RESUMO
Radioiodinated N-benzyloxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[l25I]-AlaCHN2) was previously shown to selectively label two (28 and 31 kDa) Leishmania mexicana cysteine proteinases common to both the promastigote and the amastigote stages. Here we have confirmed the specificity of the compound towards two similar enzymes of axenic L. mexicana amastigotes and demonstrated that lesion amastigotes, axenic amastigotes and stationary promastigotes internalized the l25I-labeled inhibitor at different rates. Uptake of Z-Tyr[l25I]-AlaCHN2 by the parasites, which was not significantly modified by changing the medium pH, was clearly correlated with the binding of the compound to the 28- and 3l-kDa cysteine proteinases, as judged by the specificity of enzyme labeling in gelatin gels and the recovery of 75 per cent or more parasite-associated radioactivity in TCA-insoluble fractions. For all three developmental stages, uptake markedly increased with time and linearly up to 60 min, but throughout the period examined, radiolabel accumulation occurred more efficiently in amastigotes. By 5 h, when values were near or at saturation, radioactivity (in cpm/mug of total protein) associated with lesion amastigotes was 1.8- and 2.9-times that recovered from axenic amastigotes and stationary promastigotes, respectively. Pulse-chase experiments, in which cysteine proteinases were fully blocked with Z-Phe-AlaCHN2 prior to the pulse with Z-Tyr[l25I]-AlaCHN2, showed that labeling of the amastigote enzymes could be partially restored, whereas labeling of promastigote proteinases could not, after a 5-h chase period in inhibitor-free medium.