Demonstration of diphtheria toxin receptors on surface membranes from both toxin-sensitive and toxin-resistant species.

A detailed binding study of 125I-labeled diphtheria toxin to isolated cell surface membrane-enriched fractions is reported. The study was undertaken to determine if toxin-resistant species exhibit a defet in either the binding step or the transport step of the intoxication process. Surface membrane fractions were obtained from liver and mammary glands of toxin-sensitive species, rabbit and giunea pig, and toxin-resistant species, rat mouse. All membrane fractions exhibited reversible binding of 125I-toxin which was competitively inhibited by unlabeled toxin. Toxin receptors from liver co-purified with plasma membranes and the plasma membrane marker 5'-nucleotidase. One-half saturation of all receptors occurred between 5 x 10(-8) and 1.8 x 10(-7) M. Scatchard plots were nonlinear and concave upwards. Total receptor sites ranged from 3.4 to 16 pmol/mg of membrane protein, tissue differences being more pronounced than difference between sensitive and nonsensitive species. Over 95% of the toxin specific binding was inhibited by removal of divalent cation from the medium or by the inclusion of 1 mM ATP, procedures which have been shown to protect sensitive cells from intoxication by diphtheria toxin. We conclude that the rat and mouse have surface membrane receptors for diphtheria toxin and that the toxin insensitivity of these species results from a defect in or a lack of the transport process.