Solum N O, Olsen T M
Biochim Biophys Acta. 1984 Jun 29;799(3):209-20. doi: 10.1016/0304-4165(84)90263-0.
Glycoprotein Ib could be demonstrated in the Triton-insoluble (cytoskeletal) fraction of platelets prepared with EGTA by SDS-polyacrylamide gel electrophoresis and staining with the periodic acid Schiff's reagent. Crossed immunoelectrophoresis showed that glycoprotein Ib could be extracted from such Triton-insoluble residues when the extraction solution contained 1% Triton X-100 plus 5 mM CaCl2, but not if it also contained leupeptin. This indicates that glycoprotein Ib was associated to structures in the cytoskeletal fraction in such a way that it could be extracted only after activation of a calcium-dependent protease, and degradation of the actin-binding protein was demonstrated. After crossed immunoelectrophoresis of platelet extracts prepared in the presence of leupeptin or EDTA, a glycoprotein Ib-related, rocket-shaped immunoprecipitate was seen originating from the application well. This was interpreted as being related to glycoprotein Ib associated to actin polymers which did not sediment at low-speed centrifugation. Incubation of platelets with 32P as sodium phosphate led to incorporation of phosphatase-sensitive 32P in all of the glycoprotein Ib-related immunoprecipitates except for that of glycocalicin. This supports the idea that glycoprotein Ib traverses the plasma membrane and can be phosphorylated at the inner surface whereas glycocalicin represents the terminal part of the glycoprotein Ib alpha-chain exposed at the outer surface.
通过SDS-聚丙烯酰胺凝胶电泳并用高碘酸希夫试剂染色，可在含有EGTA制备的血小板的Triton不溶性（细胞骨架）组分中检测到糖蛋白Ib。交叉免疫电泳显示，当提取液含有1% Triton X-100加5 mM氯化钙时，糖蛋白Ib可从此类Triton不溶性残渣中提取，但如果提取液中还含有亮抑酶肽则不能提取。这表明糖蛋白Ib与细胞骨架组分中的结构相关联，以至于只有在钙依赖性蛋白酶激活后才能提取，并且证明了肌动蛋白结合蛋白的降解。在存在亮抑酶肽或EDTA的情况下制备的血小板提取物进行交叉免疫电泳后，可见一种与糖蛋白Ib相关的火箭状免疫沉淀物从加样孔中产生。这被解释为与未在低速离心时沉淀的肌动蛋白聚合物相关的糖蛋白Ib有关。用32P作为磷酸钠孵育血小板导致除糖萼素外的所有与糖蛋白Ib相关的免疫沉淀物中均掺入了对磷酸酶敏感的32P。这支持了糖蛋白Ib穿过质膜并可在内表面磷酸化的观点，而糖萼素代表暴露在外表面的糖蛋白Ibα链的末端部分。
