Korsgren C, Cohen C M
Department of Biomedical Research, St. Elizabeth's Hospital, Boston, Massachusetts 02135.
J Biol Chem. 1988 Jul 25;263(21):10212-8.
We have examined the associations of purified red cell band 4.2 with red cell membrane and membrane skeletal proteins using in vitro binding assays. Band 4.2 bound to the purified cytoplasmic domain of band 3 with a Kd between 2 and 8 X 10(-7) M. Binding was saturable and slow, requiring 2-4 h to reach equilibrium. This finding confirms previous work suggesting that the principal membrane-binding site for band 4.2 lies within the 43-kDa cytoplasmic domain of band 3 (Korsgren, C., and Cohen, C. M. (1986) J. Biol. Chem. 261, 5536-5543). Band 4.2 also bound to purified ankyrin in solution with a Kd between 1 and 3.5 X 10(-7) M. As with the cytoplasmic domain of band 3, binding was saturable and required 4-5 h to reach equilibrium. Reconstitution with ankyrin of inside-out vesicles stripped of all peripheral proteins had no effect upon band 4.2 binding to membranes; similarly, reconstitution with band 4.2 had no effect upon ankyrin binding. This shows that ankyrin and band 4.2 bind to distinct loci within the 43-kDa band 3 cytoplasmic domain. Coincubation of ankyrin and band 4.2 in solution partially blocked the binding of both proteins to the membrane. Similarly, coincubation of bands 4.1 and 4.2 in solution partially blocked binding of both to membranes. In all cases, the data suggest the possibility that domains on each of these proteins responsible for low affinity membrane binding are principally affected. The data also provide evidence for an association of band 4.2 with band 4.1. Our results show that band 4.2 can form multiple associations with red cell membrane proteins and may therefore play an as yet unrecognized structural role on the membrane.
我们使用体外结合试验研究了纯化的红细胞带4.2与红细胞膜及膜骨架蛋白之间的关联。带4.2与纯化的带3胞质结构域结合，解离常数（Kd）在2至8×10⁻⁷ M之间。结合具有饱和性且速度缓慢，需要2 - 4小时才能达到平衡。这一发现证实了先前的研究工作，表明带4.2的主要膜结合位点位于带3的43 kDa胞质结构域内（科尔斯格伦，C.，和科恩，C. M.（1986年）《生物化学杂志》261，5536 - 5543）。带4.2也与溶液中的纯化锚蛋白结合，Kd在1至3.5×10⁻⁷ M之间。与带3的胞质结构域一样，结合具有饱和性，需要4 - 5小时才能达到平衡。用锚蛋白重建去除了所有外周蛋白的外翻小泡，对带4.2与膜的结合没有影响；同样，用带4.2重建对外周蛋白的结合也没有影响。这表明锚蛋白和带4.2结合于43 kDa带3胞质结构域内的不同位点。在溶液中共同孵育锚蛋白和带4.2会部分阻断这两种蛋白与膜的结合。同样，在溶液中共同孵育带4.1和带4.2也会部分阻断它们与膜的结合。在所有情况下，数据表明这些蛋白中负责低亲和力膜结合的结构域可能主要受到影响。数据还为带4.2与带4.1之间的关联提供了证据。我们的结果表明，带4.2可以与红细胞膜蛋白形成多种关联，因此可能在膜上发挥尚未被认识到的结构作用。
