Lux S E, John K M, Ukena T E
J Clin Invest. 1978 Mar;61(3):815-27. doi: 10.1172/JCI108996.
We measured spectrin "extractability" in erythrocytes which were metabolically depleted by incubation at 37 degrees C in plasma or glucose-free buffers. Membranes were extracted with 1 mM EDTA (pH 8, 40 h, 4 degrees C) and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This procedure solubilized 85--90% of the spectrin, actin, and residual hemoglobin from ghosts of fresh erythrocytes. In incubated erythrocytes, inextractable spectrin rapidly accumulated when ATP concentrations fell below 0--15% of normal. In severely depleted cells, 60--90% of the total ghost spectrin became inextractable. Inextractability was not abolished by physically disrupting the ghost before extraction, but was reversed when erythrocyte ATP was replenished with adenosine. The accumulation of inextractable spectrin correlated temporally with the increase in apparent membrane deformability and the increases in erythrocyte vicosity, calcium content, sodium gain, and potassium loss characteristic of ATP-depleted erythrocytes. No change in integral membrane protein topography (assessed by the distribution of intramembranous particles and concanavalin A surface-binding sites) was detected in depleted cells. Analogous changes were observed in erythrocytes exposed to extremes of pH and temperature. When the pH in the erythrocyte interior fell below 5.5, a pH where spectrin was aggregated and isoelectrically precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrin extractability. Similarly above 49 degrees C, a temperature where spectrin was denatured and precipitated, erythrocyte viscosity rose as inextractable spectrin accumulated. These observations provide direct evidence of a change in the physical state of spectrin associated with a change in erythrocyte shape and deformability. They support the concept that erythrocyte shape and deformability are largely determined by the shape and deformability of the spectrin-actin protein meshwork which laminates the inner membrane surface.
我们测定了红细胞中血影蛋白的“可提取性”，这些红细胞通过在37℃下于血浆或无葡萄糖缓冲液中孵育而代谢耗尽。用1 mM乙二胺四乙酸（pH 8，40小时，4℃）提取膜，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行分析。该程序可溶解新鲜红细胞幽灵中85%-90%的血影蛋白、肌动蛋白和残留血红蛋白。在孵育的红细胞中，当ATP浓度降至正常水平的0%-15%以下时，不可提取的血影蛋白迅速积累。在严重耗尽的细胞中，幽灵中总血影蛋白的60%-90%变得不可提取。在提取前通过物理破坏幽灵并不能消除不可提取性，但当用腺苷补充红细胞ATP时，不可提取性会逆转。不可提取血影蛋白的积累在时间上与ATP耗尽的红细胞特有的表观膜变形性增加以及红细胞粘度、钙含量、钠摄取和钾丢失的增加相关。在耗尽的细胞中未检测到完整膜蛋白拓扑结构的变化（通过膜内颗粒和伴刀豆球蛋白A表面结合位点的分布评估）。在暴露于极端pH值和温度的红细胞中也观察到了类似的变化。当红细胞内部的pH值降至5.5以下时，血影蛋白在此pH值下会聚集并发生等电沉淀，红细胞和幽灵的粘度增加，同时血影蛋白的可提取性显著降低。同样，在49℃以上，血影蛋白在此温度下会变性并沉淀，随着不可提取血影蛋白的积累，红细胞粘度升高。这些观察结果提供了血影蛋白物理状态变化与红细胞形状和变形性变化相关的直接证据。它们支持这样一种概念，即红细胞的形状和变形性在很大程度上由覆盖内膜表面的血影蛋白-肌动蛋白蛋白网络的形状和变形性决定。
