Seelert Holger, Dencher Norbert A, Müller Daniel J
Physical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany.
J Mol Biol. 2003 Oct 17;333(2):337-44. doi: 10.1016/j.jmb.2003.08.046.
Three fundamentally different chloroplast ATP synthase samples of increasing complexity were visualized by atomic force microscopy. The samples are distinguishable in respect to the isolation technique, the detergent employed, and the final subunit composition. The homo-oligomer III was isolated following SDS treatment of ATP synthase, the proton-turbine III+IV was obtained by blue-native electrophoresis, and complete CFO was isolated by anion exchange chromatography of NaSCN splitted ATP synthase. In all three ATP synthase subcomplexes 14 and only 14 circularly arranged subunits III composed the intact transmembrane rotor. Therefore, 14 protomers built the membrane-resident proton turbine. The observed stoichiometry of 14 is not a biochemical artifact or affected by natural growth variations of the spinach, as previously suggested. A correlation between the presence of subunit IV in the imaged sample and the appearance of a central protrusion in the narrower orifice of the oligomeric cylinder III14 has been observed. In contrast to current predictions, in chloroplast FO the subunit IV can be found inside the cylinder III14 and not at its periphery, at least in the reconstituted 2D arrays imaged.
通过原子力显微镜观察了三种复杂性递增、本质上不同的叶绿体ATP合酶样本。这些样本在分离技术、所用去污剂以及最终的亚基组成方面存在差异。同型寡聚体III是在对ATP合酶进行SDS处理后分离得到的，质子涡轮III + IV是通过蓝色非变性电泳获得的，而完整的CFO是通过对经NaSCN裂解的ATP合酶进行阴离子交换色谱分离得到的。在所有这三种ATP合酶亚复合物中，14个且仅14个呈环状排列的亚基III组成了完整的跨膜转子。因此，14个原聚体构成了膜结合质子涡轮。观察到的14的化学计量比并非生化假象，也不像之前所认为的那样受菠菜自然生长变化的影响。在成像样本中观察到亚基IV的存在与寡聚圆柱体III14较窄孔口中中央突起的出现之间存在相关性。与当前预测相反，在叶绿体F O中，至少在成像的重构二维阵列中，亚基IV可在圆柱体III14内部而非其周边找到。
