Yamaguchi T, Yamada S, Kimoto E
Department of Chemistry, Faculty of Science, Fukuoka University.
J Biochem. 1994 Apr;115(4):659-63. doi: 10.1093/oxfordjournals.jbchem.a124392.
To study the effect of cross-linking of membrane proteins on vesiculation of human erythrocytes by dimyristoylphosphatidylcholine (DMPC), red cells were treated with diamide at atmospheric pressure or 100 MPa and then incubated with DMPC in buffers of pH 6.5-8.5. Irrespective of buffer pH, the amount of released vesicles increased upon cross-linking of membrane proteins but approached the control level upon reduction of the cross-linking by dithiothreitol. Similar enhancement of vesicle release was also observed in N-ethylmaleimide-treated red cells. Hemolysis during vesiculation was observed only in red cells treated with diamide at 100 MPa. Furthermore, the composition of membrane proteins in released vesicles was analyzed by SDS-PAGE. Membrane vesicles released from intact red cells or the cells treated with diamide at atmospheric pressure contained band 3 as a major membrane protein. On the other hand, membrane vesicles from red cells treated with diamide at 100 MPa contained protein 4.1 in addition to band 3 and the orientation of these proteins was similar to that in intact cells. These results indicate that the amount and membrane protein composition of DMPC-induced vesicles are much affected by chemical modification of SH-groups in red cell membrane proteins.
为研究二肉豆蔻酰磷脂酰胆碱（DMPC）对人红细胞膜蛋白交联及囊泡化的影响，将红细胞在常压或100 MPa下用二酰胺处理，然后在pH 6.5 - 8.5的缓冲液中与DMPC孵育。无论缓冲液pH如何，膜蛋白交联后释放的囊泡数量增加，但用二硫苏糖醇降低交联程度后接近对照水平。在N - 乙基马来酰亚胺处理的红细胞中也观察到类似的囊泡释放增强。仅在100 MPa下用二酰胺处理的红细胞中观察到囊泡化过程中的溶血现象。此外，通过SDS - PAGE分析释放囊泡中的膜蛋白组成。从完整红细胞或常压下用二酰胺处理的细胞释放的膜囊泡中，带3是主要的膜蛋白。另一方面，100 MPa下用二酰胺处理的红细胞释放的膜囊泡除带3外还含有蛋白4.1，且这些蛋白的取向与完整细胞中的相似。这些结果表明，DMPC诱导的囊泡的数量和膜蛋白组成受红细胞膜蛋白中SH基团化学修饰的显著影响。
