Two Gears of Pumping by the Sodium Pump

¹ The University of Sydney F-11 School of Chemistry
² Max-Planck-Institute for Biophysics

^* To whom correspondence should be addressed. E-mail: r.clarke{at}chem.usyd.edu.au.

Submitted on April 26, 2007
Revised on June 6, 2007
Accepted on 31 July 2007

	Abstract

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°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 (F/F₀) increased to 0.98 at 50 µM ATP. F/F₀ then decreased to 0.82 at 500 µM. 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 couldn't 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 ()₂ dimeric model, in which the enzyme cycles at a low rate with ATP hydrolysis by one -subunit or at a high rate with ATP hydrolysis by both -subunits. Thus, we propose a two-gear bicyclic model to replace the classical monomeric Albers-Post model for kidney Na⁺,K⁺-ATPase.

Key Words: ATP, Na+,K+-ATPase, dimerisation, fluorescence, stopped-flow kinetics, voltage-sensitive styryl dye