Middaugh C R, Ji T H
Eur J Biochem. 1980 Sep;110(2):587-92. doi: 10.1111/j.1432-1033.1980.tb04902.x.
The subunit structure of membrane-associated spectrin was investigated by crosslinking human erythrocyte membranes with a variety of cleavable photosensitive heterobifunctional reagents by flash photolysis millisecond crosslinking. Crosslinked complexes were analyzed on agarose-polyacrylamide gels whose high exclusion limit the resolution of molecular with Mr larger than 10(6). Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420 000, 170 000 and 910 000, as well as a band at the top of the gels. No significant bands were detectable between the 910 000-Mr band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tatramer, suggesting a limiting tetrameric stoichiometry for membrane-associated spectrin. The rate of formation of spectrin oligomers was examined by increasing crosslinking time. Dimers accumulate before trimer and tetramer, and as tetramer increased, dimer diminished. A simple kinetic model is employed which is also consistent with spectrin tetramer existing on the cytoplasmic surface of the erythrocyte membrane as the basic structural unit.
通过闪光光解毫秒交联法，用多种可裂解的光敏异双功能试剂使人类红细胞膜交联，从而研究膜相关血影蛋白的亚基结构。交联复合物在琼脂糖 - 聚丙烯酰胺凝胶上进行分析，该凝胶的高排阻极限可分辨分子量大于10^6的分子。膜空壳的交联产生了一系列新条带，其表观分子量约为420000、170000和910000，以及凝胶顶部的一条带。在910000分子量的条带和凝胶顶部的物质之间未检测到明显条带。这三条带的分子量分别对应血影蛋白二聚体、三聚体和四聚体，表明膜相关血影蛋白的化学计量比限制为四聚体。通过增加交联时间来检测血影蛋白寡聚体的形成速率。二聚体在三聚体和四聚体之前积累，随着四聚体增加，二聚体减少。采用了一个简单的动力学模型，该模型也与作为基本结构单元存在于红细胞膜细胞质表面的血影蛋白四聚体一致。
