通过配体对内在和外在荧光的影响研究ATP和质子对（Na⁺ + K⁺）-ATP酶的Na⁺:K⁺选择性的影响。
Effects of ATP and protons on the Na : K selectivity of the (Na+ + K+)-ATPase studied by ligand effects on intrinsic and extrinsic fluorescence.
作者信息
Skou J C, Esmann M
出版信息
Biochim Biophys Acta. 1980 Sep 18;601(2):386-402. doi: 10.1016/0005-2736(80)90543-x.
The effect of pH and of ATP on the Na : K selectivity of the (Na+ + K+)-ATPase has been tested under equilibrium conditions. The Na+ : K+-induced change in intrinsic tryptophan fluorescence and in fluorescence of eosin maleimide bound to the system has been used as a tool. 1 mol of eosin maleimide per mol of enzyme gives no loss in either ATPase or phosphatase activity and the fluorescence in the presence of Na+ is about 30% higher than in the presence of K+. Choline, protonated Tris, protonated histidine and Mg2+ have an 'Na+' effect on the extrinsic fluorescence, while Rb+, Cs+ and NH4+ have a 'K+' effect. Choline and protonated Tris have an Na+ effect on intrinsic fluorescence. A close correlation between the effect of Na+ compared to K+ on the fluorescence change and on Na+ activation of hydrolysis indicates that the observed changes in fluorescence are due to an effect of Na+ and of K+ on the internal sites of the system. The equilibrium between the two conformations, which are reflected by the difference in fluorescence with Na+ and K+, respectively, is highly influenced by the concentration of protons. At a given Na+ : K+ ratio, an increase in the proton concentration shifts the equilibrium towards the 'K+' fluorescence form while a decrease shifts the equilibrium towards the 'Na+' fluorescence form, i.e., protons increase the apparent affinity for K+ and vice versa, K+ increases pK values of importance for the Na+ : K+ selectivity. Conversely, a decrease in protons increases the apparent affinity for Na+ and vice versa, Na+ decreases the pK. ATP decreases the apparent pK for the protonation-deprotonation, i.e., ATP facilitates the deprotonation which accompanies Na+ binding. The results suggest two effects of ATP for the hydrolysis in the presence of Na+ and K+ : (i) at low ATP concentrations (K0.5 < 10 microM) on the K+-Na+ exchange on the internal sites and (ii) at higher, substrate, concentrations on the activation by K+ on the external sites.
在平衡条件下测试了pH值和ATP对（Na⁺ + K⁺）-ATP酶的Na⁺:K⁺选择性的影响。利用Na⁺:K⁺诱导的内在色氨酸荧光变化以及与该系统结合的曙红马来酰亚胺的荧光变化作为一种工具。每摩尔酶1摩尔的曙红马来酰亚胺不会导致ATP酶或磷酸酶活性丧失，并且在存在Na⁺时的荧光比存在K⁺时高约30%。胆碱、质子化的Tris、质子化的组氨酸和Mg²⁺对外部荧光有“Na⁺”效应，而Rb⁺、Cs⁺和NH₄⁺有“K⁺”效应。胆碱和质子化的Tris对内在荧光有Na⁺效应。与K⁺相比，Na⁺对荧光变化和Na⁺激活水解的影响之间存在密切相关性，这表明观察到的荧光变化是由于Na⁺和K⁺对系统内部位点的影响。两种构象之间的平衡，分别由Na⁺和K⁺存在时荧光的差异反映，受到质子浓度的高度影响。在给定的Na⁺:K⁺比例下，质子浓度的增加会使平衡向“K⁺”荧光形式移动，而降低则会使平衡向“Na⁺”荧光形式移动，即质子增加了对K⁺的表观亲和力（反之亦然，K⁺增加了对Na⁺:K⁺选择性重要的pK值）。相反，质子的减少增加了对Na⁺的表观亲和力（反之亦然，Na⁺降低了pK）。ATP降低了质子化-去质子化的表观pK，即ATP促进了伴随Na⁺结合的去质子化。结果表明，在存在Na⁺和K⁺时，ATP对水解有两种作用：（i）在低ATP浓度（K₀.₅ < 10 μM）下对内部位点上的K⁺-Na⁺交换有作用，以及（ii）在较高的底物浓度下对外部位点上K⁺的激活有作用。
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