Matsudaira P T, Burgess D R
J Cell Biol. 1982 Mar;92(3):648-56. doi: 10.1083/jcb.92.3.648.
The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.
从鸡肠上皮细胞分离得到的刷状缘含有相对分子质量为95,000（M(r)）的多肽——绒毛蛋白。本报告描述了绒毛蛋白作为一种依赖Ca(++)的肌动蛋白成束/解聚蛋白的纯化及特性。从分离的刷状缘的Ca(++)提取物中获得的100,000g上清液由三种多肽组成，分别为95,000（绒毛蛋白）、68,000（丝束蛋白）和42,000 M(r)（肌动蛋白）。通过硫酸铵分级沉淀和离子交换色谱从该提取物中纯化得到的绒毛蛋白，与骨骼肌F-肌动蛋白混合。对这些绒毛蛋白-肌动蛋白混合物的负染制剂进行电子显微镜观察，结果显示存在细丝束。细丝束的这种粘度、沉降性和超微结构形态取决于绒毛蛋白与肌动蛋白的摩尔比、pH值以及溶液中的游离Ca(++)浓度。在低游离Ca(++)（小于10(-6) M）时，通过沉降测量，束中蛋白质的量随着绒毛蛋白与肌动蛋白的摩尔比增加而增加，并在约4:10的比例时达到平台期。这种行为与电子显微镜检测到的单根肌动蛋白丝转化为细丝束相关。在高游离Ca(++)（大于10(-6) M）时，绒毛蛋白-肌动蛋白混合物的表观粘度降低至缓冲液测量的水平。此外，这些Ca(++)效应与沉降蛋白质的损失、细丝束的消失以及细丝短片段的出现相关。束的形成也对pH敏感，在轻度酸性pH下更有利。pH从7.6降至6.6会导致可沉降蛋白质增加，同时也会使松散结合的肌动蛋白丝转化为紧密的肌动蛋白束。这些结果与以下观点一致，即绒毛蛋白是微绒毛中的一种成束蛋白，负责微绒毛细胞骨架的Ca(++)敏感拆卸。因此，绒毛蛋白可能在细胞质中作为调节微绒毛形状的主要细胞骨架元件发挥作用。
