Kinetics of the Phosphorylation of Na,K-ATPase by Inorganic Phosphate Detected by a Fluorescence Method

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Abstract
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 μM (mean ± sem) at pH 7.1 and 21 °C. The dye fluorescence increased in a biphasic manner on addition of 500 μM Pi to the enzyme: a rapid phase (t1/2 < 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 μM. Fluorescence records vs time were exponential, and a plot of the rate constant versus [Pi] had a slope of 1.47 × 105 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: E1 E2 + Pi P−E2 + H2O, where k+1 = 0.059 s-1, k-1 = 0.023 s-1, k+2 = 1.47 × 105 M-1 s-1, and k-2 = 3.1 s-1. 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.