Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources/College of Agriculture, Hainan University, Haikou, China.
Arch Biochem Biophys. 2013 Sep 1;537(1):125-32. doi: 10.1016/j.abb.2013.07.005. Epub 2013 Jul 19.
The characteristics of the Ca(2+)/H(+) exchange were directly investigated in functionally inverted (inside-out) plasma membrane vesicles isolated from yeast using an aqueous two-phase partitioning method. Results showed that following the generation of an inside-acid pH gradient (fluorescence quenching), addition of Ca(2+) caused movement of H(+) out of the vesicles (fluorescence recovery). The Ca(2+)/H(+) exchange displayed saturation kinetics with respect to extravesicular Ca(2+) and ATP concentrations in the plasma membrane, and showed specificity for Ca(2+). The protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), abolished the fluorescence quenching and consequently inhibited Ca(2+)/H(+) exchange in plasma membrane vesicles. Vanadate, which is known to inhibit the plasma membrane H(+)-ATPase, significantly decreased the Ca(2+)-dependent transport of H(+) out of vesicles. When the electrical potential across the plasma membrane was dissipated with valinomycin and potassium, the rate of Ca(2+)/H(+) exchange increased compared to that of the control without valinomycin, indicating that the stoichiometric ratio for this exchange is greater than 2H(+):Ca(2+). These data suggest that Ca(2+) is transported out of yeast cells through a Ca(2+)/H(+) exchange system that is driven by the proton-motive force generated by the plasma membrane H(+)-ATPase.
采用双水相分配法从酵母中分离功能反转（外翻）的质膜囊泡，直接研究 Ca(2+)/H(+) 交换的特性。结果表明，在产生内部酸性 pH 梯度（荧光猝灭）后，添加 Ca(2+) 会导致 H(+)从囊泡中流出（荧光恢复）。Ca(2+)/H(+) 交换对质膜中胞外 Ca(2+)和 ATP 浓度具有饱和动力学特性，并对 Ca(2+)具有特异性。质子载体 FCCP（羰基氰化物对三氟甲氧基苯腙）消除荧光猝灭，从而抑制质膜囊泡中的 Ca(2+)/H(+) 交换。已知钒酸盐抑制质膜 H(+)-ATP 酶，显著降低 Ca(2+)依赖性 H(+)从囊泡中的转运。当用缬氨霉素和钾耗散质膜的跨膜电势时，与没有缬氨霉素的对照相比，Ca(2+)/H(+)交换的速率增加，表明该交换的化学计量比大于 2H(+):Ca(2+)。这些数据表明，Ca(2+)通过由质膜 H(+)-ATP 酶产生的质子动力势驱动的 Ca(2+)/H(+) 交换系统从酵母细胞中转运出来。
