Studies on glutathione reductases from T. brucei, C. fasciculata, L. mexicana and T. cruzi have uncovered an unusual difference between the trypanosomatid enzyme and that of the mammalian host. Glutathione reductase from these parasitic organisms require a thiol-containing low molecular weight co-factor for activity. This co-factor would appear to mediate transfer of electrons from NADPH to the final electron acceptors GSSG, CoASSG or cystine, presumably by thiol-disulphide transhydrogenation reactions. The trypanosomatid enzyme is absolutely dependent on co-factor for activity, as activity in crude extracts is completely abolished by dialysis and restored by addition of dialysate. Significantly, dialysate. Significantly, dialysate extracts from a wide range of other biological materials did not restore enzyme activity, suggesting that the co-factor and enzyme are unique to trypanosomatids. Furthermore, co-factor does not serve as a substrate for yeast glutathione reductase. It is proposed to purify and determine the structure of the co-factor and, in the light of this information, determine the metabolic pathway by which these organisms synthesize this compound. In addition, glutathione reductase will be purified from C. fasciculata, characterized and its properties compared with mammalian enzyme and other trypanosomatid glutathione reductases. Glutathione metabolism in trypanosomatids will be re-evaluated to determine the relative concentrations GSH, GSSG, co-factor and other low molecular weight thiols in these organisims and to establish whether co-factor plays a role in other areas of metabolism, particularly as a substrate for """"""""glutathione peroxidase"""""""". Glutathione reductase in extracts of T. brucei is 70 times more sensitive to inhibition by melarsen oxide than the mammalian enzyme, and the mechanism of inhibition will be studied. Trypanosomatids are more sensitive to oxidant stress than their mammalian hosts and these studied should lead to a better understanding of the differences in glutathione metabolism of parasite and host and point to new potential targets for chemotherapeutic attack.
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