Leishmania amazonensis: specific labeling of amastigote cysteine proteinases by radioiodinated N-benzyloxycarbonyl-tyrosyl-alanyl diazomethane.
Living Leishmania amazonensis amastigotes were incubated with radioiodinated N-benzyloxycarbonyl-L-tyrosyl-L-alanyl diazomethane (Z-Tyr-AlaCHN2), an irreversible inhibitor of mammalian cathepsins B and L. Parasite lysates were subjected to electrophoresis in gelatin-containing sodium dodecyl sulfate-acrylamide gels to detect regions of proteolytic activity, and the distribution of the inhibitor was ascertained by autoradiography. Of the three main bands of proteolysis associated with cysteine proteinases, two, with apparent molecular weights of 28 and 31 kDa, were shown to be labeled. The third enzyme activity, detected at the 35-kDa region in substrate gels, was only faintly labeled. The distribution of labeled bands was similar when lysates of untreated parasites were electrophoresed and the gels incubated with the radioiodinated inhibitor. Under reducing conditions, the inhibitor bound to polypeptides of 29, 31, 32, and 34 kDa, of which the first and the last were the most intensely labeled. Polypeptides with the same apparent molecular weights were labeled when amastigote lysates were incubated with the 125I inhibitor. Uptake of radioactivity by the parasites was time and concentration-dependent and more than 80% of the total counts could be precipitated with trichloroacetic acid. Radioactivity associated with the amastigotes was quite stable after they were pulsed with labeled inhibitor and chased for up to 24 hr in inhibitor-free medium. Both total uptake and labeling of cysteine proteinases were markedly reduced in parasites preincubated with Z-Phe-AlaCHN2 prior to exposure to Z-Tyr(125I)-AlaCHN2. However, more radioiodinated inhibitor was taken up by parasites preincubated with cold inhibitor and chased in inhibitor-free medium, suggesting de novo synthesis or processing of inactive enzyme precursors.
Alfieri
S C
SC
Departamento de Parasitologia, Universidade de São Paulo, Brazil.
Pral
E M
EM
Shaw
E
E
Ramazeilles
C
C
Rabinovitch
M
M
D000818:Animals; D001345:Autoradiography; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D008970:Molecular Weight
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007457:Iodine Radioisotopes; C024918:benzyloxycarbonylphenylalanylalanine diazomethyl ketone; C057752:benzyloxycarbonyltyrosylalanine diazomethane; D003978:Diazomethane; D003546:Cysteine Endopeptidases