INBORN ERRORS OF REDOX PROTEINS--GLUTARIC ACIDEMIA II

拟议研究的目的是研究戊二酸。 II型酸血症--人类先天的氨基酸和脂肪酸错误 新陈代谢，以及这种疾病中缺乏的蛋白质， 即电子转移黄素蛋白(ETF)和ETF：泛醌 氧化还原酶(ETF-QO)。这些蛋白质催化电子 从线粒体黄素蛋白脱氢酶转移到 主呼吸链。这些调查代表着 继续我们正在进行的研究 实验室工作十年以上，应填补专业空白 在我们对这些蛋白质如何与一种 另一种从初级黄蛋白转移电子的方法 线粒体基质脱氢酶生成泛醌 (辅酶Q)在内膜中，以及具体缺陷是如何 这些蛋白质扰乱正常功能并导致戊二酸 酸血症II型。我们现在手头有纯净的制剂 通用(中链)酰辅酶A脱氢酶(GAD)、ETF和 ETF-QO，抗三种蛋白的免疫血清，单克隆性 针对ETFα和β亚基的抗体，以及推定的 编码ETF-QO的部分人cDNA序列和其中一种 这两个ETF子单位。我们的具体目标是克隆完整 ETF-QO和两个ETF亚基的长度cDNA序列， 并使用这些探针确定特定的突变 导致戊二酸II型。单抗将是 针对所有三种多肽产生以定义其结构域 与初级脱氢酶和ETF-QO反应的ETF，并 定义与ETF反应的ETF-QO的域 泛醌；结构域的氨基酸序列将是 下定决心。关于功能和结构的信息 从这些方法发展而来的ETF和ETF-QO领域 然后被用来构建一系列突变蛋白，通过 体外诱变及其在大肠杆菌中的表达 关于初级氨基酸结构如何的具体假设 这些蛋白质中的一部分会影响它们的功能。

The objective of the proposed research is to investigate glutaric acidemia type II, a human inborn error of amino- and fatty-acid metabolism, and the proteins that are deficient in this disorder, namely electron transfer flavoprotein (ETF) and ETF: ubiquinone oxidoreductase (ETF-QO). These proteins catalyze electron transfer from mitochondrial flavoprotein dehydrogenases to the main respiratory chain. These investigations represent a continuation of the research that has been ongoing in our laboratories for more than ten years, and should fill majors gaps in our understanding of how these proteins interact with one another to transfer electrons from primary flavo-protein dehydrogenases of the mitochondrial matrix to ubiquinone (coenzyme Q) in the inner membrane, and how specific defects in these proteins perturb normal function and lead to glutaric acidemia type II. We now have on hand pure preparations of general (medium-chain) acyl-CoA dehydrogenase (GAD), ETF, and ETF-QO, immune serum to all three proteins, monoclonal antibodies against ETF alpha- and beta-subunits, and putative partial human cDNA sequences that encode ETF-QO and one of the two ETF subunits. Our specific objectives are to clone full length cDNA sequences for ETF-QO and the two ETF subunits, and to use these probes to determine the specific mutations that cause glutaric acidamia type II. Monoclonal antibodies will be produced against all three polypeptides to define the domains of ETF that react with primary dehydrogenases and ETF-QO, and to define the domains of ETF-QO that react with ETF and ubiquinone; the amino acid sequences of the domains will then be determined. The information on the functional and structural domains of ETF and ETF-QO developed from these approaches will then be utilized to construct a series of mutant proteins by in vitro mutagenesis and expression in E. coli, in order to address specific hypotheses about how the primary amino acid structure of these proteins influences their function.