Yamakose H, Sato Y, Suzuki Y
Pharmaceutical Institute, Tohoku University, Sendai, Japan.
Biol Pharm Bull. 1993 Dec;16(12):1282-7. doi: 10.1248/bpb.16.1282.
The interaction between EMI (eosin-5-maleimide) and band 3 of human erythrocytes was studied under various conditions. It was found that the effects of the ionic strength on the EMI-band 3 interaction strongly depended on pH. At pH 6.0, the ionic strength had remarkable effects on the EMI-bound band 3, whereas at pH 7.4, the EMI-band 3 interaction was independent of ionic strength. From the change in the circular dichroism spectra of the EMI-bound band 3, it was revealed that the conformation or the structure of the EMI-binding sites in the cytoplasmic domain of band 3 was strongly dependent on ionic strength. The thermodynamic parameters for the covalent-binding between EMI and band 3 were calculated on the basis of the difference spectra of the EMI and ghost system. The values of activation energy and activation entropy change at pH 6.0 were extraordinarily small compared with those values at pH 7.4. These findings represent the characteristics of the EMI-binding sites in the cytoplasmic domain of band 3. The interaction of EMI with an isolated fragment of the cytoplasmic domain, 43k fragment, was also examined. The circular dichroism spectra of the EMI-bound 43k fragments was significantly different from those of the EMI-bound band 3. This may indicate that the quaternary structure of the EMI-binding site in the cytoplasmic domain of band 3 is altered by an allosteric connection with the membrane-spanning domain of band 3. Further, from the pH titration of the 43k fragment, it was suggested that lysine residues are responsible for the ionic interaction between EMI and the 43k fragment.
在不同条件下研究了EMI（ eosin - 5 - 马来酰亚胺）与人红细胞带3之间的相互作用。发现离子强度对EMI - 带3相互作用的影响强烈依赖于pH值。在pH 6.0时，离子强度对与EMI结合的带3有显著影响，而在pH 7.4时，EMI - 带3相互作用与离子强度无关。从与EMI结合的带3的圆二色光谱变化可知，带3细胞质结构域中EMI结合位点的构象或结构强烈依赖于离子强度。基于EMI与血影系统的差光谱计算了EMI与带3共价结合的热力学参数。与pH 7.4时的值相比，pH 6.0时的活化能和活化熵变的值非常小。这些发现代表了带3细胞质结构域中EMI结合位点的特征。还研究了EMI与细胞质结构域的一个分离片段43k片段的相互作用。与EMI结合的带3相比，与EMI结合的43k片段的圆二色光谱有显著差异。这可能表明带3细胞质结构域中EMI结合位点的四级结构因与带3跨膜结构域的变构连接而改变。此外，通过对43k片段的pH滴定表明，赖氨酸残基负责EMI与43k片段之间的离子相互作用。
