Apell H J, Roudna M, Corrie J E, Trentham D R
Department of Biology, University of Konstanz, Germany.
Biochemistry. 1996 Aug 20;35(33):10922-30. doi: 10.1021/bi960238t.
Phosphorylation by Pi of the Na,K-ATPase from rabbit kidney in the absence of Na+ ions but in the presence of Mg2+ ions has been studied. In the absence of K+ ions, unphosphorylated and phosphorylated states induce different fluorescence levels in the membrane-bound styryl dye RH421, and hence transitions between the two states were monitored. Transient kinetic studies of phosphorylation were initiated by manual addition of Pi or by photochemical release of Pi from 1-(2-nitrophenyl)ethyl phosphate (caged Pi) using laser flash photolysis at 308 nm. Equilibrium studies of phosphorylation showed that the apparent Km for Pi was 23.0 +/- 0.3 microM (mean +/- sem) at pH 7.1 and 21 degrees C. The dye fluorescence increased in a biphasic manner on addition of 500 microM Pi to the enzyme: a rapid phase (t 1/2 < 1 s) and a slower exponential phase at 0.059 +/- 0.003 s-1. The rate of the rapid phase was studied by fast concentration-jump experiments and exhibited first-order kinetics in Pi up to 60 microM. Fluorescence records vs time were exponential, and a plot of the rate constant versus [Pi] had a slope of 1.47 x 10(5) M-1 s-1 and ordinate [Pi] = 0) intercept of 3.1 s-1. Addition of 50 mM NaCl to the phosphorylated enzyme induced an exponential decay in the dye fluorescence from which a rate constant of 0.10 +/- 0.005 s-1 was determined. These data were interpreted in terms of transformations between conformational states E1 and E2, and the phosphorylated state P-E2 defined in the Post-Albers mechanism of the Na,K-ATPase [Läuger, P., (1991) Electrogenic Ion Pumps, Sinauer Associates Inc., Sunderland, MA] as follows: [formula: see text] The RH421 fluorescence of state P-E2 was studied over the pH range 6-8.5. Fluorescence was greatest at pH 8.5 and lowest at pH 6.0 in a simple binding isotherm with pK 7.5. The apparent Km for Pi rose cooperatively with increasing pH (pKa 8.6 and a Hill coefficient of 2). Therefore in the absence of monovalent metal ions, occupation of the cation (K+) binding sites by protons promotes phosphorylation by Pi.
研究了在不存在Na⁺离子但存在Mg²⁺离子的情况下，兔肾Na,K-ATP酶被无机磷酸（Pi）磷酸化的情况。在不存在K⁺离子时，未磷酸化和磷酸化状态在膜结合的苯乙烯基染料RH421中诱导出不同的荧光水平，因此监测了两种状态之间的转变。通过手动添加Pi或使用308nm激光闪光光解从1-(2-硝基苯基)乙基磷酸酯（笼形Pi）光化学释放Pi来启动磷酸化的瞬态动力学研究。磷酸化的平衡研究表明，在pH 7.1和21℃时，Pi的表观Km为23.0±0.3μM（平均值±标准误）。向酶中加入500μM Pi时，染料荧光以双相方式增加：一个快速相（t 1/2<1s）和一个在0.059±0.003s⁻¹的较慢指数相。通过快速浓度跃变实验研究了快速相的速率，在高达60μM Pi时表现出对Pi的一级动力学。荧光记录随时间呈指数变化，速率常数对[Pi]的图斜率为1.47×10⁵M⁻¹s⁻¹，纵坐标[Pi]=0时的截距为3.1s⁻¹。向磷酸化酶中加入50mM NaCl会导致染料荧光呈指数衰减，由此确定的速率常数为0.10±0.005s⁻¹。这些数据根据Na,K-ATP酶的Post-Albers机制[Läuger, P., (1991) Electrogenic Ion Pumps, Sinauer Associates Inc., Sunderland, MA]中定义的构象状态E1和E2以及磷酸化状态P-E2之间的转变进行解释如下：[公式：见原文]研究了状态P-E2在pH 6-8.5范围内的RH421荧光。在简单结合等温线中，荧光在pH 8.5时最大，在pH 6.0时最小，pK为7.5。Pi的表观Km随着pH升高而协同增加（pKa 8.6，希尔系数为2）。因此，在不存在单价金属离子的情况下，质子占据阳离子（K⁺）结合位点会促进Pi的磷酸化。
