Tomasetig Lara, Di Pancrazio Francesca, Harris David A, Mavelli Irene, Lippe Giovanna
Department of Biomedical Sciences and Technologies, University of Udine, p.le Kolbe 4, 33100, Udine, Italy.
Biochim Biophys Acta. 2002 Dec 2;1556(2-3):133-41. doi: 10.1016/s0005-2728(02)00344-4.
Solubilization of heavy bovine heart mitochondria with Triton X-100 leads to the selective extraction of F0F1ATP synthase monomer and dimer in a 2:1 ratio, as revealed by blue native gel electrophoresis (BN-PAGE). Second dimensional SDS-PAGE and immunoblotting with IF1 and F1 antibodies following BN-PAGE show that both aggregation states of the ATP synthase contain IF1. The monomer/dimer ratio does not change in extracts from mitochondria subjected to different energy conditions accompanied by IF1 binding modulation or from submitochondrial particles differing in IF1 content. In addition, the usual monomer/dimer ratio is observed even in submitochondrial particles deprived of IF1. Histochemical staining for ATPase activity demonstrates that the dimer is inactive, irrespective of its IF1 content. It is concluded that in the membrane of bovine heart mitochondria the ATP synthase dimer is a stable inactive structure, whose formation is not mediated by IF1 binding.
用Triton X-100增溶重牛心线粒体，通过蓝色非变性凝胶电泳（BN-PAGE）发现，F0F1ATP合酶单体和二聚体以2:1的比例被选择性提取。BN-PAGE后的二维SDS-PAGE以及用IF1和F1抗体进行免疫印迹显示，ATP合酶的两种聚集状态均含有IF1。在经历不同能量条件并伴有IF1结合调节的线粒体提取物中，或在IF1含量不同的亚线粒体颗粒提取物中，单体/二聚体比例不变。此外，即使在缺乏IF1的亚线粒体颗粒中也观察到了通常的单体/二聚体比例。ATP酶活性的组织化学染色表明，二聚体无活性，无论其IF1含量如何。得出的结论是，在牛心线粒体膜中，ATP合酶二聚体是一种稳定的无活性结构，其形成不是由IF1结合介导的。
