The binding sites of cytochalasin D. I. Evidence that they may be peripheral membrane proteins

Binding sites for tritiated cytochalasin D (3H-CD) on the isolated plasma membrane from HEp-2 cells were reversibly inactivated, but not dissociated from the membrane, by dialysis in 0.6 M KCl. Activity was restored by subsequent dialysis in 0.06 M KCl. Treatment with 0.2 mM ATP at low ionic strength also inactivated these sites, apparently irreversibly. Extraction of the membrane with 6% Triton X-100 removed 75% of its protein, resulting in a two-fold increase in specific binding activity for 3H-CD. Both high and low affinity binding sites were retained by the detergent-extracted membrane; at least 60% of the high affinity sites were resistant to this treatment. Evidence is presented for the attachment to the HEp-2 plasma membrane of both actin and myosin. The results support the tentative conclusion that plasma membrane binding sites for 3H-CD are peripheral proteins on the cytoplasmic face of the membrane. They are consistent with the hypothesis that myosin may be the location of the high affinity binding site and actomyosin may be the low affinity site. Comparison of these observations with those reported for the congeneric drug, cytochalasin B, suggests that CD binding sites differ from the high affinity site for cytochalasin B.