Pinder J C, Pekrun A, Maggs A M, Brain A P, Gratzer W B
Medical Research Council Muscle and Cell Motility Unit, King's College, London, UK.
Blood. 1995 May 15;85(10):2951-61.
We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.
我们研究了人类红细胞的主要跨膜蛋白、阴离子通道——带3蛋白的缔合状态，以及它与锚蛋白相互作用的异常化学计量和动力学，锚蛋白是膜骨架网络中血影蛋白的连接物。带3蛋白在良性非离子去污剂溶液中以二聚体形式存在。在操作过程中，尤其是在离子交换色谱中，会不可逆地形成四聚体。溶液中的二聚体结合锚蛋白而不会自我缔合。在无锚蛋白的内翻膜囊泡中以及掺入磷脂酰胆碱脂质体后，只有约10%至15%的带3蛋白链在饱和时结合锚蛋白。此外，在脂质体中，这与1:2至1:40之间的蛋白质与脂质比例无关。带3蛋白的结合部分保留在去污剂提取的膜细胞骨架中，但如果在去污剂处理前用胰凝乳蛋白酶切割锚蛋白，则会释放出来；因此，与膜细胞骨架的附着完全通过锚蛋白，而不是通过其他成分如蛋白4.1。该复合物中带3蛋白与锚蛋白的比例表明，至少在去污剂提取后，它由两条带3蛋白链和一条锚蛋白链组成。膜中带3蛋白的结合态和游离态可自由交换。在人工脂质体膜中，锚蛋白与带3蛋白二聚体的结合会导致带3蛋白缔合成更高的聚集体，如冷冻断裂电子显微镜所见。红细胞膜的连续操作，包括制备血影、内翻囊泡以及去除外周蛋白的内翻囊泡，冷冻断裂电子显微镜判断，这些操作会伴随着膜内颗粒的逐渐聚集。因此，膜内颗粒显然在完整细胞中由外周蛋白网络和胞质环境稳定。聚集可能会限制功能性锚蛋白结合位点的数量。然而，尽管外来的锚蛋白能与完整血影膜中血影蛋白四聚体上未占据的结合位点结合，但原位几乎没有或没有锚蛋白能与未占据的带3蛋白二聚体结合，这可能是由于膜骨架网络遮挡了结合位点。
