Tannenbaum J, Tanenbaum S W, Godman G C
J Cell Physiol. 1977 May;91(2):225-37. doi: 10.1002/jcp.1040910208.
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.
在0.6 M KCl中透析可使来自HEp - 2细胞的分离质膜上的氚化细胞松弛素D（3H - CD）结合位点可逆性失活，但不会从膜上解离。随后在0.06 M KCl中透析可恢复活性。在低离子强度下用0.2 mM ATP处理也会使这些位点失活，显然是不可逆的。用6% Triton X - 100提取膜可去除75%的蛋白质，导致3H - CD的特异性结合活性增加两倍。去污剂提取的膜保留了高亲和力和低亲和力结合位点；至少60%的高亲和力位点对这种处理具有抗性。有证据表明肌动蛋白和肌球蛋白都附着在HEp - 2质膜上。结果支持了一个初步结论，即3H - CD的质膜结合位点是膜细胞质面的外周蛋白。它们与肌球蛋白可能是高亲和力结合位点的位置以及肌动球蛋白可能是低亲和力位点的假设一致。将这些观察结果与关于同类药物细胞松弛素B的报道进行比较，表明CD结合位点与细胞松弛素B的高亲和力位点不同。
