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pubmed:abstractText |
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 Mg(2+), 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 (DeltaF/F(0)) increased to 0.98 at 50 microM ATP. DeltaF/F(0) then decreased to 0.82 at 500 microM. 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 (alphabeta)(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.
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