USING ANTIBODIES AGAINST DICTYOSTELIUM MEMBRANES TO IDENTIFY AN ACTIN-BINDING MEMBRANE-PROTEIN
USING ANTIBODIES AGAINST DICTYOSTELIUM MEMBRANES TO IDENTIFY AN ACTIN-BINDING MEMBRANE-PROTEIN
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DOI：
10.1083/jcb.104.3.513
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发表时间：
1987-03-01
影响因子：
7.8
通讯作者：
PETZOLD, AS
中科院分区：
文献类型：
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作者：
BROWN, SS;PETZOLD, AS
用多克隆抗体阻断盘基网柄菌与肌动蛋白的相互作用。正如预期的那样，肌动蛋白主要与膜的内表面相互作用，事实证明整个细胞只能吸收一小部分封闭抗体。用该抗体表明，与肌动蛋白相互作用的膜成分(S)可能是完整的；它们可以用洗涤剂提取，但不能用设计用于提取外周膜蛋白的溶液提取。为了确定负责的蛋白质(S)，将膜的蛋白质转移切割成几个组分，测试它们吸收抗体封闭活性的能力。我们观察到一个相对分子质量为.apprx的单峰。20,000摩尔重量的蛋白质，从而得出结论：20,000摩尔重量的蛋白质是网柄苔藓中主要的整合膜肌动蛋白结合蛋白。这种鉴定参与肌动蛋白-膜相互作用的蛋白质的方法使我们能够首次鉴定肌动蛋白结合膜蛋白，这种鉴定是基于其在天然膜中的活性。
Polyclonal antibodies made against Dictyostelium discoideum membranes were used to block the interaction of those membranes with actin. As expected, actin interacted mostly with the internal surface of the membrane, demonstrated by the fact that whole cells could only absorb out a minor fraction of the blocking antibody. The antibody was used to show that the membrane component(s) which interacted with actin were probably integral; they could be extracted with detergent but not with solutions designed to extract peripheral membrane proteins. To identify the responsible protein(s), Western transfers of membranes were cut into fractions which were tested for their ability to absorb out the blocking activity of the antibody. We observed a single peak at a molecular weight of .apprx. 20,000, and thus conclude that a 20,000-mol-wt protein is a major integral membrane actin-binding protein in Dictyostelium. This approach to the identification of proteins involved in actin-membrane interaction has allowed us to make the first identification of an actin-binding membrane protein which is based on its activity in native membranes.