Purification and immunologic characterization of a 30-kDa cysteine proteinase of Entamoeba histolytica.
A 30-kDa cysteine proteinase was purified from extracts of axenically grown trophozoites of Entamoeba histolytica strain HM1:IMSS. The purification procedure involved two consecutive chromatographic steps. Sequence analysis revealed high similarity with histolysin and with other 27-kDa cysteine proteinase. Western-blot analysis using F(ab')2 fragments of a polyclonal antibody raised against the purified enzyme revealed that when the amebic extract was prepared in the absence of proteinase inhibitors there were many positive bands ranging in relative molecular weight from 115 to 12.5 kDa, but when the extract was prepared in the presence of proteinase inhibitors there was only a single 30-kDa positive band. Similar results were obtained with immunoprecipitates. This phenomenon would suggest the formation of multimer aggregates of the 30-kDa cysteine proteinase after partial proteolysis.
Department of Experimental Medicine, Facultad de Medicina, Universidad Nacional Autónoma de México, México City.
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Montfort
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Pérez-Tamayo
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Pérez-Montfort
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González Canto
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Olivos
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004748:Entamoeba histolytica / Q000201:enzymology*; D007140:Immunoglobulin Fab Fragments; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D000913:Antibodies, Protozoan; D015853:Cysteine Proteinase Inhibitors; D007140:Immunoglobulin Fab Fragments; D003546:Cysteine Endopeptidases