in the bile of patients with variegate porphyria. Variegate porphyria shares the biochemical feature of excess biliary and fecal protoporphyrin excretion with protoporphyria, yet the clinical manifestations are very different. The bile of patients with variegate porphyria has shown the presence of a porphyrin moiety which has a retention time on HPLC intermediate to that of protoporphyrin and coproporphyrin. This may be a protoporphyrinpeptide conjugate. The compound will be isolated and partially purified from bile by HPLC, with structural analysis by mass spectrometry and NMR. The fluorescence spectrum of the compound will also be compared with the plasma porphyrin fluorescence marker which is unique for variegate porphyria. The identification and study of this compound may provide an understanding as to the differences in clinical manifestations between protoporphyria and variegate porphyria.

该研究项目的持续努力是定义发病机理 卟啉症中的生化和临床表现。这 重点是原态，其中铁胆管酶的缺乏（FC） 活性会导致原源性过多的积累和排泄。 第一个目的是确定发生在 具有原态的人类受试者，将基因型与表型相关。 假设是，压缩阳性突变将导致最大的突变 FC活性的降低是因为FC的功能状态 线粒体是二聚体的。大量患者 可以检查一系列升高的原磷脂水平 假设。将在患者上建立淋巴细胞细胞系 并用于测量FC活性和免疫反应性FC的水平 蛋白质。 FC cDNA将通过特定的逆转录酶-PCR扩增 方法，PCR产物将被测序以定义基因 突变。 FC mRNA也将进行量化。突变的FC cDNA克隆将是 在表达载体中检查以确认FC蛋白 酶活性失去了。该分析应定义Crim阳性和 在大多数情况下，识别FC基因 发生的缺陷，并允许基因型与 表型。第二个目的是识别牛中的FC基因突变 原质畸形。这是一种天然发生的动物模型 仅在纯合状态表达的原质畸形。使用 人类FC cDNA的放射标记的片段，阳性克隆已经是 在牛胎盘FC cDNA库中鉴定，并正在纯化 测序。牛FC与人类足球俱乐部的预期同源性应该 确定牛FC cDNA是否已克隆。当牛的序列 已经确定了FC cDNA，牛原畸形中的FC基因缺陷 将在纯合动物的细胞中检查。一旦已经 定义，将开发快速基因检测，以便杂合子 可以识别动物。大多数牛在联合 国家应具有相同的FC基因突变。 祖先。第三个目的是识别假定的卟啉肽 杂草斑点患者的胆汁中的结合物。斓 斑岩具有多余的胆道和粪便的生化特征 原源性原源性用原畸形排泄，但临床 表现非常不同。杂色患者的胆汁 卟啉症显示出存在卟啉部分的存在 HPLC中间的保留时间， 副磷脂。这可能是一种原磷脂肽结合物。这 化合物将被隔离并通过HPLC从胆汁中部分纯化， 质谱和NMR的结构分析。荧光 该化合物的光谱也将与血浆卟啉进行比较 荧光标记物对于杂色斑岩是独特的。这 对该化合物的识别和研究可能会提供理解 与原畸形和 Variegate Porphyria。