Leishmania amazonensis: involvement of cysteine proteinases in the killing of isolated amastigotes by L-leucine methyl ester.
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.
Alfieri
S C
SC
Institut Pasteur, Paris, France.
Shaw
E
E
Zilberfarb
V
V
Rabinovitch
M
M
D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003978:Diazomethane / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007894:Leishmania mexicana / Q000187:drug effects / Q000201:enzymology*; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D011480:Protease Inhibitors; C024918:benzyloxycarbonylphenylalanylalanine diazomethyl ketone; C030365:leucine methyl ester; D003978:Diazomethane; D003546:Cysteine Endopeptidases; D007930:Leucine; C024974:E 64