Effect of ATP on the Occlusion and Deocclusion Kinetics of Rb+ in Na+/K+-ATPase through the direct route

ALEJANDRO J. SPIAGGI; JOSÉ L. E. MONTI; MÓNICA R. MONTES; PATRICIO J. GARRAHAN; ROLANDO C. ROSSI

Los Cocos (Córdoba), Argentina

Congreso; XXXVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2009

Sociedad Argentina de Biofísica

The aim of this work is to characterize the effect of adenosine-5?-triphosphate (ATP) on the reactions involved in the occlusion and deocclusion of Rb+ by the Na+/K+?ATPase in the absence of phosphorylation (direct route). All experiments were performed at 25ºC and pH 7.4 using a partially purified membrane preparation of pig?kidney Na+/K+?ATPase. Time courses of Rb+ occlusion in media containing RbCl 0.25mM and ATP (imidazole salt) in concentrations ranging from 0 to 0.55mM were best fitted by the sum of two increasing exponential functions of time plus a time-independent term. While the slower phase appears to be accelerated by ATP, the initial rate showed a 3-fold decrease along a rectangular hyperbola whose K0.5 was 506±196μM. However, when Rb+ occlusion took place in media containing 1mM ATP or more, only one phase was distinguishable. Deocclusion of Rb+ in the presence of RbCl 0.25mM and up to 2.2mM ATP was adequately described by the sum of two decreasing exponential functions of time with similar amplitudes plus a constant term. The nucleotide caused a hyperbolic increase of the rate coefficients of both phases and of the initial rate of deocclusion, to an upper limit either 40 times (initial rate and fast phase) or even 170 times (slow phase) higher than in its absence. The apparent affinity for ATP was in each case 352±86μM (fast phase), 302±23μM (slow phase) and 353±82μM (initial rate). The equilibrium amount of occluded Rb+ in media lacking ATP showed a hyperbolic response to [Rb+] (K0.5 = 5.25±0.47μM). On the contrary, in the presence of ATP the increase in [Rb+] led to a sigmoidal raise in the amount of occluded cation, remaining the maximum occlusion capacity unchanged regardless of [ATP]. These results confirm previous ones obtained by our group [1]. All these results agree with a model where ATP binds to states of Na+/K+?ATPase that might or might not carry occluded Rb+ and promotes the formation of a state that occludes Rb+ at a lower rate. This process could involve a change, induced by ATP, of the frequency at which the enzyme exposes its transport sites to the intracellular face of the membrane.