Connor J, Gillum K, Schroit A J
Department of Cell Biology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Biochim Biophys Acta. 1990 Jun 11;1025(1):82-6. doi: 10.1016/0005-2736(90)90193-r.
The maintenance of lipid asymmetry in the plasma membrane of human red blood cells (RBC) was investigated by assessing the equilibrium distribution of exogenously inserted NBD-labeled phosphatidylserine (PS) and endogenous PS in RBC and hypotonically lysed ghosts. PS distribution was determined by the ability to 'back-exchange' NBD-lipids into acceptor membranes and bovine serum albumin, and by prothrombinase complex assay for endogenous PS. To maintain the normal asymmetric distribution of PS in RBC, ghosts required Mg2+ in the lysis buffer. The inclusion of Ca2+, even in the presence of Mg2+ resulted in complete randomization of endogenous and exogenously inserted PS. These results indicate that NBD-labeled PS analogs faithfully monitor the distribution of endogenous PS during ghost preparation. In contrast, treatment of RBC with bovine serum albumin had no effect on the distribution of endogenous PS, although it resulted in a time-dependent movement of NBD-labeled PS from the inner to the outer leaflet (flop). This phenomenon was dependent on continuous incubation in the presence of albumin and could not be duplicated when pure acceptor membranes were used.
通过评估外源性插入的NBD标记磷脂酰丝氨酸（PS）和内源性PS在人红细胞（RBC）及低渗裂解的红细胞影中的平衡分布，研究了人红细胞质膜中脂质不对称性的维持情况。PS的分布通过将NBD - 脂质“反向交换”到受体膜和牛血清白蛋白中的能力以及对内源性PS的凝血酶原酶复合物测定来确定。为了维持RBC中PS的正常不对称分布，红细胞影在裂解缓冲液中需要Mg2 +。即使存在Mg2 +，加入Ca2 +也会导致内源性和外源性插入的PS完全随机化。这些结果表明，NBD标记的PS类似物在红细胞影制备过程中忠实地监测内源性PS的分布。相比之下，用牛血清白蛋白处理RBC对内源性PS的分布没有影响，尽管它导致NBD标记的PS从内膜小叶向外侧小叶（翻转）的时间依赖性移动。这种现象依赖于在白蛋白存在下的持续孵育，并且当使用纯受体膜时无法复制。
