Purification of a HeLa cell high molecular weight action binding protein and its identification in HeLa cell plasma membrane ghosts and intact HeLa cells.
Purification of a HeLa cell high molecular weight action binding protein and its identification in HeLa cell plasma membrane ghosts and intact HeLa cells.
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HeLa 细胞高分子量作用结合蛋白的纯化及其在 HeLa 细胞质膜鬼影和完整 HeLa 细胞中的鉴定。
DOI：
10.1021/bi00277a015
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Weihing,RR
中科院分区：
文献类型：
--
作者：
Weihing,RR
摘要：高分子量蛋白质(HMWP)是以肌动蛋白为基础的凝胶的主要成分，通过在25℃下孵育HeLa细胞的细胞质提取物而形成[魏兴，RR(1977)J.Cell Biol]。75，95-103]现已通过0.6MKc1沉淀凝胶提取物的凝胶过滤得到纯化。从4×109细胞中可以分离出几百微克的HMWP，纯度约为90%。HMWP可以凝胶肌肉肌动蛋白，并将其交联成细丝束。其亚基相对分子质量为250000，斯托克斯半径为125A，沉降系数为9S，用后两个参数计算的天然相对分子质量为48万，表明天然分子为二聚体。它的氨基酸分析几乎与巨噬细胞肌动蛋白结合蛋白以及哺乳动物和禽丝蛋白的氨基酸分析没有区别。所有这些发现表明，HMWP与后一种蛋白同源。然而，HeLa细胞HMWP和禽丝蛋白的初级序列一定不同，因为它们的部分肽谱是不同的，而且抗HMWP的抗血清只与丝蛋白反应很弱。为了研究HMWP的细胞内定位，针对纯化的HMWP真核细胞的山羊抗血清含有多种肌动蛋白结合蛋白，这些蛋白被认为影响基于肌动蛋白的微丝系统的组织和功能[见Schliwa(1981)和Weeds(1982)]。本实验室通过对HeLa细胞胞浆提取液肌动蛋白凝胶的研究，研究了HeLa细胞的肌动蛋白结合蛋白。这些研究产生了三条证据，表明凝胶化至少部分可以通过一种名为HMWP的蛋白质将肌动蛋白交联成三维网络来解释。1首先，电泳法分析表明肌动蛋白和HMWP是分离凝胶的主要多肽成分(魏兴，1976a，b，1977)。第二，淡化
Robert R. Weihing abstract: The high molecular weight protein (HMWP) which was previously observed to be a major component of the actin based gels formed by incubating cytoplasmic extracts of HeLa cells at 25 C [Weihing, RR (1977) J. Cell Biol. 75, 95-103] has now been purified by gel filtration of 0.6 M KC1 extracts of precipitated gels. A few hundred micrograms of HMWP, which is about 90% pure, can be isolated from 4 X 109 cells. HMWP can gel muscle actin and cross-link it into filament bundles. Its subunit molecular weight is 250000, its Stokes radius is 125 A, and its sedimentation coefficient is 9 S. A native molecular weight of 480 000 was calculated by using the latter two parameters, and therefore the native molecule is a dimer. Its amino acid analysis is nearly indistinguishable from thatof macrophage actin binding protein and of mammalian and avian filamins. All of these findings indicate that HMWP is homologous to the latter proteins. However, HeLa cell HMWP and avian filamin must differ in their primary sequences because their partial peptidemaps are distinct and because an antiserum against HMWP reacts only weakly with filamin. For studies on the intracellular location of HMWP, a goat antiserum against purified HMWP^ Eukaryotic cells contain a variety of actin binding proteins that are believed to influence the organization and function of the actin-based microfilament system [reviewed in Schliwa (1981) and Weeds (1982)]. This laboratory has investigated an actin binding protein of HeLa cells through studies of the actin-based gelation of cytoplasmic extracts of HeLa cells. These studies produced three lines of evidence suggesting that gelation could be explained, at least in part, by cross-linking of actin into a three-dimensional network by a protein designated HMWP. 1 First, electrophoretic analysis showed that actin and HMWP are prominent polypeptide components of isolated gels (Weihing, 1976a, b, 1977). Second, dilution of
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影响因子：
64.5
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1083/jcb.68.3.602
发表时间：
1976-03
期刊：
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影响因子：
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影响因子：
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影响因子：
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