Institute of Biophysics, Biological Research Centre, Temesvári krt. 62, 6726, Szeged, Hungary.
Eur Biophys J. 2013 Mar;42(2-3):147-58. doi: 10.1007/s00249-012-0871-z. Epub 2012 Nov 16.
The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6-32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8-48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment.
在标准条件下，从酿酒酵母的天然液泡膜小泡中估计了酵母液泡质子-ATP 酶（V-ATPase）的转子相对于定子或酶的稳定部分的旋转速度。在暴露于渗透压冲击后，纯化的酵母液泡自发形成膜小泡。通过使用该酶的强效和特异性抑制剂康纳霉素 A 来确定源自 V-ATPase 的总 ATP 酶活性的分数。在 20°C 下进行 10 分钟的 ATP 酶活性期间，在液泡膜小泡系统中从 ATP 释放的无机磷酸盐通过分光光度法在不同的康纳霉素 A 浓度下进行测定。对于单体 V-ATPase 上的单个康纳霉素 A 结合位点（我们的数据与假设多个结合位点的模型不兼容），假设二次结合方程的拟合，对抑制剂滴定曲线进行拟合确定了酶的浓度。将这与 V-ATPase 的已知 ATP/旋转化学计量学以及通过 V-ATPase 释放的无机磷酸盐的测定浓度相结合，导致全 360°旋转的平均速率约为 10 Hz（考虑到酶浓度的±标准偏差，范围为 6-32 Hz），从活性的时间依赖性推断，在反应开始时，约为 14 Hz（8-48 Hz）。这些都是下限估计值。据我们所知，这是首次报道在未经过遗传或化学修饰且未固定在固体支撑物上的 V-ATPase 中的旋转速度；相反，它在其天然膜环境中发挥作用。
