Isolation and partial characterization of human platelet vinculin.
Isolation and partial characterization of human platelet vinculin.
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人血小板质蛋白的隔离和部分表征。
DOI：
10.1083/jcb.100.3.669
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发表时间：
1985-03
影响因子：
7.8
通讯作者：
Bryan, J
中科院分区：
文献类型：
--
作者：
Rosenfeld, G C;Hou, D C;Dingus, J;Meza, I;Bryan, J
用DEAE-Sephacel和SepharoseCl-4 B连续层析从人血小板中分离到一个130，000 Mr的蛋白质。低剪切粘度测量表明，富集蛋白后，DEAE-Sephacel层析抑制肌动蛋白聚合。在EGTA的存在下，这种效果比在钙的存在下稍大。通过Sepharose Cl-4 B层析进一步纯化导致这种抑制作用完全丧失。用荧光肌动蛋白的研究在DEAE-Sephacel-或SepharoseCl-4 B-纯化的黏着斑蛋白中没有检测到成核或“+”封端活性。在小鼠中产生的针对130，000-mol-wt蛋白质的抗体显示出与鸡砂囊粘着斑蛋白交叉反应，并且在WI 38细胞和Madin-Darby犬肾细胞中检测到类似分子量的蛋白质。Madin-Darby犬肾细胞的裂解实验表明，大多数黏着斑蛋白可溶于Triton X-100，尽管发现一些与不溶性细胞骨架残留物相关。如其他人所述，通过免疫荧光，WI 38细胞中的黏着斑蛋白定位于粘附斑。血小板中的离散定位也被检测到，似乎取决于它们的粘附和扩散状态。这些实验的结果表明，人类血小板含有一种类似于黏着斑蛋白的蛋白质。目前尚不清楚血小板黏着斑蛋白是否与其他细胞类型中发现的粘附斑类似的结构有关。这些数据表明，以前报道的影响非肌肉纽蛋白肌动蛋白聚合可能是由于污染物或污染物。
A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE- Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.