Proteinase activities of Entamoeba histolytica cytotoxin☆

Abstract 

The proteinase activity of the low molecular weight cytotoxin of Entamoeba histolytica was correlated with its cytotoxicity. Gel-filtered amebal toxin (mol wt 10–30,000) proteinase activities could be assayed on azocasein at pH 6 or on hemoglobin at pH 4.5. Proteinase activity was inhibited by serum fractions, thiol reagents, heavy metals, leupeptin, and antipain. The cytotoxic activity of gel-filtered amebal toxin was inhibited by serum fractions, leupeptin, and antipain. Increased proteinase and cytotoxic activity was produced by treatment with cysteine. These data support the action of a thiol proteinase in the production of cytopathic effects by gel-filtered amebal toxin in vitro. The cytotoxic and proteinase activities were further purified using a combination of column chromatography and preparative isoelectric focusing. Two low molecular weight cytotoxins with proteinase activity on both substrates were isolated. The major cytotoxin had an isoelectric point of 4.5 and a molecular weight of 22,000; the other cytotoxin had a basic isoelectric point. These substances may be cathepsin B-like proteinase and elastase or cathepsin G-like proteinases of E. histolytica. The major proteinase activity in the high molecular weight fraction was not cytotoxic. The isoelectric points of the high molecular weight proteinase activities corresponded to that of mammalian cathepsin D. The major cell rounding cytotoxic activity of E. histolytica extracts in vitro is probably due to the activity of a thiol-containing cathepsin B-like proteinase.

Abbreviations:  α₂M, α₂-macroglobulin, α₁AP, α₁-antiprotease, CPE, cytopathic effect, CPEep, cytopathic effect titration end point, DEAE, diethylaminoethyl, I_D50, 50% inhibition, I_A50, 50% activation, IgC, immunoglobulin C, MEM, minimal essential medium, PCMB, p-chloromercuribenzoate, PCMPS, p-chloromercuriphenyl sulfonate, PSF, Puck's normal saline F, TU, unit of cytotoxic activity

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