Cohen A M, Liu S C, Derick L H, Palek J
Blood. 1986 Oct;68(4):920-6.
Spectrin was shown previously to interact with phosphatidylserine and phosphatidylethanolamine, which are preferentially localized in the inner half of the membrane lipid bilayer, but this interaction is not well characterized. In the present study we used electron microscopy of rotary-shadowed platinum replicas of spectrin dimer-phosphatidylserine complexes to study the interaction of spectrin with phosphatidylserine vesicles. At a spectrin concentration of 0.6 mg/mL, 60% of spectrin dimers were associated with phosphatidylserine vesicles and at a spectrin concentration of 1.2 mg/mL, some vesicles were crosslinked by spectrin dimers. The length of the protruding segment of spectrin dimer from the liposome edge ranged from 400 to 960A degrees and the contact region to phosphatidylserine extended 272 +/- 144A degrees from either end of the molecule. Therefore, these data are consistent with multiple binding sites to phosphatidylserine throughout the spectrin dimer molecule. Spectrin tetramers, when bound to phosphatidylserine liposomes, extended 1804 +/- 79A degrees from the liposome edge and crosslinked liposomes, suggesting that some of the binding sites to phosphatidylserine vesicles is in the proximity of the tail end of spectrin. The association between spectrin dimers to phosphatidylserine was demonstrated by nondenaturing gel electrophoresis. The complexes were separated into multiple bands with molecular weight of 1.4 X 10(6), 1.8 X 10(6), and 2.3 X 10(6). These bands did not represent self-associated spectrin oligomers, since postincubation treatment with Triton-X-100 dissociated them into spectrin dimers. Furthermore, these spectrin high molecular weight bands, as visualized by Coomassie blue absorbance, closely corresponded to the 14C-phosphatidylserine distribution. These data provide ultrastructural and biochemical evidence that spectrin binds to phosphatidylserine at multiple sites including the tail end region.
此前研究表明，血影蛋白可与磷脂酰丝氨酸和磷脂酰乙醇胺相互作用，这两种物质优先定位于膜脂双层的内层，但这种相互作用尚未得到充分表征。在本研究中，我们利用血影蛋白二聚体 - 磷脂酰丝氨酸复合物的旋转阴影铂复制品的电子显微镜来研究血影蛋白与磷脂酰丝氨酸囊泡的相互作用。在血影蛋白浓度为0.6 mg/mL时，60%的血影蛋白二聚体与磷脂酰丝氨酸囊泡相关联；在血影蛋白浓度为1.2 mg/mL时，一些囊泡被血影蛋白二聚体交联。血影蛋白二聚体从脂质体边缘突出部分的长度范围为400至960埃，与磷脂酰丝氨酸的接触区域从分子的任一端延伸272±144埃。因此，这些数据与血影蛋白二聚体分子上存在多个磷脂酰丝氨酸结合位点一致。血影蛋白四聚体与磷脂酰丝氨酸脂质体结合时，从脂质体边缘延伸1804±79埃并交联脂质体，这表明磷脂酰丝氨酸囊泡的一些结合位点位于血影蛋白尾端附近。通过非变性凝胶电泳证明了血影蛋白二聚体与磷脂酰丝氨酸之间的关联。复合物被分离为多条带，分子量分别为1.4×10⁶、1.8×10⁶和2.3×10⁶。这些条带并不代表自缔合的血影蛋白寡聚体，因为用Triton-X-100孵育后处理会将它们解离为血影蛋白二聚体。此外，通过考马斯亮蓝吸光度观察到的这些血影蛋白高分子量条带与¹⁴C-磷脂酰丝氨酸分布密切对应。这些数据提供了超微结构和生化证据，表明血影蛋白在包括尾端区域在内的多个位点与磷脂酰丝氨酸结合。
