Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica.
We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion.
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004549:Elastin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data
D003094:Collagen; D004549:Elastin; D003546:Cysteine Endopeptidases; C056423:histolysain