Vigneron L, Scarborough G A, Ruysschaert J M, Goormaghtigh E
Laboratoire des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
Biochim Biophys Acta. 1995 May 24;1236(1):95-104. doi: 10.1016/0005-2736(95)00028-2.
The purified H(+)-ATPase of the Neurospora crassa plasma membrane has been reconstituted by a gel filtration method into lipidic vesicles using sodium deoxycholate as the detergent. Reconstitution was performed for lipid/ATPase ratios ranging from 1000:1 to 5:1 (w/w). Whatever the lipid/ATPase ratio, the ATPase molecules completely associate with the lipid vesicles. The ATPase specific activity is identical for all proteoliposomes regardless of the lipid/ATPase ratio, but the H+ transport decreases at high protein/lipid ratios, suggesting that the proteoliposomes are more leaky to H+ as the amount of protein inserted into the lipidic membrane increases. Analysis of the fragments generated by trypsin proteolysis in the presence and in the absence of MgATP+ vanadate indicate that most of the reconstituted ATPase molecules are able to assume the transition state of the enzyme dephosphorylation reaction, and are therefore functional. The orientation (inside-out or rightside-out) of the ATPase molecules in the vesicles is independent of the lipid/ATPase ratio chosen for the reconstitution. For all the lipid/ATPase ratios tested, most of the ATPase molecules (> 99%) expose their cytoplasmic side to the outside of the reconstituted proteoliposomes. The size of the vesicles increases parallel to the ATPase amount. Although the H+ leakiness of our preparation at low lipid/protein ratios prevents proton pumping measurements, the reconstitution procedure described here has the main advantage on other procedures to allow the obtention of vesicles at high protein-to-lipid ratios, facilitating further structural characterization of the ATPase by biochemical and biophysical techniques. Therefore, the procedure described here could be of general interest in the field of membrane protein study.
粗糙脉孢菌质膜纯化的H(+)-ATP酶已通过凝胶过滤法，以脱氧胆酸钠为去污剂重构成脂质体。脂质/ATP酶的比例范围为1000:1至5:1（w/w）时进行了重构。无论脂质/ATP酶的比例如何，ATP酶分子都与脂质体完全结合。所有蛋白脂质体的ATP酶比活性相同，与脂质/ATP酶比例无关，但在高蛋白/脂质比时H+转运减少，这表明随着插入脂质膜的蛋白量增加，蛋白脂质体对H+的渗漏性更高。在有和没有MgATP+钒酸盐存在的情况下，对胰蛋白酶蛋白水解产生的片段进行分析表明，大多数重构的ATP酶分子能够呈现酶去磷酸化反应的过渡态，因此具有功能。脂质体中ATP酶分子的取向（内翻或外翻）与重构时选择的脂质/ATP酶比例无关。对于所有测试的脂质/ATP酶比例，大多数ATP酶分子（>99%）将其细胞质侧暴露于重构的蛋白脂质体外部。脂质体的大小与ATP酶量平行增加。尽管我们的制剂在低脂质/蛋白质比时的H+渗漏性妨碍了质子泵浦测量，但此处描述的重构程序相对于其他程序具有主要优势，即允许获得高蛋白与脂质比的脂质体，便于通过生化和生物物理技术对ATP酶进行进一步的结构表征。因此，此处描述的程序可能在膜蛋白研究领域具有普遍意义。
