Saito S Y, Watabe S, Ozaki H, Kobayashi M, Suzuki T, Kobayashi H, Fusetani N, Karaki H
Department of Veterinary Pharmacology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113.
J Biochem. 1998 Apr;123(4):571-8. doi: 10.1093/oxfordjournals.jbchem.a021975.
We compared the effects of dimeric marine toxins, bistheonellide A, and swinholide A, on actin polymerization. Bistheonellide A and swinholide A possess two identical side chains with similar structures to those of other marine toxins, mycalolide B, and aplyronine A. By monitoring changes in fluorescent intensity of pyrenyl-actin, bistheonellide A was found to inhibit polymerization of G-actin and to depolymerize F-actin in a concentration-dependent manner. The relationship between the concentration of bistheonellide A and its inhibitory activity on actin polymerization suggested that one molecule of bistheonellide A binds two molecules of G-actin. We demonstrated by SDS-PAGE that the complex of G-actin with bistheonellide A, swinholide A, or mycalolide B could not interact with myosin. No evidence was found that bistheonellide A severs F-actin at the concentrations examined (molar ratio to actin; 0. 025-2.5), while swinholide A showed severing activity, although it was weaker than that of mycalolide B. We also demonstrated that the depolymerizing effect of bistheonellide A or mycalolide B is irreversible. Bistheonellide A increased, while swinholide A decreased, the rate of nucleotide exchange in G-actin, suggesting that binding of these toxins induces different conformational changes in the actin molecule. These results suggest that bistheonellide A intervenes between two actin molecules, forms a tertiary complex with each of its side chains bound to G-actin, and inhibits polymerization by sequestering G-actin from incorporation into F-actin. A difference in structure at the end of the side chain between dimeric macrolides and mycalolide B may account for the weak severing activity of the former.
我们比较了二聚体海洋毒素双西奈利德A和西加毒素A对肌动蛋白聚合的影响。双西奈利德A和西加毒素A拥有两条相同的侧链，其结构与其他海洋毒素、海绵大环内酯B和软海绵素A的侧链结构相似。通过监测芘基肌动蛋白荧光强度的变化，发现双西奈利德A以浓度依赖的方式抑制G-肌动蛋白的聚合并使F-肌动蛋白解聚。双西奈利德A的浓度与其对肌动蛋白聚合的抑制活性之间的关系表明，一个双西奈利德A分子结合两个G-肌动蛋白分子。我们通过SDS-PAGE证明，G-肌动蛋白与双西奈利德A、西加毒素A或海绵大环内酯B的复合物不能与肌球蛋白相互作用。在所检测的浓度（与肌动蛋白的摩尔比为0.025 - 2.5）下，未发现双西奈利德A切断F-肌动蛋白的证据，而西加毒素A表现出切断活性，尽管其活性比海绵大环内酯B弱。我们还证明，双西奈利德A或海绵大环内酯B的解聚作用是不可逆的。双西奈利德A增加了G-肌动蛋白中的核苷酸交换速率，而西加毒素A降低了该速率，这表明这些毒素的结合在肌动蛋白分子中诱导了不同的构象变化。这些结果表明，双西奈利德A插入两个肌动蛋白分子之间，形成一种三级复合物，其每条侧链都与G-肌动蛋白结合，并通过隔离G-肌动蛋白使其不掺入F-肌动蛋白来抑制聚合。二聚体大环内酯类化合物和海绵大环内酯B在侧链末端的结构差异可能解释了前者较弱的切断活性。
