Polymerization-induced changes in the fluorescence of actin labeled with iodoacetamidotetramethylrhodamine.
Polymerization-induced changes in the fluorescence of actin labeled with iodoacetamidotetramethylrhodamine.
复制标题
聚合诱导的用碘乙酰胺四甲基罗丹明标记的肌动蛋白的荧光变化。
DOI：
10.1016/0003-9861(82)90197-7
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发表时间：
1982
影响因子：
3.9
通讯作者：
Frieden,C
中科院分区：
文献类型：
--
作者：
Tait,JF;Frieden,C
兔骨骼肌肌动蛋白已标记荧光巯基试剂碘乙酰胺四甲基罗丹明（罗丹明）。标记可能位于Cys-373上，因为用N-乙基马来酰亚胺预先处理肌动蛋白阻止了罗丹明的掺入。当罗丹明-肌动蛋白与MgSO 4或KCl聚合时，荧光增加约1.5倍。荧光的变化与单体球状肌动蛋白掺入聚合物相关，因此可以用作聚合的定量测量。痕量的罗丹明-肌动蛋白可用于跟踪未标记的肌动蛋白的聚合动力学而不干扰样品，并且表明使用毛细管粘度计加速聚合速率。荧光光漂白恢复实验测量了若丹明标记的肌动蛋白的扩散，表明在聚合过程中出现了不可扩散（表观扩散系数< 10− 10 cm 2/s）的细丝，但在剪切后这些细丝立即就很容易扩散了。这些结果表明流体动力学剪切应力对聚合肌动蛋白的破坏性。
Rabbit skeletal muscle actin has been labeled with the fluorescent sulfhydryl reagent iodoacetamidotetramethylrhodamine (rhodamine). The label is probably located on Cys-373 because prior treatment of the actin withN-ethylmaleimide prevents incorporation of rhodamine. When the rhodamine-actin is polymerized with MgSO4or KCl, there is approximately a 1.5-fold increase in fluorescence. The change in fluorescence is correlated with incorporation of monomeric globular actin into polymer and can therefore be used as a quantitative measure of polymerization. Trace quantities of rhodamine-actin can be used to follow the kinetics of polymerization of unlabeled actin without disturbing the sample, and it is shown that the use of a capillary viscometer accelerates the rate of polymerization. Fluorescence photobleaching recovery experiments, which measure the diffusion of rhodamine-labeled actin, show that nondiffusible (apparent diffusion coefficient < 10−10cm2/s) filaments appear during the polymerization process but that immediately after shearing these filaments are readily diffusible. These results demonstrate the destructive nature of hydrodynamic shear stress on polymerized actin.