Two gears of pumping by the sodium pump

The kinetics of the phosphorylation and subsequent conformational change of Na+, K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na+)(3) state. On mixing with ATP in the presence of Mg2+, a fluorescence increase occurred, due to enzyme conversion into the E2P state. The fluorescence change accelerated with increasing ATP concentration until a saturating limit in the hundreds of micromolar range. The amplitude of the fluorescence change (Delta F/F-0) increased to 0.98 at 50 mu M ATP. Delta F/F-0 then decreased to 0.82 at 500 mu M. The decrease was attributed to an ATP-induced allosteric acceleration of the dephosphorylation reaction. The ATP concentration dependence of the time course and the amplitude of the fluorescence change could not be explained by either a one-site monomeric enzyme model or by a two-pool model. All of the data could be explained by an (alpha beta)(2) dimeric model, in which the enzyme cycles at a low rate with ATP hydrolysis by one alpha-subunit or at a high rate with ATP hydrolysis by both alpha-subunits. Thus, we propose a two-gear bicyclic model to replace the classical monomeric Albers-Post model for kidney Na+, K+-ATPase.