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  • 1
    ISSN: 1573-7284
    Keywords: Cellular cations ; Tissue culture cells ; Clostridium perfringens ; Enterotoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The morphological alterations (bleb-balloon formation) induced by Clostridium perfringens type A enterotoxin in HeLa and Vero cells were studied under defined extracellular conditions. The action of enterotoxin was found to depend on the temperature but not on energy metabolism. The morphological alterations by the enterotoxin occurred in phosphate buffered saline containing Ca2+ and Mg2+. Of the constituents of the buffered saline, Ca2+ was essential for the morphological alterations and other ions were interchangeable. The morphological alterations by the enterotoxin occurred also in 10 mM Hepes-Na buffer, pH 7.2 containing NaCl, KCl or choline chloride at a concentration of over ca. 50 mM and in 10 mM Hepes-Ca buffer, pH 7.2 containing CaCl2 at a concentration of over ca. 50 mM. Addition of sucrose to the medium prevented induction of the morphological alterations. The amount of sucrose necessary to protect the cells increased with increase in NaCl, KCl or CaCl2 concentration in the medium. A calcium ionophore A23187 mimicked the action of enterotoxin. Examination of the cation contents of the cells by atomic absorption spectrophotometry showed early and rapid increase of Ca 2+ during intoxication with concomitant changes in Na+, K+ and Mg2+ that reduced the ion concentration gradients between inside and outside of the cell present before toxin treatment. The mechanism of action of C. perfringens type A enterotoxin is discussed on the basis of these findings.
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  • 2
    ISSN: 1573-7284
    Keywords: Purification ; High performance liquid chromatography ; Clostridium perfringens ; Enterotoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract High enterotoxin-producing substrains of Clostridium perfringens type A were selected reproducibly as colonies having toxin-antitoxin haloes on agar plates of Duncan-Strong medium containing antitoxin serum. Enterotoxin from these sub strains was subjected to rapid purification by high performance liquid chromatography (HPLC). For this, the toxin was extracted by sonication from sporulating bacteria grown in Duncan-Strong sporulation medium, fractionated by ammonium sulfate (40% saturation) precipitation and differential solubilization and then purified by HPLC: gel permeation chromatography through a G2000SW column and ion-exchange chromatography on a Mono Q column. Purified toxin preparations had a similar specific activity (4.2 x 102 mouse MLD/mg protein) and homogeneity on polyacrylamide gel-electrophoresis to preparations obtained by conventional gel permeation through a Sephadex-G200 column. By further HPLC on a Mono Q column, minor nontoxin proteins were separated from the toxin without loss of the toxicity on a protein basis. The final yield of the purified toxin was about 15° of that in the bacterial extract. The two HPLC procedures each took only one hour.
    Type of Medium: Electronic Resource
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