Purification and characterization of Band 3, the major intrinsic membrane protein of the bovine erythrocyte membrane.
Purification and characterization of Band 3, the major intrinsic membrane protein of the bovine erythrocyte membrane.
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Band 3（牛红细胞膜的主要内在膜蛋白）的纯化和表征。
DOI：
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发表时间：
1980
期刊：
影响因子：
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通讯作者：
S. Makino
中科院分区：
文献类型：
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作者：
H. Nakashima;S. Makino
在非离子去污剂九乙二醇正十二烷基醚（C12 E9）存在下，通过以下步骤从牛红细胞膜中分离高纯度的条带3：（1）通过用2%C12E9溶解从血影中选择性去除条带2.6（2）用4%C12E9从2%C12E9处理的膜残余物中提取富含条带3的级分，和（3）通过氨乙基缀合的Sepharose 4 B柱层析纯化条带3。用1%C12E9溶解红细胞膜蛋白并用2，3-二甲基马来酸酐处理，通过氨乙基偶联的Sepharose 4 B柱层析，也以良好的产率纯化了人带3。牛和人带3之间在C12 E9的CD光谱、氨基酸组成和十二烷基硫酸钠凝胶电泳中的迁移率方面没有显著差异。平均疏水性和判别函数的计算表明，牛带3可以归类为一个典型的完整的膜蛋白。牛条带3显示在0.1% C12 E9中形成二聚体和更高聚集体的趋势;这些在十二烷基硫酸钠溶液中抗解离成单体，此外，蛋白质在高浓度盐酸胍溶液中保留残留二级结构，表明可能存在疏水氨基酸残基的延伸序列。
Band 3 from bovine erythrocyte membranes was isolated in a state of high purity by the following steps in the presence of a nonionic detergent, nonaethyleneglycol n-dodecyl ether (C12E9): (1) selective removal of Band 2.6 from ghosts by solubilization with 2% C12E9 (2) extraction of Band 3-rich fraction with 4% C12E9 from 2% C12E9-treated membrane residues, and (3) purification of Band 3 by aminoethyl-conjugated Sepharose 4B column chromatography. Human Band 3 was also purified in good yield by aminoethyl-conjugated Sepharose 4B column chromatography of erythrocyte membrane proteins solubilized with 1% C12E9 and treated with 2,3-dimethymaleic anhydride. There were no significant differences in CD spectra in C12E9, amino acid compositions, and migration mobilities in sodium dodecyl sulfate-gel electrophoresis between bovine and human Band 3. Calculations of average hydrophobicity and discriminant function demonstrated that bovine Band 3 could be categorized as a typical integral membrane protein. Bovine Band 3 showed a tendency to form a dimer and higher aggregates in 0.1% C12E9; these were resistant to dissociation into monomers in sodium dodecyl sulfate solution and, further, the protein retained residual secondary structure in highly concentrated guanidine hydrochloride solution, indicating the possible presence of an extended sequence of hydrophobic amino acid residues.