Detergent-mediated reconstitution of a glycosyl-phosphatidylinositol-protein into liposomes
Detergent-mediated reconstitution of a glycosyl-phosphatidylinositol-protein into liposomes
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DOI：
10.1111/j.1432-1033.1997.t01-1-00168.x
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发表时间：
1997-11-15
期刊：
影响因子：
--
通讯作者：
Roux, B
中科院分区：
文献类型：
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作者：
Angrand, M;Briolay, A;Roux, B
已应用三步去污剂介导的重构将糖基磷脂酰肌醇蛋白掺入脂质体中。研究的蛋白质是来自牛肠的碱性磷酸酶。通过透析制备的脂质体用不同量的两种去污剂（正辛基β-D-葡萄糖苷或Triton X-100）进行处理。在溶解过程的不同步骤中，添加蛋白质并通过疏水性树脂去除去污剂。最有效的重构是用对应于脂质体溶解开始的辛基葡萄糖苷浓度和导致脂质体部分溶解的Triton X-100浓度获得的。磷酸肌醇特异性磷脂酶C-水解碱性磷酸酶无法掺入脂质体，证明了糖基磷脂酰肌醇锚参与碱性磷酸酶重建。与脂质体相关的蛋白质的 70-85% 锚定在双层的外叶中，朝向脂质体的外部。其余部分被捕获在脂质体的内腔内。
A three-step detergent-mediated reconstitution has been applied to the incorporation of a glycosylphosphatidylinositol-protein into liposomes. The protein studied was alkaline phosphatase from bovine intestine. Liposomes prepared by dialysis were treated with various amounts of two detergents, either n-octyl beta-D-glucoside or Triton X-100. At different steps of the solubilization process, protein was added and the detergent was removed by hydrophobic resins. The most efficient reconstitutions were obtained with an octyl glucoside concentration corresponding to the: onset of liposome solubilization and with a Triton X-100 concentration leading to partial solubilization of the liposomes. The involvement of the glycosyl-phosphatidylinositol anchor in alkaline phosphatase reconstitution was demonstrated by the inability of phosphoinositol-specific phospholipase C-hydrolysed alkaline phosphatase to incorporate into liposomes. Between 70-85% of the protein associated with liposomes were anchored in the outer leaflet of the bilayer, oriented towards the outside of the liposome, The remainder was trapped within the lumen of the liposomes.