Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, IL 60064, USA.
J Bioenerg Biomembr. 2011 Aug;43(4):333-47. doi: 10.1007/s10863-011-9364-5. Epub 2011 Jul 12.
The mitochondrial ATP synthase from yeast S. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. The ATP synthase has been modified on the N-terminus of the β-subunit to include a His(6) tag for Ni-chelate affinity purification. The enzyme is purified by a two-step procedure from submitochondrial particles and the resulting enzyme demonstrates lipid dependent oligomycin sensitive ATPase activity of 50 units/mg. The yeast ATP synthase shows a strong lipid selectivity, with cardiolipin (CL) being the most effective activating lipid and there are 30 moles CL bound per mole enzyme at saturation. Green Fluorescent Protein (GFP) has also been fused to the C-terminus of the ε-subunit to create a steric block for rotation of the central stalk. The ε-GFP fusion peptide is imported into the mitochondrion, assembled with the ATP synthase, and inhibits ATP synthetic and hydrolytic activity of the enzyme. F(1)F(o) ATP synthase with ε-GFP was purified to homogeneity and serves as an excellent enzyme for two- and three-dimensional crystallization studies.
已对来自酿酒酵母的线粒体 ATP 合酶进行基因修饰,以功能性形式进行纯化,并对其脂质需求、与多种去污剂的兼容性以及中央柄旋转的空间限制进行了特征描述。已在β亚基的 N 端对 ATP 合酶进行修饰,以包含用于 Ni-螯合亲和纯化的 His(6)标签。该酶通过从亚线粒体颗粒进行两步纯化程序进行纯化,所得酶显示出寡霉素敏感的依赖脂质的 ATP 酶活性为 50 单位/毫克。酵母 ATP 合酶显示出强烈的脂质选择性,心磷脂(CL)是最有效的激活脂质,在饱和时每摩尔酶结合 30 摩尔 CL。还已将绿色荧光蛋白(GFP)融合到ε-亚基的 C 末端,以形成中央柄旋转的空间位阻。ε-GFP 融合肽被导入线粒体,与 ATP 合酶组装,并抑制酶的 ATP 合成和水解活性。ε-GFP 的 F(1)F(o)ATP 合酶已被纯化至均一性,是进行二维和三维结晶研究的极好的酶。