猪血小板中分子量5.4×10^4钙结合蛋白的分离、纯化和鉴定

PURIFICATION AND CHARACTERIZATION OF MW 5.4×10CALCIUM BINDING PROTEIN FROMPORCINE PLATELET

从猪血中分离得到血小板，经反复洗涤后用Triton X-100（1％）溶解血小板，以DEAE纤维素作离子交换层析，经梯度洗脱后，洗脱组分以Dot-blotting放射自显影法进行钙结合活性测定，收集钙结合活性最强的洗脱组分，再以羟基磷灰石作吸附层析，用磷酸缓冲液作梯度洗脱，分离纯化了一种具有较强钙结合活性的蛋白质。SDS－聚丙烯酰胺凝胶电泳（PAGE）测定其分子量为5.4×10^4。该蛋白质能够促进天然磷脂酰丝氨酸（PS）脂质体聚集，并且这种作用依赖钙离子的存在。提示该蛋白质可能属于一类新的钙结合蛋白－Annexins，其功能可能与钙调节的血小板分泌有关，或在血小板骨架成分与膜相互作用时起关键的桥梁作用。

Porcine platelet was washed repeatedly with PWS B, then dissolved in Triton X-100. The platelet lysate was applied on DEAE ion-exchange column and eluated with linear gradient eluation. The calcium binding activity of the fraction was assayed by Dot-blotting autoradiography. The active fraction was purified further on hydroxy apatite column, eluated with linear gradient eluation of phosphate buffer. A calcium binding protein was found in this eluant. The molecular weight of this protein is MW 5.4×104 as determined by SDS-polyacrylamide gel electrophoresis (PAGE). This protein was shown to enhance Ca2+-induced aggregation of phosphatidylserine (PS) lipoSome. It indicates thatthis protein may be a member of a new class of calcium binding protein-----Anne-xin. Its function may be related to the calcium regulated platelet secretion, or to play an important role in the interaction between platelet cytoskeleton and membrane.