Kawamura Yukio
Department of Cell Biology & Molecular Genetics, University of Maryland, HJ Patterson Hall, College Park, MD 20742-5815, USA.
Anal Biochem. 2007 Oct 15;369(2):137-48. doi: 10.1016/j.ab.2007.06.037. Epub 2007 Jun 28.
To adapt to environmental changes, plant cells very likely possess a biochemical system, using vacuoles, for maintaining cytoplasmic pH homeostasis. A simple approach is to estimate the active H(+) influx and H(+) efflux of isolated vacuolar vesicles, although there is no good mathematical model to describe H(+) flux. To establish a new quantitative model, vacuolar vesicles were isolated from hypocotyls of mung bean (Vigna radiata L.), and pyrophosphate (PPi)- or ATP-dependent acidification was monitored using acridine orange. The change of pH inside the vesicles (pH(in)) was calculated using a pH calibration curve relating fluorescence quenching with DeltapH. After formation of a steady state DeltapH, passive H(+) efflux was monitored after terminating pumping with ethylenediaminetetraacetate, and the relative H(+) permeability coefficient (p(H+)) was calculated. The H(+) efflux simulated using the p(H+) corresponded to the H(+) efflux determined experimentally. H(+) influx was then calculated by subtracting the predicted H(+) efflux from the experimental net H(+) influx. H(+) influx into vesicles driven by H(+)-PPase or H(+)-ATPase decreased exponentially as the intravesicular pH(in) decreased, suggesting modulation of pumping by DeltapH, pH(in), or both. Finally, the PPi- or ATP-dependent H(+) accumulation determined experimentally was closely simulated by the predicted H(+) influx and H(+) efflux. The ability to predict H(+) flux under different conditions provides a powerful tool for studying pH homeostasis.
为了适应环境变化，植物细胞很可能拥有一个利用液泡来维持细胞质pH稳态的生化系统。一种简单的方法是估计分离的液泡囊泡的活性H⁺内流和H⁺外流，尽管目前还没有很好的数学模型来描述H⁺通量。为了建立一个新的定量模型，从绿豆（Vigna radiata L.）下胚轴中分离出液泡囊泡，并用吖啶橙监测焦磷酸（PPi）或ATP依赖性酸化。利用将荧光猝灭与ΔpH相关联的pH校准曲线计算囊泡内部的pH变化（pH(in)）。在形成稳态ΔpH后，在用乙二胺四乙酸终止泵浦后监测被动H⁺外流，并计算相对H⁺渗透系数（p(H+)）。使用p(H+)模拟的H⁺外流与实验测定的H⁺外流相对应。然后通过从实验净H⁺内流中减去预测的H⁺外流来计算H⁺内流。随着囊泡内pH（pH(in)）的降低，由H⁺-PPase或H⁺-ATPase驱动的H⁺流入囊泡呈指数下降，这表明ΔpH、pH(in)或两者对泵浦有调节作用。最后，预测的H⁺内流和H⁺外流紧密模拟了实验测定的PPi或ATP依赖性H⁺积累。预测不同条件下H⁺通量的能力为研究pH稳态提供了一个强大的工具。
