Abbott R E, Schachter D
J Biol Chem. 1976 Nov 25;251(22):7176-83.
Maleimides impermeant to human erythrocyte membranes have been synthesized and applied to studies of the sulfhydryl groups of the membrane. Reaction of radioactive dextran-maleimide and glutathione-maleimide with either intact erythrocytes or ghosts yields sulfhydryl titers for the outer (exofacial) and inner (endofacial) surfaces, respectively, of 1.5 to 1.7 and 27 to 28 amol/cell. Corresponding values for sulfhydryl groups within the membrane interior, as estimated with radioactive N-ethylmaleimide, are 16 to 22 amol/cell. After exofacial labeling of intact cells with [35S]glutathione-maleimide, autoradiography of sodium dodecyl sulfate-polyacrylamide gels demonstrates four bands (alpha, beta, gamma, and delta) containing, respectively, 13%, 63%, 11%, and 13% of the radioactivity. The major beta-band corresponds in position to polypeptides of molecular weight 40,000 to 70,000 and to Coomassie brilliant blue-stained Band 5. Selective extraction demonstrates that the major Band 5 protein is not identical with the labeled beta-band polypeptides. Following endofacial labeling of ghosts with [35S]glutathione-maleimide, autoradiography reveals radioactivity in all of the major Coomassie brilliant blue bands. The impermeant maleimides described are also applicable to studies of discrete functional proteins of the erythrocyte membrane, including the hexose transport mechanism and the major Rho antigenic site.
已合成对人红细胞膜不可渗透的马来酰亚胺，并将其应用于膜巯基的研究。放射性葡聚糖 - 马来酰亚胺和谷胱甘肽 - 马来酰亚胺与完整红细胞或血影反应，分别得出外表面（外侧面）和内表面（内侧面）的巯基滴定值为1.5至1.7和27至28 amol/细胞。用放射性N - 乙基马来酰亚胺估计的膜内部巯基的相应值为16至22 amol/细胞。在用[35S]谷胱甘肽 - 马来酰亚胺对完整细胞进行外表面标记后，十二烷基硫酸钠 - 聚丙烯酰胺凝胶的放射自显影显示出四条带（α、β、γ和δ），分别含有13%、63%、11%和13%的放射性。主要的β带在位置上对应于分子量为40,000至70,000的多肽以及考马斯亮蓝染色的第5带。选择性提取表明主要的第5带蛋白与标记的β带多肽不同。在用[35S]谷胱甘肽 - 马来酰亚胺对血影进行内表面标记后，放射自显影显示所有主要的考马斯亮蓝带都有放射性。所述的不可渗透马来酰亚胺也适用于红细胞膜离散功能蛋白的研究，包括己糖转运机制和主要的Rho抗原位点。
