Lévy D, Gulik A, Bluzat A, Rigaud J L
Service de Biophysique, Département de Biologie et URA-CNRS (D 1290), CEN Saclay, Gif-sur-Yvette, France.
Biochim Biophys Acta. 1992 Jun 30;1107(2):283-98. doi: 10.1016/0005-2736(92)90415-i.
The Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into sealed phospholipid vesicles using the method recently developed for bacteriorhodopsin (Rigaud, J.L., Paternostre, M.T. and Bluzat, A. (1988) Biochemistry 27, 2677-2688). Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, sodium cholate or dodecyl octa(oxyethylene) glycol ether (C12E8) and protein incorporation was studied at each step of the liposome solubilization process by each of these detergents. After detergent removal by SM-2 Bio-Beads the resulting vesicles were analyzed with respect to protein incorporation by freeze-fracture electron microscopy, sucrose density gradients and Ca2+ pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With octyl glucoside, direct incorporation of Ca(2+)-ATPase into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes homogeneous in regard to protein distribution. With the other detergents, optimal Ca(2+)-ATPase pumping activities were obtained when starting from Ca(2+)-ATPase/detergent/phospholipid micellar solutions. However, the homogeneity of the resulting recombinants was shown to be dependent upon the detergent used and in the presence of Triton X-100 or C12E8 different populations were clearly evidenced. It was further demonstrated that the rate of detergent removal drastically influenced the composition of resulting proteoliposomes: upon slow detergent removal from samples solubilized with Triton X-100 or C12E8, Ca(2+)-ATPase was found seggregated and/or aggregated in very few liposomes while upon rapid detergent removal compositionally homogeneous proteoliposomes were obtained with high Ca2+ pumping activities. The reconstitution process was further analyzed by centrifugation experiments and the results demonstrated that the different mechanisms of reconstitution were driven predominantly by the tendency for self-aggregation of the Ca(2+)-ATPase. A model for Ca(2+)-ATPase reconstitution was proposed which accounted for all our results. In summary, the advantage of the systematic studies reported in this paper was to allow a rapid and easy determination of the experimental conditions for optimal detergent-mediated reconstitution of Ca(2+)-ATPase. Proteoliposomes prepared by the present simple method exhibited the highest Ca2+ pumping activities reported to date in Ca(2+)-ATPase reconstitution experiments performed in the absence of Ca2+ precipitating agents.
利用最近为细菌视紫红质开发的方法（里戈，J.L.，帕特诺斯特雷，M.T.和布卢扎特，A.（1988年）《生物化学》27卷，2677 - 2688页），将骨骼肌肌浆网的Ca(2 +)-ATP酶重组到密封的磷脂囊泡中。通过反相蒸发制备的脂质体用不同量的 Triton X - 100、辛基葡糖苷、胆酸钠或十二烷基八(氧乙烯)二醇醚（C12E8）处理，并且在这些去污剂使脂质体溶解过程的每个步骤中研究蛋白质掺入情况。在用SM - 2生物珠去除去污剂后，通过冷冻断裂电子显微镜、蔗糖密度梯度和Ca2 +转运测量对所得囊泡进行蛋白质掺入分析。结果表明，用于重组的去污剂的性质对于确定蛋白质插入机制很重要。使用辛基葡糖苷时，观察到Ca(2 +)-ATP酶直接掺入预先形成的、因该去污剂达到饱和水平而不稳定的脂质体中，并且得到蛋白质分布均匀的蛋白脂质体。使用其他去污剂时，从Ca(2 +)-ATP酶/去污剂/磷脂胶束溶液开始时可获得最佳的Ca(2 +)-ATP酶转运活性。然而，所得重组体的均匀性显示取决于所使用的去污剂，并且在存在 Triton X - 100或C12E8时，明显可以看出存在不同的群体。进一步证明，去污剂去除速率极大地影响所得蛋白脂质体的组成：在用 Triton X - 100或C12E8溶解的样品中缓慢去除去污剂时，发现Ca(2 +)-ATP酶在极少数脂质体中分离和/或聚集，而快速去除去污剂时可获得具有高Ca2 +转运活性且组成均匀的蛋白脂质体。通过离心实验进一步分析了重组过程，结果表明不同的重组机制主要由Ca(2 +)-ATP酶的自聚集倾向驱动。提出了一个Ca(2 +)-ATP酶重组模型，该模型解释了我们所有的结果。总之，本文报道的系统研究的优点是能够快速、轻松地确定最佳去污剂介导的Ca(2 +)-ATP酶重组的实验条件。通过本简单方法制备的蛋白脂质体在不存在Ca2 +沉淀剂的Ca(2 +)-ATP酶重组实验中表现出迄今为止报道的最高Ca2 +转运活性。
