Hu V W, Esser A F, Podack E R, Wisnieski B J
J Immunol. 1981 Jul;127(1):380-6.
We have utilized a membrane-restricted, photoactivable glycolipid probe to investigate the protein-lipid interactions involved in complement (C) mediated lysis of a target membrane. The purified C proteins C5b-6, C7, C8, and C9 were added to artificial membrane vesicles containing the 14C-labeled photoreactive probe anchored in the outer monolayer of the membrane, and 6-carboxyfluorescein trapped in the lumen as an indicator for effective lysis. Irradiation of the membrane samples at different stages of functional complex assembly resulted in labeling of each of the 5 terminal C proteins, indicating that all 5 proteins become inserted into the hydrophobic milieu of the membrane during some stage of complex assembly. However, at the final stage of complex assembly, only C9 appeared to be labeled. Because we can demonstrate that the photoreactive probe has no strong affinity for C9 over the other terminal components (C5b-C8), the extensive change in labeling specificity during assembly is evidence for substantial changes in protein-lipid and possibly protein-protein interactions during formation of the C lesion.
我们使用了一种膜限制的、可光活化的糖脂探针来研究补体（C）介导的靶膜裂解过程中涉及的蛋白质-脂质相互作用。将纯化的补体蛋白C5b-6、C7、C8和C9添加到人工膜泡中，该膜泡含有锚定在膜外单层的14C标记的光反应性探针，以及被困在膜泡腔内的6-羧基荧光素作为有效裂解的指示剂。在功能复合物组装的不同阶段对膜样品进行辐照，结果表明5种末端补体蛋白中的每一种都被标记，这表明在复合物组装的某个阶段，所有5种蛋白都插入到膜的疏水环境中。然而，在复合物组装的最后阶段，似乎只有C9被标记。因为我们可以证明光反应性探针相对于其他末端成分（C5b-C8）对C9没有很强的亲和力，所以组装过程中标记特异性的广泛变化证明了在补体损伤形成过程中蛋白质-脂质以及可能的蛋白质-蛋白质相互作用发生了实质性变化。
