ACTIN FILAMENT STRUCTURE PROBED WITH MONOCLONAL-ANTIBODIES
ACTIN FILAMENT STRUCTURE PROBED WITH MONOCLONAL-ANTIBODIES
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DOI：
10.1002/cm.970250109
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发表时间：
1993-01-01
影响因子：
--
通讯作者：
WINKELMANN, DA
中科院分区：
文献类型：
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作者：
MONCMAN, CL;PENG, I;WINKELMANN, DA
两个单抗(MAbs)与肌动蛋白的相互作用已被用来确定这些mAbs所定义的表位，并确定这些位置在肌动蛋白细丝(F-肌动蛋白)中的可及性。在放射免疫分析和免疫印迹分析以及免疫沉淀中，这两种单抗都能与肌动蛋白发生特异性反应。肌动蛋白一级结构中表位的位置已经通过有限的肌动蛋白和Western blotts的蛋白分解，或使用在细胞自由翻译实验中合成的截短的肌动蛋白片段的免疫沉淀来确定。这两种单抗都与胰凝乳酶裂解产生的肌动蛋白的C末端片段(残基68375)结合。一个表位进一步定位为9.9kD的多肽，对应于残基5-93。因此，该单抗(2G11.4)确定的表位位于肌动蛋白的LyS6g和Glu93残基之间。通过体外合成的肌动蛋白片段的免疫沉淀法确定另一个表位的位置。去掉肌动蛋白C末端的356-365残基，完全取消了单抗4E3.adl的结合。因此，该单抗定义了一个包含Trp356和Ala365之间残基的表位。用溶液结合试验确定这些表位在天然F-肌动蛋白中的可及性，并用免疫电子显微镜进行表征。单抗4E3.adl与微丝强烈结合，导致F-肌动蛋白的结合或修饰取决于单抗的价态，这表明F-肌动蛋白中的表位很容易获得。相反，mAb2G11.4破坏了F-肌动蛋白结构，导致无定形免疫沉淀物的形成。这些结果对肌动蛋白细丝结构的模型提出了限制。
The interaction of two monoclonal antibodies (mAbs) with actin has been characterized to map die epitopes defined by these mAbs and to determine the accessibility of these sites in the actin filament (F-actin). Both mAbs react specifically with actin in radioimmunoassays and Western blot assays, and by immunoprecipitation. The location of the epitopes within the primary structure of actin has been determined using limited proteolysis of actin and Western blots, or using immunoprecipitation of truncated actin fragments synthesized in a cell free translation assay. Both mAbs bind to the C-terminal fragment of actin (residues 68375) produced by chymotrypsin cleavage. One epitope is further localized to a 9.9 kD peptide corresponding to residues 5-93. Therefore, the epitope defined by this mAb (2G11.4) lies between residues LyS6g and Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal of residues 356-365 from the C-terminus of actin completely abolished the binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of these epitopes in native F-actin was determined with solution binding assays and characterized by immunoelectron microscopy. Monoclonal antibody 4E3.adl binds strongly to filaments, resulting in bundling or decoration of F-actin depending on the valency of the mAb, and indicating that the epitope is readily accessible in F-actin. In contrast, mAb 2G11.4 disrupts F-actin structure, resulting in the formation of an amorphous immunoprecipitate. These results place constraints on models of actin filament structure.