Haest C W, Kamp D, Deuticke B
Institut für Physiologie, Medizinische Fakultät der RWTH, Universitätsklinikum, Aachen, Germany.
Biochim Biophys Acta. 1997 Apr 3;1325(1):17-33. doi: 10.1016/s0005-2736(96)00239-8.
In order to characterize in more detail the previously observed (Dressler et al. (1983) Biochim. Biophys. Acta 732, 304-307) increases in transbilayer mobility of phospholipids in the erythrocyte membrane following electroporation at 0 degrees C and subsequent resealing at 37 degrees C of the cells, we have studied rates of flip and flop as well as steady state distributions of the fluorescent N-(NBD)-aminohexanoyl-analogues of the four major membrane phospholipids. Measurements comprised the passive non-mediated components as well as those mediated by specific translocases (flippase and floppase). The major new findings and insights can be summarized as follows. (1) The enhancement of passive transbilayer mobility which increases with the strength, duration, and number of field pulses at 0 degrees C, cannot be fully reversed by subsequent resealing at 37 degrees C. Flip-flop remains considerably elevated relative to the original values.(2) Enhanced mobilities induced by electroporation differ for the probes studied in the sequence SM <<< PS << PC < PE. Other membrane perturbations going along with enhanced flip-flop share only in part this pattern. (3) Mediated, ATP-dependent components of flip and flop of the probes are suppressed in electroporated/resealed cells, partly due to loss of cellular Mg2+, partly - in case of flippase - due to competition by externalized endogenous PS. (4) Electroporated/resealed cells provide an elegant means to demonstrate the contribution of various components of flip and flop to the steady state transbilayer distribution of phospholipids, in particular the role of passive mobility. The new, detailed information on the displacements of phospholipid between the two leaflets of the membrane bilayer in porated/resealed cells will help to understand erythrocyte shape changes following poration and during resealing (Henszen et al. (1993) Biol. Chem. Hoppe-Seyler 374, 114).
为了更详细地表征先前观察到的（德雷斯勒等人，1983年，《生物化学与生物物理学报》732卷，304 - 307页）在0℃进行电穿孔并随后于37℃对细胞进行重新封闭后红细胞膜中磷脂跨双层流动性的增加，我们研究了四种主要膜磷脂的荧光N -（NBD）-氨基己酰类似物的翻转和摆动速率以及稳态分布。测量包括被动非介导成分以及由特定转位酶（翻转酶和摆动酶）介导的成分。主要的新发现和见解可总结如下。（1）在0℃时随着场脉冲强度、持续时间和数量增加而增强的被动跨双层流动性，在随后于37℃重新封闭时不能完全恢复。翻转相对于原始值仍显著升高。（2）电穿孔诱导的流动性增强对于所研究的探针而言，顺序为SM <<< PS << PC < PE。与增强的翻转相关的其他膜扰动仅部分具有这种模式。（3）在电穿孔/重新封闭的细胞中，探针的介导的、ATP依赖的翻转和摆动成分受到抑制，部分原因是细胞内Mg2 +的丧失，部分原因（对于翻转酶而言）是由于外化的内源性PS的竞争。（4）电穿孔/重新封闭的细胞提供了一种巧妙的方法来证明翻转和摆动的各种成分对磷脂稳态跨双层分布的贡献，特别是被动流动性的作用。关于在穿孔/重新封闭的细胞中膜双层两个小叶之间磷脂位移的新的详细信息将有助于理解穿孔后和重新封闭期间红细胞的形状变化（亨森等人，1993年，《生物化学杂志》霍普 - 赛勒374卷，114页）。
