Detection by HPLC of a trypanothione synthetase activity in vitro from Entamoeba histolytica

We have previously demonstrated the presence of glutathione-spermidine (Csp ) and trypanothione [T(SH)(2)] from Entamoeba histolytica trophozoites, on the basis of results obtained with acid extracts purified by Florisil and D EAE-cellulose, derivatized with the fluorescent reagent monobromobimane and separated by HPLC, Gsp was originally found in Escherichia coli and later in trypanosomatids such as Trypanosoma cruzi, T, brucei, T. congolense and the insect trypanosomatid Crithidia fasciculata, along with the novel compo und T(SH)(2), N-1,N-8-bis(glutathionyl)-spermidine. Here we demonstrate the presence of a T(SH)(2) synthetase activity in partly purified extracts fro m Entamoeba histolytica HK9, incubated at two pH values (6.5 and 7.5) with reduced glutathione (GSH), spermidine and ATP, in the presence of Mg2+ at d ifferent time intervals, The thiol products were detected by HPLC in picomo le amounts and compared with commercial Gsp and T(SH)(2) standards. We have used also an extract of Crithidia luciliae as a reference, to compare our results with C, fasciculata, in which the presence of this enzyme has previ ously been demonstrated and was later purified and separated into two synth etase activities from the same source: one for Csp and the other for T(SH)( 2). The presence of a T(SH)(2) synthetase activity in Entamoeba histolytica means that this protozoan has a similar metabolism to that of the trypanos omatids and opens the possibility of establishing a rational drug design ag ainst this human parasite.