Mooseker M S, Graves T A, Wharton K A, Falco N, Howe C L
J Cell Biol. 1980 Dec;87(3 Pt 1):809-22. doi: 10.1083/jcb.87.3.809.
The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.
肠上皮细胞微绒毛内的细丝束包含五种主要蛋白质，包括肌动蛋白、钙调蛋白以及105、95和70千道尔顿的亚基。先前的研究表明（豪，C.L.，M.S.穆斯克，和T.A.格雷夫斯。1980年。刷状缘钙调蛋白：分离的微绒毛核心的主要成分。《细胞生物学杂志》85：916 - 923），向微绒毛核心添加Ca++（>10^(-6) M）会导致这种肌动蛋白丝束迅速、剧烈但至少部分可逆的破坏。对用Ca++处理的微绒毛核心进行高速离心表明，几种核心蛋白可溶解，包括30 - 50%的肌动蛋白和钙调蛋白，以及大部分95和70千道尔顿的亚基。在有和没有Ca++的情况下对这种Ca++提取物进行凝胶过滤表明，被Ca++“分离”的微绒毛肌动蛋白处于寡聚状态，可能与95千道尔顿的亚基复合。去除Ca++会导致F - 肌动蛋白重新组装，通过凝胶过滤、共沉降、粘度测定和电子显微镜测定，可能仍然与95千道尔顿的亚基复合。95千道尔顿的亚基（95K）通过DEAE - 葡聚糖凝胶色谱从Ca++提取物中纯化，并通过粘度测定、共沉降以及在有和没有Ca++的情况下的电子显微镜观察来表征其与肌动蛋白的相互作用。在有Ca++但没有Ca++时不存在的情况下，95K抑制肌动蛋白组装（在95K与肌动蛋白比例为1:50 - 60时抑制50%），并且还降低F - 肌动蛋白溶液的粘度。同样，95K抑制肌动蛋白的沉降，但在Ca++存在下形成的一个小的、可能是寡聚的肌动蛋白 - 95K复合物在长期离心后可沉淀。在没有Ca++的情况下，95K与F - 肌动蛋白共沉降。95K - 肌动蛋白混合物的电子显微镜观察表明，在Ca++存在下，95K将肌动蛋白“断裂”成小的丝状片段。一旦去除Ca++，细丝就会重新组装。在没有Ca++的情况下，95K对细丝结构没有影响，并且在95K与肌动蛋白的比例相对较高（1:2 - 6）时，这种核心蛋白会将肌动蛋白丝聚集成束。
