NADH Dehydrogenase (Complex I) of the African Parasite Trypanosoma brucei

9728124 Beattie和Kiaira本研究的总体目标是表征假定的鱼藤酮敏感性NADH脱氢酶或复合物I，其已被证明存在于原生动物布氏锥虫的胞体中。t.布氏杆菌在哺乳动物宿主和昆虫载体的血流中都具有双重生命周期，在哺乳动物宿主的血流中，生物体的能量需求通过糖酵解得到满足，在昆虫载体中，存在含有细胞色素的电子传递链。在缺乏氰化物敏感性呼吸的细长血流形式中存在低活性的鱼藤酮敏感性NADH脱氢酶的观察结果表明，在血流形式中存在NADH脱氢酶;但是，在此情况下，用特异性抗体进行的免疫印迹研究表明，复合物I的一个亚单位位于膜上，复合物的跨越区域不存在于血流形式的线粒体中，而存在位于复合物的外周区域中的两个亚基。这些结果导致了新的假设，即具有部分酶活性但缺乏具有质子泵送能力的膜部分的不完全NADH脱氢酶可能在形成完整的质子泵送复合物I之前组装在血流形式中。为了验证这一假设，我们在细长的血流和T。将建立和比较布鲁塞。将针对从基因序列预测的抗原性合成肽产生抗体，并将其用于免疫印迹线粒体膜和从放射性标记的锥虫中免疫沉淀这些亚基。NADH脱氢酶的亚基结合成多聚体复合物将通过复合物中相关亚基与抗亚基7的抗体的免疫共沉淀来检查，亚基7是定位于复合物的连接片段中的蛋白质。 本文比较了T.布氏杆菌将指示在形成完整的质子泵NADH脱氢酶之前，在细长血流形式中是否已经形成不完全复合物。 更深入地了解代谢，特别是T。布鲁氏菌病的研究将有助于设计更好和更适当的方法来控制锥虫病，这是一种对包括肯尼亚在内的撒哈拉以南非洲造成严重经济后果的牛疾病。此外，这些拟议的研究结果可能会提供有关这个最复杂的线粒体电子传递链复合体的生物起源的信息。

9728124 Beattie and Kiaira The overall goal of this research is to characterize the putative rotenone-sensitive NADH dehydrogenase, or complex I, that has been shown to be present in the mitochondrion of the protozoan Trypanosoma brucei. T. brucei has a dual life cycle in both the bloodstream of the mammalian host, where the energy needs of the organism are met by glycolysis, and the insect vector, where a cytochrome containing electron transport chain is present. The observation that low activities of rotenone-sensitive NADH dehydrogenase are present in the long slender bloodstream forms that lack cyanide-sensitive respiration suggested the presence of NADH dehydrogenase in the bloodstream forms; however, immunoblotting studies with specific antibodies indicated that a subunit of complex I located in the membrane-spanning region of the complex was not present in the mitochondria of the bloodstream forms while two of the subunits located in the peripheral region of the complex were present. These results have led to the novel hypothesis that an incomplete NADH dehydrogenase with partial enzymatic activity but lacking the membrane portion with proton pumping capability may be assembled in the bloodstream forms prior to the formation of the complete proton-pumping complex I. To test this hypothesis the presence of subunits localized in the peripheral and membrane arms of NADH dehydrogenase in the long slender bloodstream and procyclic forms of T. brucei will be established and compared. Antibodies will be generated against antigenic synthetic peptides predicted from the gene sequence and will be used to immunoblot mitochondrial membranes and to immunoprecipitate these subunits from radiolabeled trypanosomes. The association of the subunits of NADH dehydrogenase into a multimeric complex will be examined by co-immunoprecipitation of associated subunits in the complex with an antibody against subunit 7, a protein localized in the connecting fragment of the complex. A comparison of the number of s ubunits present in the complex of the long slender and procyclic forms of T. brucei will indicate whether an incomplete complex has been formed in the long slender bloodstream form prior to the formation of the complete proton pumping NADH dehydrogenase. A greater understanding of the metabolism, and especially the bioenergetics of T. brucei, will permit the design of better and more appropriate approaches for control of trupanosomiasis, a disease of cattle with severe economic consequences to sub-Saharan Africa including Kenya. Moreover, the results of these proposed studies may provide information about the biogenesis of this most complicated complex of the mitochondrial electron transport chain.