carbobenzoxycarbonyl-l-phenylalanyl-l-alanine-d-diazomethane and leucine-methyl-ester

Trypanosoma cruzi epimastigotes were subjected to the lysosomotropic agents L-leucine methyl ester and ammonium chloride to determine their effects on the ultrastructure of the parasite. The lysosomotropic agents applied to epimastigotes caused a time-dependent alteration in the morphology of the cells marked by a 5-fold increase in the number of lysosomes. Continued exposure to ammonium chloride caused slight disruption of the reservosomes. The amino acid ester, however, while causing the parasite to swell after prolonged exposure (e.g., 24 h), had little effect on the reservosomes, the kinetoplast, or even the mitochondrion. A specific inhibitor of cysteine proteinases provided some protection for lysosomes from the effects of the amino acid ester. Although it is agreed that reservosomes are similar to endosomes, no lysosomal fusion with the reservosomes was observed. Acid phosphatase activity was observed only in lysosomes.

Topics: Ammonium Chloride; Animals; Cysteine Proteinase Inhibitors; Diazomethane; Leucine; Lysosomes; Microscopy, Electron; Organelles; Trypanosoma cruzi

L-leucine-methyl ester (Leu-OMe) kills Leishmania mexicana amazonensis amastigotes by a mechanism which requires proteolytic cleavage of the ester. N-Benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-AlaCHN2), a specific and irreversible inhibitor of cysteine proteinases, was used to characterize the enzymes involved in parasite destruction. It was shown that (1) amastigotes preincubated with micromolar concentrations of Z-Phe-AlaCHN2 survived challenge with Leu-OMe concentrations lethal to control parasites; (2) the proteolytic activity of 25- to 33-kDa cysteine proteinases in parasite lysates subjected to electrophoresis in gelatin-containing acrylamide gels was selectively inhibited in parasites pretreated with Z-Phe-AlaCHN2 and chased in inhibitor-free medium; and (3) cysteine proteinase activity was also inhibited in gels incubated with amino acid and dipeptide esters, possibly because the compounds were acting either as substrates (e.g., Leu-Leu-OMe) or as inhibitors (e.g., Ile-OMe) of the enzyme. The results support the involvement of low molecular weight cysteine proteinases in the destruction of amastigotes by Leu-OMe. Characterization of the structure and substrate specificity of the enzymes may permit the rational development of more selectively leishmanicidal amino acid derivatives.

Topics: Animals; Cysteine Endopeptidases; Diazomethane; Electrophoresis, Polyacrylamide Gel; Leishmania mexicana; Leucine; Macrophages; Mice; Protease Inhibitors; Substrate Specificity