Lutter R, Saraste M, van Walraven H S, Runswick M J, Finel M, Deatherage J F, Walker J E
Medical Research Council, Laboratory of Molecular Biology, Cambridge, U.K.
Biochem J. 1993 Nov 1;295 ( Pt 3)(Pt 3):799-806. doi: 10.1042/bj2950799.
A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.
已开发出一种从牛线粒体中分离ATP合酶的新方法，其主要目的是制备适合结晶试验的酶。用十二烷基-β-D-麦芽糖苷从线粒体膜中提取蛋白质，然后在相同去污剂存在的情况下，通过离子交换、凝胶过滤色谱和硫酸铵沉淀相结合的方法从提取物中纯化ATP合酶。这种简单快速的方法可产生20 - 30毫克高度纯净且单分散的酶，该酶显然由14种不同亚基组成，其中亚基e与已确定的亚基以明显的化学计量比存在，亚基e是最近发现的功能未知的亚基。该酶制剂具有对寡霉素敏感的ATP水解活性，因此F1结构域在功能上与膜结构域F0相关联。与牛线粒体F1 - ATP酶某些亚基的N端不同，F1F0 - ATP合酶的N端在分离过程中不会被蛋白酶降解。因此，该制剂满足结晶试验的先决条件。
