African trypanosomes are the causative agents of sleeping sickness in man and of nagana in domestic livestocks in tropical Africa. Their development in host blood depends entirely on glycolysis in the microbody organelles, the glyscosomes, for energy. In Trypanosoma brucei, all the glycolytic enzymes in the organelle can be crosslinked together without loss of activities. There is, however, little substrate channeling among the crosslinked enzymes. The glysocomal membrane compartmentalizes the glycolytic process, its selective permeabilities to sugar phosphates, ADP and NAD+ performs unique functions, such as synthesis of intraglycosomal ATP from exogeneous ADP and alpha-glycerol phosphate. The glycosomal membrane and two integral membrane proteins of 26K and 24K were recently purified. An in vitro assay of insertion of precursor proteins into glycosome has been established. The process is dependent on ATP and inhibitable by suramin. The chemically synthesized C-terminal peptide of glycosomal 3-phosphoglycerate kinase (PGK) postulated as essential for PGK insertion, was, however, without effect. Glycosomal hexokinase, glycerol kinase and phosphoglucoisomerase were purified to homogeneity. They and phosphoenolpyruvate carboxykinase and malate dehydrogenase of the procyclic form glycosome will be partly sequenced for gene cloning. The cloned genes will be sequenced, transcribed and translated in vitro for the insertion assay. Antibodies generated against the purified enzymes will be used to monitor precursor insertions. Monoclonal antibodies to glycosomal membrane proteins will be made and tested against protein insertion. A recent indication that polyamine deficiency may trigger transformation of T. brucei from long-slender to short-stumpy form prompted us to clone and sequence the T. brucei ornithine decarboxylase (ODC) gene. The gene was transcribed and translated in vitro and the protein product identified by ODC antibody. The in vitro transformation of T. brucei will be established, and polyamines and ODC inhibitors will be tested. Cloned ODC genes and ODC antibodies will monitor the expression of ODC during transformation. Similarly, the genes and antibodies of the glycolytic enzymes will examine the mechanisms of genetic control of glycosomal composition during T. brucei transformation. By understanding the regulation and mechanism of glycosomal biogenesis one can try to interfere and confuse these processes in the parasite for antitrypanosomal therapy.
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