Ishikawa R, Yamashiro S, Matsumura F
Department of Molecular Biology and Biochemistry, Nelson Laboratory, Rutgers University, Piscataway, New Jersey 08854-1059.
J Biol Chem. 1989 Oct 5;264(28):16764-70.
Previous results from our laboratory have shown that 1) cultured rat cells contain two classes of tropomyosin (TM), one (high Mr TMs) with higher Mr values and greater affinity for actin than the other (low Mr TMs); 2) presaturation of F-actin with high Mr TMs, but not with low Mr TMs, inhibits both actin-severing and actin binding activities of gelsolin; and 3) nonmuscle caldesmon not only enhances the inhibitory effects of high Mr TMs but also makes low Mr TMs capable of inhibiting the severing activity of gelsolin (Ishikawa, R., Yamashiro, S., and Matsumura, F. (1989) J. Biol. Chem. 264, 7490-7497). These results suggest that gelsolin has much lower affinity for F-actin-TM-caldesmon complexes than for pure F-actin. We have therefore examined whether addition of TM and/or caldesmon to gelsolin-severed actin filaments can make gelsolin dissociate from barbed ends of actin filaments, resulting in annealing of short actin filaments into long ones. Flow birefringence and electron microscopic studies have suggested that high Mr TMs slowly and partially anneal gelsolin-severed actin fragments in 3 h, whereas low Mr TMs have no effects. Nonmuscle caldesmon greatly potentiates the effects of high Mr TMs and accelerates the process to 20 min, whereas nonmuscle caldesmon alone shows no effects. Furthermore, nonmuscle caldesmon makes low Mr TMs capable of reversing gelsolin-severing action. Actin binding assay has shown that gelsolin (or a gelsolin-actin complex) is dissociated from these annealed actin filaments. Smooth muscle TM and smooth muscle caldesmon also appear to anneal gelsolin-severed actin fragments as do high Mr TMs and nonmuscle caldesmon. Calmodulin decreases the potentiation effects of caldesmon as calmodulin inhibits actin binding of caldesmon. These results suggest that tropomyosin and caldesmon may regulate both capping and severing activities of gelsolin.
1）培养的大鼠细胞含有两类原肌球蛋白（TM），一类（高分子量TM）的分子量较高，对肌动蛋白的亲和力高于另一类（低分子量TM）；2）用高分子量TM而非低分子量TM对F-肌动蛋白进行预饱和，可抑制凝溶胶蛋白的肌动蛋白切断和肌动蛋白结合活性；3）非肌肉钙调蛋白不仅增强了高分子量TM的抑制作用，还使低分子量TM能够抑制凝溶胶蛋白的切断活性（石川，R.，山城，S.，和松村，F.（1989年）《生物化学杂志》264，7490 - 7497）。这些结果表明，凝溶胶蛋白对F-肌动蛋白-TM-钙调蛋白复合物的亲和力远低于对纯F-肌动蛋白的亲和力。因此，我们研究了向凝溶胶蛋白切断的肌动蛋白丝中添加TM和/或钙调蛋白是否能使凝溶胶蛋白从肌动蛋白丝的带刺末端解离，从而导致短肌动蛋白丝退火成长肌动蛋白丝。流动双折射和电子显微镜研究表明，高分子量TM在3小时内缓慢且部分地使凝溶胶蛋白切断的肌动蛋白片段退火，而低分子量TM则无此作用。非肌肉钙调蛋白极大地增强了高分子量TM的作用，并将这一过程加速至20分钟，而单独的非肌肉钙调蛋白则无作用。此外，非肌肉钙调蛋白使低分子量TM能够逆转凝溶胶蛋白的切断作用。肌动蛋白结合试验表明，凝溶胶蛋白（或凝溶胶蛋白-肌动蛋白复合物）从这些退火的肌动蛋白丝上解离。平滑肌TM和平滑肌钙调蛋白似乎也像高分子量TM和非肌肉钙调蛋白一样，使凝溶胶蛋白切断的肌动蛋白片段退火。钙调蛋白降低了钙调蛋白的增强作用，因为钙调蛋白抑制钙调蛋白与肌动蛋白的结合。这些结果表明，原肌球蛋白和钙调蛋白可能调节凝溶胶蛋白的封端和切断活性。
