Proton transport coupled ATP synthesis by the purified yeast H+-ATP synthase in proteoliposomes
Proton transport coupled ATP synthesis by the purified yeast H+-ATP synthase in proteoliposomes
复制标题
DOI：
10.1016/j.bbabio.2010.07.013
复制
发表时间：
2010-11-01
影响因子：
4.3
通讯作者：
Petersen, Jan
中科院分区：
文献类型：
--
作者：
Foerster, Kathrin;Turina, Paola;Petersen, Jan
从酵母线粒体，MF 0 F1的H+/ATP合酶，纯化和重组到脂质体制备的磷脂酰胆碱和磷脂酸。通过质谱分析揭示了除K-亚基之外的酵母酶的所有亚基的存在。MF 0 F1脂质体通过酸碱转换（Δ pH）和K+/缬氨霉素扩散电位（Δ phi）供能。ATP的合成被完全废除，除了解偶联剂，以及抑制剂寡霉素。ATP合成速率作为各种参数的函数进行优化，并在跨膜pH差为3.2单位（pH（in）= 4.8和pH（out）= 8.0）和Ay为133 mV（能斯特电位）时达到最大值（周转数）120 s（-1）。功能研究表明，单体MF 0 F1在ATP合成中具有完全活性。营业额增加，在一个S形的方式增加内部和外部质子浓度的降低。周转率对磷酸盐浓度的依赖性和KM对pHout的依赖性表明，ATP合成的底物是单阴离子磷酸盐物种H2 PO 4-。(C)2010 Elsevier B. V.保留所有权利。
The H+/ATP synthase from yeast mitochondria, MF0F1, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF0F1 liposomes were energized by acid-base transitions (Delta pH) and a K+/valinomycin diffusion potential (Delta phi). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120 s(-1) at a transmembrane pH difference of 3.2 units (at pH(in) = 4.8 and pH(out) = 8.0) and a Ay of 133 mV (Nernst potential). Functional studies showed that the monomeric MF0F1 was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of KM on pHout indicated that the substrate for ATP synthesis is the monoanionic phosphate species H2PO4-. (C) 2010 Elsevier B.V. All rights reserved.