Che A, Morrison I E, Pan R, Cherry R J
Department of Biological and Chemical Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
Biochemistry. 1997 Aug 5;36(31):9588-95. doi: 10.1021/bi971074z.
Rotational diffusion of eosin-5-maleimide-labeled band 3 was measured in erythrocyte membranes at pH 9.4-10.4. Band 3 was found to be more mobile in this pH range than at pH 7.5. Similar results were obtained with spectrin-actin-depleted membranes, where it was further shown that ankyrin is the only detectable protein released from the membrane at pH 10. Further experiments were performed at pH 7.5 to investigate the effects of rebinding purified ankyrin and/or band 4.1 to ghosts stripped of skeletal proteins. Ankyrin was found to reduce band 3 rotational mobility, but band 4.1 had no effect. A fluorescence binding assay revealed that fluorescein isothiocyanate-labeled ankyrin had similar binding parameters to those reported previously using 125I labeling. Finally, the rotational mobility of purified band 3 reconstituted into lipid bilayers was determined before and after ankyrin binding. The results of these reconstitution experiments were globally analyzed, assuming the existence of two populations of band 3 with different correlation times. The faster correlation time is consistent with that expected for either dimers or compact tetramers of band 3. Ankyrin binding reduces the proportion of band 3 contributing to the faster component. This result demonstrates that ankyrin promotes the association of band 3 into more slowly rotating complexes independently of any other components of the erythrocyte membrane. It has been reported that ankyrin contains two binding sites for band 3 [Michaely, P., & Bennett, V. (1995) J. Biol. Chem. 270, 22050-22057]. The results of the present study are thus explained by the ability of ankyrin to cross-link band 3 into larger diameter complexes. Cross-linking by ankyrin in part accounts for the slow components in the anisotropy decays of band 3 in the erythrocyte membrane. Other factors which probably influence band 3 aggregation include the membrane "fluidity" and protein concentration.
在pH 9.4 - 10.4的红细胞膜中测量了用嗜酸性-5-马来酰亚胺标记的带3的旋转扩散。发现带3在此pH范围内比在pH 7.5时更具流动性。对于去除血影蛋白-肌动蛋白的膜也获得了类似的结果，进一步表明锚蛋白是在pH 10时从膜中释放的唯一可检测到的蛋白质。在pH 7.5下进行了进一步的实验，以研究将纯化的锚蛋白和/或带4.1重新结合到去除骨架蛋白的血影上的效果。发现锚蛋白会降低带3的旋转流动性，但带4.1没有影响。荧光结合试验表明，异硫氰酸荧光素标记的锚蛋白具有与先前使用125I标记报道的相似的结合参数。最后，在锚蛋白结合前后测定了重构到脂质双层中的纯化带3的旋转流动性。假设存在具有不同相关时间的两个带3群体，对这些重构实验的结果进行了整体分析。较快的相关时间与带3的二聚体或紧密四聚体预期的时间一致。锚蛋白结合降低了对较快成分有贡献的带3的比例。该结果表明，锚蛋白促进带3缔合形成旋转更慢的复合物，而与红细胞膜的任何其他成分无关。据报道，锚蛋白含有两个与带3结合的位点[米凯利，P.，& 贝内特，V.（1995年）《生物化学杂志》270，22050 - 22057]。因此，本研究的结果可以通过锚蛋白将带3交联成更大直径复合物的能力来解释。锚蛋白的交联部分解释了红细胞膜中带3各向异性衰减中的慢成分。可能影响带3聚集的其他因素包括膜的“流动性”和蛋白质浓度。
