Dynamics of the holes in human erythrocyte membrane ghosts.
Dynamics of the holes in human erythrocyte membrane ghosts.
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DOI：
10.1016/s0021-9258(18)33813-4
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发表时间：
1982-10
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影响因子：
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通讯作者：
M. Lieber;T. Steck
中科院分区：
文献类型：
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作者：
M. Lieber;T. Steck
溶血缓冲液对孔大小的影响-从渗透溶解过程中固定的细胞的显微照片可以看出，溶血孔暂时非常大，半径超过1 μ m（4-7）。由于通过溶血制备并在5 mM NaPi（pH 8.0）、10-LM MgS 〇 4中洗涤的血影通常具有140 A（I）的孔，因此它们必须快速且几乎完全地从渗透应力中恢复。因此，我们问什么参数决定了静止时的孔半径。在一个代表性实验中，将红细胞在0 ℃下悬浮在40倍体积的各种低渗缓冲液中，静置15分钟，立即测定鬼孔。5 m NaPi（pH 8）中的溶血产生的平均孔半径为56 8。（在该缓冲器中重复清洗重影会将孔尺寸扩大到100-150 A。）通过添加45 m蔗糖将裂解培养基的渗透活性增加至60 mosm，产生孔半径为52 A的鬼影，这是一个微不足道的变化。在20 mM Nap（pH 8）中以相同渗透活性裂解使孔半径达到18埃。在pH 6（10 mosm）的5 m Nap中溶血产生的平均孔半径为20 A。在溶血缓冲液中存在300 μ L甘油不会改变孔的大小.我们还观察到，通过透析逐渐溶血产生的孔大小与快速稀释到5 mM Nap中所产生的孔大小相同（1）。我们的结论是，离子强度和pH值的裂解缓冲液，而不是其渗透活性确定孔的大小。因此，我们进一步详细探讨了这些离子参数。阳离子和阴离子对孔尺寸的影响-人们早就知道，鬼对大分子的渗透性可以以温度、盐和pH敏感的方式逆转（8-14）。我们已经表明，密封的动力学过程代表了每个幻影（1）中单个孔的逐渐闭合。进一步研究了该过程的两个特征：孔尺寸的初始变化率和延长孵育后达到的最终（平台）孔尺寸。将清洗过的鬼状物悬浮在不同NaCl浓度的温溶液中，并跟踪孔半径直到其稳定（见参考文献1的图10）。如图1所示，在5 μ M NaPi中，平台半径通常从> 10 ° 8减小到在50 mM Na+存在下达到接近20 ° 8的最小值。
RESULTSEffect of Hemolysis Buffer on Hole Size-It appears from micrographs of cells fixed during osmotic lysis that the hemolytic hole is transiently quite large, exceeding 1 pm in radius (4-7). Since ghosts prepared by hemolysis and washed in 5 mM NaPi (pH 8.0), 10~ LM MgS04 typically have holes of 140 A (I), they must recover rapidly and nearly completely from the osmotic stress. We therefore asked what parameters determine the hole radius at rest. In a representative experiment, red cells were suspended in 40 volumes of various hypotonic buffers at 0 “C, allowed to stand for 15 min, and ghost hole sue assayed immediately. Hemolysis in 5 m NaPi (pH 8) created a mean hole radius of 56 8.(Repeated washing of the ghosts in this buffer enlarges the hole size to 100-150 A.) Increasing the osmotic activity of the lysis medium to 60 mosm by the addition of 45 m sucrose produced ghosts with a hole radius of 52 A, an insignificant change. Lysis at the same osmotic activity in 20 mM Nap,(pH 8) brought the hole radius to 18 A. Hemolysis in 5 m Nap, at pH 6 (10 mosm) produced a mean hole radius of 20 A. The presence of 300 lll~ glycerol in the hemolysis buffer did not alter hole size. We have also observed that gradual hemolysis by dialysis produced holes of identical size with those caused by rapid dilution into 5 mM Nap,(1). We conclude that the ionic strength and pH of the lysis buffer rather than its osmotic activity determined hole size. We therefore explored these ionic parameters in further detail. Effect of Cations and Anions on Hole Size-It has long been known that the permeability of ghosts to macromolecules can be reversed in a temperature, salt, and pH-sensitive fashion (8-14). We have shown that the kinetic course of sealing represents a gradual closure of the single hole in each ghost (1). Two features of this process were further investigated: the initial rate of change of hole size and the final (plateau) hole sue achieved following prolonged incubation. Washed ghosts were suspended in warm solutions of varied NaCl concentration and the hole radius followed until it stabilized (see Fig. 10 of Ref. 1). As shown in Fig. 1, the plateau radius typically decreased from> lo0 8, in 5 II~ M NaPi to reach a minimum near 20 8, in the presence of 50 mM Na’.