Birchmeier W, Lanz J H, Winterhalter K H, Conrad M J
J Biol Chem. 1979 Sep 25;254(18):9298-304.
ATP-induced endocytosis in human erythrocyte ghosts has been studied, and a procedure for the isolation of the endocytotic vesicles is described. Under isotonic conditions and 37 degrees C, optimal endocytosis occurs with concentrations of 4 to 10 mM MgATP. Within 30 min, up to 45% of the membrane is removed from the surface and converted into sealed inside-out vesicles. Local anesthetics, such as chlorpromazine, potentiate ATP-induced endocytosis in ghosts. Forcing cells containing endocytotic vesicles through a hypodermic needle leads to the exclusive fragmentation of the outermost plasma membrane. The endocytosed vesicles can then be separated from these fragments by centrifugation on a gradient of dextran T70. Biochemical analyses indicate that endocytotic vesicles contain full complements of the major membrane proteins (i.e. also spectrin and actin), common phospholipids, fatty acids, and cholesterol. Furthermore, they exhibit a fully intact spectrin component 2 phosphorylation machinery. In contrast, MgATPase activity is largely excluded from these vesicles. The novel inside-out vesicles described have properties different from those of previously analyzed fragments of the erythrocyte membrane. They will permit a detailed study of a native spectrin-actin network now exposed to the outside.
已对人红细胞血影中ATP诱导的内吞作用进行了研究，并描述了一种分离内吞小泡的方法。在等渗条件和37摄氏度下，4至10 mM的MgATP浓度可实现最佳内吞作用。在30分钟内，高达45%的膜从表面脱离并转化为密封的外翻小泡。局部麻醉剂，如氯丙嗪，可增强血影中ATP诱导的内吞作用。迫使含有内吞小泡的细胞通过皮下注射针头会导致最外层质膜的特异性破碎。然后可以通过在葡聚糖T70梯度上离心将内吞小泡与这些碎片分离。生化分析表明，内吞小泡含有主要膜蛋白（即血影蛋白和肌动蛋白）、常见磷脂、脂肪酸和胆固醇的完整成分。此外，它们还具有完全完整的血影蛋白成分2磷酸化机制。相比之下，MgATP酶活性在很大程度上被排除在这些小泡之外。所描述的新型外翻小泡具有与先前分析的红细胞膜片段不同的特性。它们将允许对现在暴露于外部的天然血影蛋白-肌动蛋白网络进行详细研究。
