A radioiodinated peptidyl diazomethane detects similar cysteine proteinases in amastigotes and promastigotes of Leishmania (L.) mexicana and L. (L.) amazonensis.
Cysteine proteinase activities were examined in lesion amastigotes as well as in stationary-phase promastigotes of Leishmania (L.) mexicana and Leishmania (L.) amazonensis isolates. Enzyme detection in gelatin gels revealed that amastigotes of three L. (L.) mexicana isolates (M379, IOC-0561, and IP) shared similar proteinases, including the multiple low-molecular-weight (25-35 kDa) cysteine proteinases. High cysteine proteinase activity was also observed in L. (L.) amazonensis amastigotes, but the banding profile was different in two of the isolates examined. Promastigotes displayed fewer low-molecular-weight proteinase bands, and these were much less intense as compared with those of lesion amastigotes. Independently of the Leishmania isolates and developmental stages examined, incubation of the parasites for 2 h with 0.2 microM radioiodinated N-benzyl-oxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[125I]-AlaCHN2) markedly and selectively labeled bands comigrating with the 28- and 31-kDa cysteine proteinases. Under reducing conditions, labeling was associated with four similar polypeptides (29-34 kDa), which were also detected when incubation with Z-Tyr[125I]-AlaCHN2 was carried out after cell lysis. Labeling was completely abolished if lysates were first incubated with 20 microM E-64 and then exposed to the 125I-tagged inhibitor, thus confirming the specificity of the compound toward cysteine proteinases.
Alfieri
S C
SC
Departamento de Parasitologia, Universidade de São Paulo, Brazil.
Balanco
J M
JM
Pral
E M
EM
D000818:Animals; D001345:Autoradiography; D003546:Cysteine Endopeptidases / Q000032:analysis*; D003978:Diazomethane / Q000031:analogs & derivatives; D004151:Dipeptides; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D007894:Leishmania mexicana / Q000201:enzymology* / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C
D004151:Dipeptides; D007457:Iodine Radioisotopes; C057752:benzyloxycarbonyltyrosylalanine diazomethane; D003978:Diazomethane; D003546:Cysteine Endopeptidases