African trypanosomes are the causative agents of sleeping sickness in humans and of nagana in domestic livestocks in tropical Africa. Their development in the host bloodstream is entirely dependent on glycolysis for production of energy. Since only two ATP molecules can be gained from glycolytic conversion of each glucose molecule a vigorous glycolysis proceeding at a rate fifty times higher than that in mammals is needed to sustain the rapid growth and has been observed in at least one of the species Trypanosoma brucei. This is apparently made possible by the aggregation of glycolytic enzymes in the small organelle, the glycosome, in the parasite. Our preliminary studies have been able to cross-link all the T. brucei glycosomal proteins across intact glycosomal membrane and still retain the glycolytic enzymic activities. This cross-linked enzyme complex is capable of converting glucose all the way to Alpha-glycerophosphate without a lag phase; suggesting direct substrate channeling among some of the glycolytic enzymes. Future research plans call for further refinement of the cross-linking techniques and in-depth kinetic studies of the cross-linked enzyme-complex-catalyzed-chain-reactions to identify the enzymes sharing true substrate channelings. Substrate analogs will be tested on the channeled enzmes to see if their products trapped in the channel will turn into blockers of the next enzymic action thus shut down glycolysis for the purpose of antitrypanosomal chemotherapy. Antibodies against the cross-linked enzyme complex will be provided for a search of precursors of the glycolytic enzymes and the m-RNA encoding the precursors in the cytoplasm, because glycosomes do not contain nucleic acids. The process of insertion of the precursors into glycomsome will be ivestigated. c-DNA copied from the m-RNA will be cloned and used to identify the glycolytic enzyme genes in the nucleus. Similar cross- linking studies will be performed on the glycosomes of the insect (procyclic) form of T. brucei, which have lower glycolytic enzymic activities but have at least two new enzymes, to detect possible cross-linking and channeling between the old and new enzymes. The old enzymes in procylic glycosome can be removed by the antibodies to leave the new proteins to make new antibodies, which, in turn, will be useful in monitoring the emergence of new glycosomal proteins in the transformation of T. brucei from bloodstream to procyclic form. Cloned glycolytic enzyme genes will be useful probes to look for possible glycolytic enzyme gene amplifications in the transition of T. brucei from insect to bloodstream.
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