Moriyama R, Ideguchi H, Lombardo C R, Van Dort H M, Low P S
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1992 Dec 25;267(36):25792-7.
To determine why deletion of the nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian ovalocytes (SAO) renders the erythrocytes rigid, we compared the structural and functional properties of SAO and normal band 3. Calorimetric data, inhibitor binding studies, and anion transport assays all reveal that the membrane-spanning domain of SAO band 3 is denatured, while proteolysis studies and circular dichroism spectroscopy suggest the mutant domain retains much secondary structure. It is concluded that the transmembrane helices of SAO band 3 are dissociated and randomized but not unfolded. The cytoplasmic domain of SAO band 3 was shown to be structurally and functionally normal based on (i) calorimetric properties, (ii) native conformational change, (iii) ability to form an intersubunit disulfide bond, (iv) affinity and capacity for binding ankyrin and protein 4.1, and (v) kinetics of association with ankyrin. However, both normal and mutant isoforms of band 3 in SAO cells were found to adhere nonspecifically to the spectrin skeleton. Further, when SAO cells were osmotically swollen, the detergent extractability of band 3 became normal. We propose that much of band 3 is nonspecifically entrapped in the spectrin network in SAO cells and that this nonspecific adhesion may be responsible for the rigidity of the SAO erythrocyte.
为了确定从东南亚椭圆形红细胞（SAO）中删除连接带3膜结构域和细胞质结构域的9个氨基酸会导致红细胞变硬的原因，我们比较了SAO和正常带3的结构和功能特性。量热数据、抑制剂结合研究和阴离子转运分析均表明，SAO带3的跨膜结构域已变性，而蛋白水解研究和圆二色光谱表明突变结构域保留了许多二级结构。得出的结论是，SAO带3的跨膜螺旋已解离并随机化，但并未展开。基于以下几点，SAO带3的细胞质结构域在结构和功能上被证明是正常的：（i）量热特性；（ii）天然构象变化；（iii）形成亚基间二硫键的能力；（iv）结合锚蛋白和蛋白4.1的亲和力和容量；（v）与锚蛋白结合的动力学。然而，在SAO细胞中发现带3的正常和突变同工型均非特异性地粘附于血影蛋白骨架。此外，当SAO细胞发生渗透性肿胀时，带3的去污剂可提取性恢复正常。我们提出，SAO细胞中大部分带3非特异性地被困在血影蛋白网络中，这种非特异性粘附可能是SAO红细胞变硬的原因。
