Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica.
Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 micrograms/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN2). Iodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 micrograms/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-beta-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of 125I-labeled protease inhibitor (10 micrograms/ml) showed as many as eight radioactive bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons). Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.
de Meester
F
F
MacArthur Center for Molecular Biology of Parasitic Diseases, Rehovot, Israel.
Shaw
E
E
Scholze
H
H
Stolarsky
T
T
Mirelman
D
D
D000345:Affinity Labels; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000472:pathogenicity; D008970:Molecular Weight
D000345:Affinity Labels; D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases