The specific interaction of the cytolytic
Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this
glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated
neuroblastoma gangliosides, and (iii) toxin fixation by
phospholipid-
cholesterol unilamellar vesicles containing either sheep
gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C
glucosamine, which inserts into the outer layer and labels
integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2
liposomes did not release entrapped 14C-glucose. Treatment of toxin with
carboxypeptidases, but not
aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with
antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.