title: Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant.
abstract: Purified Escherichia coli Shiga-like toxin II variant (SLT-IIv) was characterized with regard to selected physical, chemical, and biological properties. N-terminal amino acid sequencing confirmed the identities of 33,000-, 27,500-, and 7,500-molecular-weight (MW) bands seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified SLT-IIv as the A subunit, A1 fragment, and B subunit, respectively. The arginine-serine bond between amino acids 247 and 248 in the A subunit was determined to be the site for proteolytic cleavage into A1 and A2 fragments. As with other SLTs, gel filtration chromatography of SLT-IIv gave estimates of the MW of holotoxin that were variable and less than predicted for a 1-A-subunit-5-B-subunit configuration. The MWs were estimated to be 40,000 and 43,000 by Sephacryl S-100 and Sephadex G-100 and less than 2,000 by Bio-Sil Sec-250 gel filtration chromatography. The isoelectric point of SLT-IIv holotoxin was 9.0. Cytotoxicity of SLT-IIv was destroyed by heating at 65 degrees C for 30 min and by incubation with 2-mercaptoethanol and dithiothreitol, but it increased 30-fold by incubation with trypsin, chymotrypsin, or pepsin and 2-fold by incubation with thermolysin. SLT-IIv cytotoxic activity was stable at neutral and alkaline pH values but was lost at pHs 3, 4, and 5. SLT-IIv was stable in fluid from the anterior and posterior small intestines of pigs but was not enterotoxic in pig intestinal loops. The smallest doses of SLT-IIv that inhibited protein synthesis in porcine endothelial cells and Vero cells were 0.1 ng and 0.1 fg, respectively.
authors.affiliation: Department of Veterinary Microbiology and Immunology, University of Guelph, Ontario, Canada.
authors.forename: D L
authors.initials: DL
authors.lastname: MacLeod
authors.forename: C L
authors.initials: CL
authors.lastname: Gyles
authors.forename: A
authors.initials: A
authors.lastname: Valdivieso-Garcia
authors.forename: R C
authors.initials: RC
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D004768:Enterotoxins / Q000737:chemistry*; D004926:Escherichia coli; D007106:Immune Sera / Q000276:immunology; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D022641:Shiga Toxin 2; D013552:Swine
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D007106:Immune Sera; D022641:Shiga Toxin 2