Oclusion and deoclusion of Rb by NaKATPase through the direct route in the presence of ATP

SPIAGGI ALEJANDRO; MONTI JOSÉ; MONTES MÓNICA; GARRAHAN PATRICIO; ROSSI ROLANDO

Rio de Janeiro

Congreso; VII Iberoamerican Congress of Biophysics; 2009

OCCLUSION AND DEOCCLUSION OF Rb+ BY Na+/K+-ATPase THROUGH THE DIRECT ROUTE IN THE PRESENCE OF ATP Alejandro J. Spiaggi**, José L. E. Monti**, Mónica R. Montes, Patricio J. Garrahan y Rolando C. RossiInstituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina 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 Na+/K+–ATPase through the 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 Rb+ 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. As ATP concentration increased, the initial rate of occlusion showed a 3-fold decrease along a rectangular hyperbola whose K0.5 was 506±196mM. When Rb+ occlusion took place in media containing more than 1mM ATP, we could distinguish only one phase whose rate coefficient was practically independent of [ATP]. The equilibrium level of occluded Rb+ also decreased with [ATP] (K0.5=406±64mM). Deocclusion of Rb+ in media containing up to 1.5mM 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, with an apparent affinity of 242±164mM (fast phase), 290±20mM (slow phase) and 227±156mM (initial rate). 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 should involve the exposure of transport sites to the intracellular face of the membrane. With grants from ANPCyT, CONICET