Identification by monoclonal antibodies and characterization of human platelet caldesmon
Identification by monoclonal antibodies and characterization of human platelet caldesmon
复制标题
人血小板钙结合蛋白的单克隆抗体鉴定和表征
作者：
J. Dingus;S. Hwo;J. Bryan
基于肌动蛋白的凝胶是通过人血小板的澄清高盐提取物通过生理盐缓冲液透析来制备的。凝胶用 0.3 M KCl 部分溶解。用肌动蛋白凝胶的 0.3 M KCl 提取物免疫小鼠，并通过脾细胞与骨髓瘤细胞融合产生杂交瘤。产生了三个分泌针对 80-kD 蛋白质的抗体的杂交瘤。这些单克隆抗体对培养细胞中的应力纤维进行染色，并与包括平滑肌在内的多种组织类型中的蛋白质发生交叉反应。鸡砂囊平滑肌中的交叉反应蛋白具有140,000的表观分子量，并被证明是钙结合蛋白，一种钙调蛋白和肌动蛋白结合蛋白(Sobue，K.，Y.Muramoto，M.Fujita，和S.Kakiuchi，Proc.Natl.Acad.Sci.USA，78：5652-5655)。使用单克隆抗体进行免疫印迹，在血小板中未检测到分子量大于 80 kD 的蛋白质。 80-kD 蛋白质是热稳定的，并且使用 Bretscher 报道的用于快速纯化平滑肌钙结合蛋白的程序的修改来纯化（Bretscher，A.，1985，J.Biol.Chem.，259：12873-12880）。 80-kD 蛋白以 Ca++ 依赖性方式与钙调蛋白-琼脂糖结合，并与肌动蛋白丝一起沉淀，但并没有大大增加 F-肌动蛋白溶液的粘度。在钙存在的情况下，肌动蛋白结合活性被钙调蛋白抑制。除了分子量差异外，80 kD 血小板蛋白在功能上与 140 kD 平滑肌钙结合蛋白相似。我们推测 80 kD 的蛋白是血小板钙结合蛋白。
Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross- reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin- Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.
DOI：
10.1016/0006-291x(80)91175-4
发表时间：
1980-01-01
影响因子：
3.1
作者：
MACLEANFLETCHER, S;POLLARD, TD
通讯作者：
POLLARD, TD