title: A radioiodinated peptidyl diazomethane detects similar cysteine proteinases in amastigotes and promastigotes of Leishmania (L.) mexicana and L. (L.) amazonensis.
abstract: 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.
authors.affiliation: Departamento de Parasitologia, Universidade de São Paulo, Brazil.
authors.forename: S C
authors.initials: SC
authors.lastname: Alfieri
authors.forename: J M
authors.initials: JM
authors.lastname: Balanco
authors.forename: E M
authors.initials: EM
authors.lastname: Pral
mesh_terms: 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
chemical_list: D004151:Dipeptides; D007457:Iodine Radioisotopes; C057752:benzyloxycarbonyltyrosylalanine diazomethane; D003978:Diazomethane; D003546:Cysteine Endopeptidases