Lutz H U, von Däniken A, Semenza G, Bächi T
Biochim Biophys Acta. 1979 Apr 4;552(2):262-80. doi: 10.1016/0005-2736(79)90282-7.
A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.
本文描述了一种从人红细胞中分离富含血型糖蛋白囊泡的方法，具体操作是用非离子去污剂 Triton X - 100 提取在 37℃、pH 4.5 条件下孵育 30 分钟的膜，并在低离子强度和高离子强度下洗涤。提取物中血型糖蛋白的富集倍数为 11.8±2.4 倍，每毫克蛋白质含有 325±69 微克唾液酸，占总唾液酸的 61±16%。去除 Triton X - 100 后，三分之一的总血型糖蛋白与共提取的内源性脂质形成富含血型糖蛋白的囊泡，沉降、葡聚糖密度梯度离心和电子显微镜观察均证实了这一点。添加外源性脂质可使富含血型糖蛋白的囊泡比例增加至 87%。使用抗 - M 血清的血凝抑制试验以及血型糖蛋白对胰蛋白酶的可及性表明血型糖蛋白已整合到膜中。冷冻断裂的囊泡未显示出膜内颗粒。提取过程的选择性并非仅仅由于蛋白质组分 3 的二硫键交联所引入的化学限制，因为只有 20%的该蛋白质发生二硫键交联。血型糖蛋白的选择性提取意味着当红细胞膜在 pH 4.5 条件下孵育时，血型糖蛋白与蛋白质组分 3 分离，从而与膜内颗粒分离。这种分离可能先于膜内颗粒的聚集。
