Actin oligomers below the critical concentration detected by fluorescence photobleaching recovery
Actin oligomers below the critical concentration detected by fluorescence photobleaching recovery
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通过荧光光漂白恢复检测到低于临界浓度的肌动蛋白寡聚物
DOI：
10.1021/bi00327a038
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
B. Ware
中科院分区：
文献类型：
--
作者：
A. Mozo;B. Ware
材料和方法通过Pardee & Spudich（1982）的方法从兔肌肉的丙酮粉末中获得肌动蛋白，并通过如别处所述的连续聚合和解聚循环进行纯化（Lanni等人，1981年）。根据Wang和Taylor（1980）的方法，通过肌动蛋白丝与5-（碘乙酰氨基）-荧光素（Molecular Probes）的反应来标记肌动蛋白。该程序在Cys-374处连接单个荧光素残基。通过Sephadex G-25-150柱实现未反应标记物与蛋白质的分离;通过使用DE-52柱实现标记肌动蛋白与未标记肌动蛋白的分离。将纯化的肌动蛋白（标记和未标记）聚合并在0 ℃下作为小球储存不超过1个月.在实验准备中，通过在适当的缓冲液中均质化和透析使均质化的肌动蛋白沉淀解聚。通过离心澄清所得溶液。通过Lowry方法测量最终的G-肌动蛋白浓度（始终约为9 µ）（Lowry等人，1951），以牛血清白蛋白为标准，应用Houk & Ue（1974）给出的校正因子。在这些实验中标记的肌动蛋白的比例总是在10%和15%之间。G-肌动蛋白在溶液中的停留时间从未超过3天。通过独立证明其可逆组装的能力，验证了G-肌动蛋白在这段时间内的稳定性。（2 mM Tris-HCl、0.2 mM CaCl 2、0.2 mM ATP和0.5 mM 2-巯基乙醇，pH 8.0）和缓冲液B（2 mM Tris-HCl、50 µ MgCl 2、0.2 mM ATP和0.5 mM 2-巯基乙醇，pH 8.0）。所有试剂级化学品均购自Sigma ChemicalCo。不经进一步纯化即可使用。我们在许多实验中改变了Ca 2+、Mg 2+和ATP的浓度。在这种情况下，游离Mg 2+、游离Ca 2+和游离ATP的浓度通过使用我们的条件（pH 8，低盐）下可用的解离常数的最佳文献值来计算。所用的解离常数值对于CaATP为100 μ（Heyde & Rimai，1971），对于MgATP为20 μ（Laget等人，1973年）。
Materials and Methods Actin was obtained by the method of Pardee & Spudich (1982) from acetone powder of rabbit muscle and was purified by successive cycles of polymerization and depolymerization as described elsewhere (Lanni et al., 1981). Actin was labeled by reaction of actin filaments with 5-(iodoacetamido)-fluorescein (Molecular Probes) according to the method of Wang & Taylor (1980). This procedure attaches a single fluorescein residue at Cys-374. Separation of unreacted label from protein was accomplished by passage through a Sephadex G-25-150 column; separation of labeled actinfrom unlabeled actin was accomplished by using a DE-52 column. Purified actins (labeled and unlabeled) were polymerized and stored as pellets at 0 C for no longer than 1 month. In preparation for experiments, the homogenized actin pellet was depolym-erized by homogenization in and dialysis against the appropriate buffer. The resulting solution was clarified by cen-trifugation. The final G-actin concentration (always around 9 µ) was measured by the Lowry method (Lowry et al., 1951) using bovine serum albumin as the standard and ap-plying the correction factor given by Houk & Ue (1974). The proportion of labeled actin in these experiments was always between 10% and 15%. G-Actin was never left in solution for more than 3 days. Stability of G-actin for this period of time was verified by independent demonstrations of its competency for reversible assembly.The buffers used in these experiments were variations of the standard buffer A (2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, and 0.5 mM 2-mercaptoethanol, pH 8.0) and buffer B (2 mM Tris-HCl, 50 µ MgCl2, 0.2 mM ATP, and 0.5 mM 2-mercaptoethanol, pH 8.0). All reagent-grade chemicals were purchased from Sigma ChemicalCo. and used without further purification. We have varied the concentrations of Ca2+, Mg2+, and ATP in a number of our experiments. In such cases the concentrations of free Mg2+, free Ca2+, and free ATP were calculated by using thebest literature values for dissociation constants available for our conditions (pH 8, low salt). The values of the dissociation constants used were 100 µ for CaATP (Heyde & Rimai, 1971) and 20 µ for MgATP (Laget et al., 1973).