Jänsch L, Kruft V, Schmitz U K, Braun H P
Institut für Genbiologische Forschung GmbH, Berlin, Germany.
Plant J. 1996 Mar;9(3):357-68. doi: 10.1046/j.1365-313x.1996.09030357.x.
Recently a powerful electrophoresis method for the native preparation and characterization of the respiratory protein complexes of mitochondria from fungi and mammals has been developed, which employs Coomassie dyes to introduce charge shifts on proteins (Schägger and von Jagow (1991) Anal. Biochem. 199, 223-231). The procedure, which is called 'blue native-polyacrylamide gel electrophoresis' (BN-PAGE), was modified and introduced for the analysis of mitochondria from higher plants. BN-PAGE of mitochondrial protein from potato allows the separation of nine distinct protein complexes between 100 and 1000 kDa and reveals novel results for their composition, molecular mass and stoichiometry. For the first time soluble mitochondrial protein complexes, like the HSP60 complex (750 kDa) and a complex of 200 kDa, which includes a formate dehydrogenase, are analysed by BN-PAGE. Complex I from potato (1000 kDa) is about 100 kDa larger than the corresponding enzyme from beef and can be resolved into more than 30 different subunits on a second gel dimension. The F1F0 ATP synthase (580 kDa) and the cytochrome c oxidase (160 kDa) from potato seem to contain more subunits than hitherto reported. Direct sequencing of subunits revealed that the F1 part of the F1F0 ATP synthase lacks the oligomycin sensitivity conferring protein (OSCP), which was reported to be present in F1 parts of dicotyledonous plants, but contains the ATPase inhibitory protein. N-terminal sequences of 16 mitochondrial proteins were obtained, several of which are presented for the first time from a plant source. BN-PAGE allows the preparation of mitochondrial protein complexes from gram amounts of plant tissue, as the procedure only requires milligram amounts of organelles. This potential of BN-PAGE is demonstrated by the separation and characterization of the mitochondrial enzyme complexes from Arabidopsis thaliana. Further analysis of organellar protein complexes by BN-PAGE will allow the generation of 'protein maps' from different tissues and developmental stages or from mutant plants.
最近，一种用于从真菌和哺乳动物中线粒体呼吸蛋白复合物的天然制备和表征的强大电泳方法被开发出来，该方法使用考马斯染料在蛋白质上引入电荷转移（施格和冯·雅戈（1991年）《分析生物化学》199卷，223 - 231页）。这个被称为“蓝色天然聚丙烯酰胺凝胶电泳”（BN - PAGE）的程序经过修改后被引入用于高等植物线粒体的分析。马铃薯线粒体蛋白的BN - PAGE能够分离出100至1000 kDa之间的9种不同的蛋白复合物，并揭示了它们在组成、分子量和化学计量方面的新结果。首次通过BN - PAGE分析了可溶性线粒体蛋白复合物，如HSP60复合物（750 kDa）和一个200 kDa的复合物，其中包括一种甲酸脱氢酶。来自马铃薯的复合物I（1000 kDa）比来自牛肉的相应酶大约大100 kDa，并且在第二维凝胶上可以分解为30多个不同的亚基。来自马铃薯的F1F0 ATP合酶（580 kDa）和细胞色素c氧化酶（160 kDa）似乎比迄今报道的含有更多亚基。亚基的直接测序表明，F1F0 ATP合酶的F1部分缺乏赋予寡霉素敏感性的蛋白（OSCP），据报道该蛋白存在于双子叶植物的F1部分，但含有ATP酶抑制蛋白。获得了16种线粒体蛋白的N端序列，其中几种是首次从植物来源获得的。BN - PAGE能够从克量的植物组织中制备线粒体蛋白复合物，因为该程序仅需要毫克量的细胞器。拟南芥线粒体酶复合物的分离和表征证明了BN - PAGE的这种潜力。通过BN - PAGE对细胞器蛋白复合物进行进一步分析将能够生成来自不同组织和发育阶段或突变植物的“蛋白质图谱”。
