DETECTION AND CHARACTERIZATION OF ACTIN MONOMERS, OLIGOMERS, AND FILAMENTS IN SOLUTION BY MEASUREMENT OF FLUORESCENCE PHOTOBLEACHING RECOVERY
DETECTION AND CHARACTERIZATION OF ACTIN MONOMERS, OLIGOMERS, AND FILAMENTS IN SOLUTION BY MEASUREMENT OF FLUORESCENCE PHOTOBLEACHING RECOVERY
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DOI：
10.1016/s0006-3495(84)84002-3
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发表时间：
1984-01-01
影响因子：
3.4
通讯作者：
WARE, BR
中科院分区：
文献类型：
--
作者：
LANNI, F;WARE, BR
荧光光漂白恢复（FPR）进行了测量，以确定在低盐缓冲液中的荧光素标记的[兔] G-肌动蛋白的扩散系数。所得结果为7.15 ±. 0.35. times. 10-7 cm ~ 2/s，与由分子量、偏比容和沉降系数计算的结果吻合较好，但高于以前得到的值。从理论上证明，在低离子强度下，静电对特性粘数的贡献导致高估G-肌动蛋白的流体动力学离心率。来自FPR、沉降和荧光偏振实验的数据都表明，真正的低盐形式的肌动蛋白单体具有轴比≤3.0.在装配阶段，在稳定状态下，以及在配体如细胞松弛素和醛缩酶的存在下，通过测量FPR也观察到肌动蛋白的G-F转化。每个FPR记录一般产生3个数据：快速和缓慢扩散的肌动蛋白的相对比例，高流动性部分的扩散系数，和低流动性部分的平均扩散系数。导出了平均低迁移率扩散系数与数均细丝长度之间的关系，并应用于FPR数据的分析。在典型的条件下，在稳定状态下平均长丝长度>> 10 μ m。发现细胞松弛素D成比例地减少细丝长度和细丝总数，而细丝总数没有受到很大影响。在所有聚合的G-肌动蛋白，在原位观察到的高流动性材料基本上是单体肌动蛋白。通过在4 ℃下在50 μ M MgCl 2中凝胶过滤分级G-AF-肌动蛋白，分离相对稳定的肌动蛋白寡聚体。C.基于扩散系数，单体和二聚体构成了在这些条件下存在的主要颗粒类型。在1.0 mM MgCl 2中聚合的标记肌动蛋白的沉淀产生了一个梯度的上清液，其中含有肌动蛋白低聚物显着大于单体。
Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled [rabbit] G-actin in low-salt buffer. The result obtained, 7.15 .+-. 0.35 .times. 10-7 cm2/s, is in good agreement with that computed from the MW, partial specific volume and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio .ltoreq. 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields 3 data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was >> 10 .mu.m in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 .mu.M MgCl2 at 4.degree. C. On the basis of the diffusion coefficient, monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.