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.
