Davis J Q, Bennett V
J Biol Chem. 1984 Feb 10;259(3):1874-81.
Polypeptides of Mr = 190,000-220,000 that cross-react with erythrocyte ankyrin were detected in immunoblots of membranes from pig lens, pig brain, and rat liver. The cross-reacting polypeptides from brain were cleaved by chymotrypsin to fragments of Mr = 95,000 and 72,000 which are the same size as fragments obtained with erythrocyte ankyrin. The brain 72,000 Mr fragment associated with erythrocyte spectrin, and the binding occurred at the same site as that of erythrocyte ankyrin 72,000 Mr fragment since (a) brain 72,000 Mr fragment was adsorbed to erythrocyte spectrin-agarose and (b) 125I-labeled erythrocyte spectrin bound to brain 72,000 Mr fragment following transfer of the fragment from a sodium dodecyl sulfate gel to nitrocellulose paper, and this binding was displaced by erythrocyte ankyrin 72,000 Mr fragment. Brain 72,000 Mr fragment was purified about 400-fold by selective extraction and by continuous chromatography on columns attached in series containing DEAE-cellulose followed by erythrocyte spectrin coupled to agarose, and finally hydroxylapatite. The brain 72,000 Mr fragment was not derived from contaminating erythrocytes since peptide maps of pig brain and pig erythrocyte 72,000 Mr fragments were distinct. The amount of brain 72,000 Mr fragment was estimated as 0.28% of membrane protein or 39 pmol/mg based on radioimmunoassay with 125I-labeled brain fragment and antibody against erythrocyte ankyrin. Brain spectrin tetramer was present in about the same number of copies (30 pmol/mg of membrane protein) based on densitometry of Coomassie blue-stained sodium dodecyl sulfate gels. The binding site on brain spectrin for both brain and erythrocyte ankyrin 72,000 Mr fragments was localized by electron microscopy to the midregion of spectrin tetramers about 90 nM from the near end and 110 nM from the far end. These studies demonstrate the presence in brain membranes of a protein closely related to erythrocyte ankyrin, and are consistent with a function of the brain ankyrin as a membrane attachment site for brain spectrin.
在猪晶状体、猪脑和大鼠肝脏细胞膜的免疫印迹中检测到分子量为190,000 - 220,000且与红细胞锚蛋白发生交叉反应的多肽。来自脑的交叉反应多肽被胰凝乳蛋白酶切割成分子量为95,000和72,000的片段，其大小与红细胞锚蛋白切割得到的片段相同。脑的分子量为72,000的片段与红细胞血影蛋白结合，且结合位点与红细胞锚蛋白分子量为72,000的片段相同，这是因为：(a) 脑的分子量为72,000的片段可吸附至红细胞血影蛋白 - 琼脂糖上；(b) 将该片段从十二烷基硫酸钠凝胶转移至硝酸纤维素纸上后，125I标记的红细胞血影蛋白可与脑的分子量为72,000的片段结合，且这种结合可被红细胞锚蛋白分子量为72,000的片段取代。通过选择性提取以及在串联的含有DEAE - 纤维素的柱上连续层析，随后在与琼脂糖偶联的红细胞血影蛋白柱上，最后在羟基磷灰石柱上进行层析，脑的分子量为72,000的片段被纯化了约400倍。脑的分子量为72,000的片段并非来自污染的红细胞，因为猪脑和猪红细胞分子量为72,000的片段的肽图不同。基于用125I标记的脑片段和抗红细胞锚蛋白抗体进行的放射免疫测定，脑的分子量为72,000的片段的量估计为膜蛋白的0.28%或39 pmol/mg。基于考马斯亮蓝染色的十二烷基硫酸钠凝胶的光密度测定，脑血影蛋白四聚体的拷贝数大致相同（30 pmol/mg膜蛋白）。通过电子显微镜将脑和红细胞锚蛋白分子量为72,000的片段在脑血影蛋白上的结合位点定位在血影蛋白四聚体的中部区域，距离近端约90 nM，距离远端约110 nM。这些研究证明了在脑细胞膜中存在一种与红细胞锚蛋白密切相关的蛋白质，并且与脑锚蛋白作为脑血影蛋白的膜附着位点的功能一致。
