Binding of Clostridium perfringens [125I]enterotoxin to rabbit intestinal cells

Abstract

125I-Labeled enterotoxin from Clostridium perfringens was utilized to characterize the association of the enterotoxin with cells isolated from rabbit intestine and tissue homogenates from liver, kidney, and brain. The enterotoxin was found to bind in a specific and saturable manner to cells from intestine and to tissue homogenates from liver and kidney but not the brain. Detailed studies of the binding were carried out with the ileal epithelial intestinal cells. The rate and amount of binding of enterotoxin to cells appeared to be temperature dependent. Apparent affinity and association and dissociation rate constants were calculated for what appeared to be two classes of saturable binding sites. The amount of enterotoxin molecules that bound per milligram of cell protein was similar in tissue of intestinal, liver, and kidney origin (approximately 10(13) molecules/mg of cell protein). Spontaneous dissociation into the supernatant medium was observed to be much slower than expected from calculations based on the rate of association. Chaotropic ions did not enhance dissociation of the enterotoxin from cells. Enterotoxin binding was demonstrated to be heat labile (binding ability was lost after the enterotoxin was heated for 10 min at 60 degrees C). A mechanism is described whereby the enterotoxin binds and then is inserted into the membrane where it becomes trapped.