Sawyer W H, Woodhouse A G, Czarnecki J J, Blatt E
Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Biochemistry. 1988 Oct 4;27(20):7733-40. doi: 10.1021/bi00420a023.
The rotational diffusion of actin was studied with the technique of time-resolved phosphorescence anisotropy using actin labeled at Cys-374 with erythrosin iodoacetamide. Immediately after the polymerization of actin was initiated, the correlation time increased sharply, passing through a maximum at 5 min and then declined to low values. F-Actin at equilibrium showed no anisotropy decay. The results were interpreted as indicating the initial formation of short mobile filaments which became increasingly immobile as elongation proceeded, leaving a decay which was dominated by shorter filaments. Some of these short filaments could have arisen by fragmentation of longer filaments. Eventually, the shorter filaments themselves became immobilized by entanglement within the gel matrix. The infinite-time anisotropy increased during polymerization, reflecting a smaller range of angular motion of the probe brought about by restricted torsional motion on the submicrosecond time scale. The results were compared with the length distribution of actin filaments revealed by electron microscopy [Kawamura, M., & Maruyama, K. (1970) J. Biochem. (Tokyo) 67, 437-457]. Polymerization in the presence of 1 microM cytochalasin B abolished the maximum in the correlation time profile and tended to prevent the immobilization of filaments by favoring shorter capped filaments which retained considerable rotational freedom. Addition of spectrin dimer to F-actin caused an increase in the time-invariant anisotropy. Subsequent additions of spectrin-binding proteins (erythrocyte bands 2.1 and 4.1) caused further increases in the anisotropy in a concentration-dependent manner, suggesting additional restriction of submicrosecond torsional motions. The results suggest that actin filaments within nonmuscle cells are rotationally immobile particularly if they are cross-linked by actin-binding proteins.
利用时间分辨磷光各向异性技术，对用碘乙酰胺荧光桃红标记半胱氨酸 - 374的肌动蛋白的旋转扩散进行了研究。肌动蛋白聚合反应一开始，相关时间急剧增加，在5分钟时达到最大值，然后下降到低值。处于平衡状态的F - 肌动蛋白没有各向异性衰减。这些结果被解释为表明短的可移动细丝最初形成，随着伸长过程其移动性越来越低，留下的衰减主要由较短的细丝主导。其中一些短细丝可能是由较长细丝断裂产生的。最终，较短的细丝自身因在凝胶基质中缠结而变得固定。在聚合过程中无限时间各向异性增加，反映了在亚微秒时间尺度上由于受限的扭转运动导致探针角运动范围变小。将这些结果与电子显微镜揭示的肌动蛋白丝长度分布进行了比较[河村，M.，&丸山，K.（1970年）《生物化学杂志》（东京）67，437 - 457]。在1微摩尔细胞松弛素B存在下聚合消除了相关时间曲线中的最大值，并倾向于通过有利于保留相当大旋转自由度的较短帽化细丝来防止细丝固定。向F - 肌动蛋白中添加血影蛋白二聚体导致时间不变各向异性增加。随后添加血影蛋白结合蛋白（红细胞带2.1和4.1）以浓度依赖方式导致各向异性进一步增加，表明亚微秒扭转运动受到额外限制。结果表明，非肌肉细胞内的肌动蛋白丝旋转固定，特别是如果它们被肌动蛋白结合蛋白交联。
