Radiolabelling studies using tritiated ornithine, arginine and lysine, together with the relevant amino acid decarboxylase enzyme assays, indicate that the epimastigote stage of Trypanosoma cruzi is unable to synthesise significant amounts of putrescine and cadaverine de novo, compared to the amounts of these diamines scavenged from the growth medium. Radiolabelled putrescine is readily incorporated into spermidine, spennine and the trypanosomatid-specific polyamine-glutathione conjugate trypanothione (W,iP-bis(g1utathionyl)spermidine). Likewise, radiolabelled cadaverine is incorporated into the analogous polyamines aminopropylcadaverine, bis(aminopropy1)cadaverine and another major unidentified component. Subsequent studies showed this major component to be a novel polyamine-thiol conjugate whose structure was confirmed by chemical synthesis to be hr , N9-bis(glutathiony1)aminopropylcadaverine (homotrypanothione). Kinetic analyses using recombinant 7: cruzi trypanothione reductase demonstrated that homotrypanothione disulphide is readily reduced by this enzyme with kinetic parameters similar to trypanothione disulphide, suggesting that it is a physiological substrate in vivo. Thus the epimastigote form of 7: cruzi differs significantly from the African trypanosomes and Leishmania in (a) being unable to synthesise significant amounts of diamines de novo, (b) converting significant amounts of putrescine and cadaverine to spermine and bis(aminopropyl)cadaverine, respectively and (c) the ability to synthesise homotrypanothione as well as trypanothione. The implications of these findings with respect to the prospective chemotherapy of Chagas' disease are discussed.

Trypanosoma cruzi epimastigotes take up exogenous [3H]putrescine and [3H]cadaverine by a rapid, high-affinity, transport system that exhibits saturable kinetics (putrescine Km 2.0 μM, Vmax 3.3 nmol/min per 108 cells; cadaverine Km 13.4 μM, Vmax 3.9 nmol/min per 108 cells). Putrescine transport is temperature dependent and requires the presence of a membrane potential and thiol groups for activity. Its activity is altered in response to extracellular putrescine levels and as the cells proceed through the growth cycle. This transporter shows high specificity for the diamines putrescine and cadaverine, but low specificity for the polyamines spermidine and spermine. The existence of rapid diamine/polyamine transport systems whose activity can be adjusted in response to the growth conditions is of particular importance, as they seem unable to synthesize their own putrescine [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019–1027].

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