Apell H J, Marcus M M, Anner B M, Oetliker H, Läuger P
J Membr Biol. 1985;85(1):49-63. doi: 10.1007/BF01872005.
A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.
描述了一种用于测量含有纯化的钠钾ATP酶的脂质小泡中ATP驱动的离子通量的荧光方法。通过使用电压敏感的吲哚菁染料测量含酶小泡的膜电压。加入缬氨霉素后，小泡膜对K+具有选择性通透性，从而使膜电压接近K+的能斯特电位。在外部K+浓度恒定的情况下，在泵激活后，随着荧光信号的变化，可以连续测量内部K+浓度的时间进程。该光学方法比示踪通量实验具有更高的时间分辨率，并且可以准确测定初始通量率。根据活性K+转运的温度依赖性，确定其活化能为115 kJ/mol。当膜电导较低时（即缬氨霉素浓度较低或为零时），ATP刺激的生电泵浦可作为快速荧光变化进行测量。正如预期的那样，快速信号变化的幅度随着小泡膜被动离子通透性的降低而增加。电荷移动的分辨率非常高，以至于可以很容易地检测到几次泵的周转。
