Takashi Uemura

Effects of Ca2+ and other cations on the action of Clostridium perfringens enterotoxin.

We investigated the role of extracellular Ca2+ in the Clostridium perfringens enterotoxin-induced alteration of the permeability of the plasma membrane. Enterotoxin released 86Rb and 51Cr from the Vero cells preloaded with the isotope. In the presence of EGTA, however, it released 86Rb but not 51Cr. The binding of enterotoxin to the cells was not influenced by Ca2+ or Mg2+. The effects of various cations on the enterotoxin-induced 51Cr release was also studied. The release depended on extracellular Ca2+ but not on Mg2+; it was inhibited by each of Zn2+, La3+ and Co2+. Zn2+ and Co2+ also inhibited 51Cr release caused by the enterotoxin previously bound to the cell membrane. In contrast, antibody against enterotoxin did not neutralize the toxin once it was bound to the Vero cells. When the cells were treated with enterotoxin, 45Ca influx occurred and reached the plateau in a few minutes, as did 86Rb release.

Trypsinization of derivatives of Clostridium perfringens enterotoxin

Three proteins, named P1, P2 and P3, were separated from purified Clostridium perfringens enterotoxin by high performance liquid chromatography (HPLC) on a DEAE‐5PW column. The relative biological activities of P1, P2 and P3 on protein basis determined by the African green monkey kidney (Vero) cell staining assay were 3·0, 1·9 and 1·0, respectively. P1, when trypsinized and re‐chromatographed, revealed the same HPLC pattern as that of untrypsinized P1, but a new peak, P2′, emerged from P2 and P3 after trypsinization, by HPLC. The relative biological activity of trypsinized P1 was of the same level as that of untreated P1, but those of P2 and P3 increased one and a half to three times upon trypsinization and finally reached the same level as those of P1 and P2′.