Ishiura M, Okada Y
J Cell Biol. 1979 Feb;80(2):465-80. doi: 10.1083/jcb.80.2.465.
Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.
艾氏腹水瘤细胞提取物在蔗糖溶液中于pH 7.0加热至25℃时形成凝胶，而冷却至0℃时该凝胶迅速变为溶胶。通过测定低速离心获得的凝胶沉淀的体积或总蛋白对这种凝胶 - 溶胶转变进行了定量研究。凝胶化取决于三磷酸核苷酸、Mg2 +、KCl和一种还原剂。0.5 microM游离Ca2 +以及25 - 50 ng/ml的细胞松弛素B或D可可逆地抑制凝胶化，但10 mM秋水仙碱对其无影响。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳表明，该凝胶由六种主要蛋白质组成，其分子量分别大于300,000、270,000、89,000、51,000、48,000和42,000道尔顿。最后一种成分被鉴定为细胞肌动蛋白，因为它与肌肉肌动蛋白具有相同的分子量，并能与肌肉肌球蛋白和原肌球蛋白结合。通过使用肌动蛋白抑制剂研究了肌动蛋白在凝胶化中的作用。凝胶化受到化学修饰的肌球蛋白亚片段 - 1的抑制，该亚片段即使在ATP存在下也能与F - 肌动蛋白结合，还受到牛胰脱氧核糖核酸酶I的抑制，后者与G - 肌动蛋白紧密结合。当以等摩尔比添加到后者时，肌肉G - 肌动蛋白中和了脱氧核糖核酸酶I的抑制作用，并且在其被脱氧核糖核酸酶I抑制后还恢复了凝胶化。这些发现表明凝胶化取决于肌动蛋白。然而，当提取物中的细胞肌动蛋白被肌肉肌动蛋白取代时，提取物显示出与原始提取物一样的温度依赖性、细胞松弛素敏感性和Ca2 +调节的凝胶化，这表明除细胞肌动蛋白外的其他成分可能是这些凝胶化特性的原因。
