Rockwell M A, Fechheimer M, Taylor D L
Cell Motil. 1984;4(3):197-213. doi: 10.1002/cm.970040305.
We have compared the meniscus depletion assay and falling ball viscometry, two means of assessing the extent of gelation in actin-based systems using mixtures of actin and the actin-binding protein filamin. We examined the effect of varying the concentrations of actin and filamin in both assays. The interaction of actin and filamin was detected only above a threshold concentration of filamin. This threshold concentration was lower for falling ball viscometry than for the meniscus depletion assay at equal actin concentrations. At constant concentrations of filamin, an increase in actin concentration caused an increase in apparent viscosity measured by the falling ball assay, but a decrease in sedimentability detected by the meniscus depletion assay. The rate of sedimentation of actin was dependent on the molar ratio of actin to filamin. At each molar ratio, the sedimentation of actin was not dependent on the specific concentrations of actin and filamin used. The apparent viscosity was dependent on both the molar ratio and the specific concentrations of actin and filamin. To relate the present results to earlier studies, we examined mixtures of actin and filamin using a macroscopic assay of gelation (tube tipping assay), and polarized light microscopy. The effect of increasing filamin concentration in the four assays was compared at three actin concentrations. Mixtures of actin and filamin whose apparent viscosities were low enough to be estimated by falling ball viscometry were optically isotropic fluids that flowed out of inverted test tubes. Mixtures of actin and filamin in the range of sensitivity of the meniscus depletion assay were either viscous fluids or gels, and were either optically isotropic or anisotropic. Thus, the four assays provide different estimates of gelation. Both the meniscus depletion assay and falling ball viscometry can be used to determine relative gelation activity, but neither can be used as a quantitative assay of gelation.
我们比较了半月板耗竭试验和落球粘度测定法，这两种方法用于评估基于肌动蛋白的系统中使用肌动蛋白和肌动蛋白结合蛋白细丝蛋白混合物的凝胶化程度。我们在两种试验中研究了改变肌动蛋白和细丝蛋白浓度的影响。仅在细丝蛋白的阈值浓度以上才能检测到肌动蛋白和细丝蛋白的相互作用。在肌动蛋白浓度相等的情况下，落球粘度测定法的该阈值浓度低于半月板耗竭试验的阈值浓度。在细丝蛋白浓度恒定的情况下，肌动蛋白浓度的增加导致落球试验测得的表观粘度增加，但半月板耗竭试验检测到的沉降性降低。肌动蛋白的沉降速率取决于肌动蛋白与细丝蛋白的摩尔比。在每个摩尔比下，肌动蛋白的沉降不取决于所用肌动蛋白和细丝蛋白的具体浓度。表观粘度取决于肌动蛋白和细丝蛋白的摩尔比以及具体浓度。为了将当前结果与早期研究联系起来，我们使用凝胶化的宏观试验（试管倾斜试验）和偏振光显微镜检查了肌动蛋白和细丝蛋白的混合物。在三种肌动蛋白浓度下比较了四种试验中细丝蛋白浓度增加的影响。表观粘度低到足以通过落球粘度测定法估计的肌动蛋白和细丝蛋白混合物是从倒置试管中流出的光学各向同性流体。在半月板耗竭试验灵敏度范围内的肌动蛋白和细丝蛋白混合物要么是粘性流体要么是凝胶，并且要么是光学各向同性要么是各向异性。因此，这四种试验提供了不同的凝胶化估计值。半月板耗竭试验和落球粘度测定法都可用于确定相对凝胶化活性，但都不能用作凝胶化的定量试验。
