GLUTARIC ACIDEMIA TYPE II--MUTANT ELECTRON TRANSFER FLAVOPROTEIN

拟议研究的目的是继续研究葡萄糖 酸血症II型，一种人类先天性氨基酸和脂肪酸氧化缺陷， 和ETF：泛醌氧化还原酶（ETF：QO）， 在某些患者中缺乏这种药物， 电子转移黄素蛋白（ETF）和 线粒体呼吸链 我们对这个问题的调查 已经持续了十多年，现在已经定位为 大大提高了我们对电子转移 影响之间的黄素和铁硫氧化还原中心的ETF：QO和 辅酶Q（泛醌）在线粒体内膜，以及如何 蛋白质的突变扰乱了它的功能并导致谷氨酸血症 第二类。 了解这种蛋白质的功能是至关重要的， 通过基因转移方法治疗疾病，当他们 开发，是要成功，有关基因的信息， 监管可能对理解为什么ETF：QO缺陷如此重要 常伴有严重的先天性肾囊肿。 我们现在 我手头上有超过95%的猪肉ETF：QO cDNA的五个克隆， BlueScript，以及两个克隆中约85%的人类ETF：QO cDNA。 本供资期的具体目标是：（a）完成猪肉分析 和人ETF：QO cDNA，（B）使用人探针来定义突变 ETF：QO缺陷型GA 2患者，以及（c）开发结构-功能图谱 的ETF：QO，确定与ETF，黄素和 铁硫氧化还原中心和泛醌。 使用定点 将进行副球菌ETF-QO的诱变， 解决不同结构域中氨基酸序列的变化 改变酶的功能。

The objective of the proposed research is to pursue studies on glutaric acidemia type II, a human inborn error of amino and fatty acid oxidation, and on ETF:ubiquinone oxidoreductase (ETF:QO), the protein which is deficient in some patients with the disease and which normally catalyzes electron transfer between electron transfer flavoprotein (ETF) and the mitochondrial respiratory chain. Our investigations on this subject have been ongoing for more than ten years, and are now positioned to substantially improve our understanding of how electron transfer is effected between the flavin and iron-sulfur redox centers of ETF:QO and coenzyme Q (ubiquinone) in the inner mitochondrial membrane, and how mutations of the protein perturb its function and lead to glutaric acidemia type II. Knowledge of how this protein functions is crucial if treatment for the disease by gene transfer approaches, when and if they are developed, is to be successful, information about the gene and its regulation may be important in understanding why ETF:QO deficiency is so frequently associated with severe congenital renal cystic disease. We now have on hand over 95 percent of pork ETF:QO cDNA in five clones in BlueScript, and about 85 percent of the human ETF:QO cDNA in two clones. Specific aims for this funding period are to (a) complete analysis of pork and human ETF:QO cDNA, (b) to use the human probe to define mutations in ETF:QO-deficient GA2 patients, and (c) to develop a structure-function map of ETF:QO, identifying domains which interact with ETF, the flavin and iron-sulfur redox centers, and ubiquinone. Experiments using site-directed mutagenesis of the ETF-QO of Paracoccus denitrificans will be performed to address issues of how changes in amino acid sequence in various domains alter enzyme function.