大豆下胚轴质膜H+-ATPase质子转运的测定

以大豆下胚轴为材料，采用改进的匀浆介质,通过两相法制得具有质子转运活力的高纯度质膜微囊.并且发现冻融处理可以促进质膜微囊的翻转而提高荧光猝灭效率.质子载体和质子转运特性分析表明,由Mg^2＋-ATP引发的荧光猝灭可以被质子载体CCCP恢复,并被质子通道抑制剂DCCD抑制;并且发现质膜H^＋-ATPase专一抑制剂钒酸钠可以完全抑制荧光猝灭,同时发现荧光猝灭依赖于Mg^2＋,并受K^＋刺激,最适pH为6.5.以上证明所测荧光猝灭是由质膜H^＋-ATPase所进行的质子转运引起的.结果同时表明,维持H^＋-ATPase合适构象和提高质膜微囊封闭性是制备具有H^＋转运活力质膜微囊的两个关键因素.

The Determination of Proton Transport by the Plasma Membrane ATPase from Soybean Hypocotyls

The transport competent plasma membrane vesicles were obtained using a modified homogenization medium by the two phase partitioning method from soybean hypocotyls. It was noted that freeze thaw treatment could increase the amount of inside out vesicles.Further experiments showed that the fluorescence quenching caused by adding of Mg 2 ATP could be dissipated by the proton ionophore CCCP, and inhibited by the proton transport inhibitor DCCD; it was also found that the plasma membrane H ATPase specific inhibitor vanadate could quench the fluorescence completely. The fluorescene quenching was Mg 2 dependent, K stimulated, with an optimum pH of 6 5. All the above results indicated that the fluorescence quenching was caused by the transport of protons by the plasma membrane H ATPase. It was also suggested that the maintaining of suitable conformation of the plasma membrane H ATPase,and the tightness of plasma membrane vescials are the two key factors for the proton transport determination.