Kitajima H, Yamaguchi T, Kimoto E
Department of Chemistry, Faculty of Science, Fukuoka University.
J Biochem. 1990 Dec;108(6):1057-62. doi: 10.1093/oxfordjournals.jbchem.a123305.
The effects of cross-linking of membrane proteins on hemolysis of human erythrocytes under high pressure (2.0 kbar) were examined. The membrane proteins were cross-linked by oxidation of their SH-groups with diamide (0.05-0.5 mM) under different pressures (1-1,000 bar) at which no hemolysis occurs. As the pressure during diamide treatment was raised, the degree of hemolysis under 2.0 kbar and the quantity of cytoskeletal proteins extracted in a low ionic strength medium were gradually decreased. However, both values were increased by reduction with dithiothreitol. From the determination of membrane SH-groups, it was found that cross-linking of membrane proteins by diamide was accelerated under pressure. Only in erythrocytes treated with diamide under pressure were parts of spectrin and ankyrin, in addition to band 3 and band 4.2 proteins, extracted by using Triton X-100. One- and two-dimensional SDS-PAGE of membrane proteins showed that cross-linking of the membrane with cytoskeletal meshwork through linking proteins, in addition to that of membrane proteins themselves, was formed only in the diamide treatment under pressure. These results indicate that pressure-induced hemolysis is greatly suppressed by the supramolecular-weight polymers formed among membrane proteins, and that the high pressure technique is useful for cross-linking membrane proteins with diamide.
研究了高压（2.0千巴）下膜蛋白交联对人红细胞溶血的影响。在不同压力（1 - 1000巴）下，用二酰胺（0.05 - 0.5 mM）氧化膜蛋白的SH基团使其交联，此压力下不会发生溶血。随着二酰胺处理时压力升高，2.0千巴下的溶血程度以及在低离子强度介质中提取的细胞骨架蛋白量逐渐降低。然而，用二硫苏糖醇还原后，这两个值均增加。通过测定膜SH基团发现，压力下二酰胺对膜蛋白的交联作用加速。仅在压力下用二酰胺处理的红细胞中，除了带3和带4.2蛋白外，血影蛋白和锚蛋白的部分也能用Triton X - 100提取。膜蛋白的一维和二维SDS - PAGE表明，除了膜蛋白自身交联外，仅在压力下的二酰胺处理中，通过连接蛋白形成了膜与细胞骨架网络的交联。这些结果表明，膜蛋白之间形成的超高分子量聚合物极大地抑制了压力诱导的溶血，并且高压技术有助于用二酰胺交联膜蛋白。
