Tsuji A, Ohnishi S
Biochemistry. 1986 Oct 7;25(20):6133-9. doi: 10.1021/bi00368a045.
The effects of incubation of erythrocyte ghosts under various conditions (ionic strength or addition of ankyrin, diamines, or ATP) on the lateral motion of band 3 in the membranes were studied by using the fluorescence photobleaching recovery technique. Incubation of ghosts with exogenous ankyrin increased the immobile fraction of band 3, from 0.6 in intact ghosts to 0.8-0.9 when an average of 0.2 mol of extra ankyrin was bound per mole of band 3. Ankyrin-free band 3 proteins were mobile, but their mobility was governed by the spectrin association state in the cytoskeletal network. The diffusion constant was 5.3 X 10(-11) cm2 s-1 at a spectrin tetramer mole fraction of 0.3-0.4 in 10 mM NaCl/5 mM sodium phosphate, pH 7.8, and decreased 1 order of magnitude when the tetramer fraction increased to 0.5 in higher NaCl concentration (150 mM NaCl). A similar decrease was observed when the spectrin tetramer fraction was increased by 0.2 mM spermine in 10 mM NaCl/10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6. On the other hand, the rotational motion of band 3 in the membranes was not affected by the spectrin association state. Trypsin treatment of ghosts cleaved off the cytoplasmic domain of band 3 and caused a marked (8-fold) increase in the lateral mobility, D = 4.0 X 10(-10) cm2 s-1. These results indicate that the lateral mobility of ankyrin-free band 3 protein is restricted by interactions of their cytoplasmic domain with the cytoskeletal network. A model is presented that band 3 can pass the network when spectrins are in dissociated dimers and cannot pass when they are tetramers. The lateral diffusion constant is thus determined by the spectrin dimer population in the network.
利用荧光光漂白恢复技术，研究了在各种条件下（离子强度或添加锚蛋白、二胺或ATP）对红细胞血影进行孵育后，血影膜中带3蛋白侧向运动的影响。用外源锚蛋白孵育血影会增加带3的固定部分，从完整血影中的0.6增加到每摩尔带3平均结合0.2摩尔额外锚蛋白时的0.8 - 0.9。不含锚蛋白的带3蛋白是可移动的，但其移动性受细胞骨架网络中血影蛋白结合状态的控制。在10 mM NaCl/5 mM磷酸钠（pH 7.8）中，当血影蛋白四聚体摩尔分数为0.3 - 0.4时，扩散常数为5.3×10⁻¹¹ cm² s⁻¹，当四聚体分数在更高的NaCl浓度（150 mM NaCl）下增加到0.5时，扩散常数降低1个数量级。当在10 mM NaCl/10 mM三（羟甲基）氨基甲烷盐酸盐（pH 7.6）中通过添加0.2 mM精胺使血影蛋白四聚体分数增加时，也观察到类似的降低。另一方面，膜中带3的旋转运动不受血影蛋白结合状态的影响。用胰蛋白酶处理血影会切割掉带3的细胞质结构域，并导致侧向移动性显著增加（8倍），D = 4.0×10⁻¹⁰ cm² s⁻¹。这些结果表明，不含锚蛋白的带3蛋白的侧向移动性受到其细胞质结构域与细胞骨架网络相互作用的限制。提出了一个模型，即当血影蛋白处于解离的二聚体状态时，带3可以穿过网络，而当它们是四聚体时则不能穿过。因此，侧向扩散常数由网络中的血影蛋白二聚体数量决定。
