Effects of ADP on the Na+/K+-ATPase

P. G. SCHVARTZ; J. L. E. MONTI; P. J. GARRAHAN; R. C. ROSSI

Rosario, ARGENTINA

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

The Na+/K+-ATPase is an intrinsic membrane protein which couples the transport of Na+ and K+ with the hydrolysis of ATP.  The pump can hydrolyze ATP in the absence or in the presence of K+ through the so-called Na+-ATPase and Na+/K+-ATPase cycles, respectively. These can be described in terms of the Post-Albers model, which postulates that, in presence of K+ or Rb+ (a K+-like cation), the Rb+ occluded intermediate can bind ATP to a regulatory, activating site [1]. In a previous work [2], we have shown that the time courses of the reaction intermediates could only be described considering a competitive effect of ADP for the ATP binding sites in E1 and in E2.  To test this hypothesis, we investigated the effects of ADP on the steady-state level of the Na+/K+-ATPase reaction intermediates during Na+-ATPase and Na+/K+-ATPase activities. We first determined the concentration of E2P, measuring the fluorescence change of the RH421 dye [3] at 25 °C in a medium containing 150 mM NaCl and 0.5 mM free Mg2, and 25 mM Imidazole-HCl, pH 7.4, at different concentrations of ATP and ADP. Results show that, during Na+-ATPase activity, ADP could act as a mixed-type inhibitor, competing with ATP in E1 and acting as a product inhibitor in the reverse reaction E1P+ADP->E1ATP. Additionally, we measured the concentration of Rb+ occluded through the standard quench flow technique [4] or by the fluorescence change of the RH421, in the same medium as that described above, plus 6 mM RbCl.  Results show that ADP, besides the effects observed in absence of Rb+, binds to the E2 form of the pump acting in lieu of ATP as a partial activator of the ATPase activity. The ability of ADP to increase the deocclusion rate was confirmed measuring the initial velocity of deocclusion at different [ADP], the results showing a hyperbolic relationship with a K0.5 for ADP of 750 mM. The results were adequately fitted by the equations derived from the Post-Albers mechanism and the values of the rate constants were estimated. We observed that the rate constant for the product inhibition caused by ADP is small enough as to be neglected at the micromolar concentrations of ATP used in the experiments described previously [2].   [1] Glynn, I. M., and Karlish, S. J., 1990, Annu Rev Biochem, 59, 171-205. [2] Schvartz P.G., Monti J.L.E.., González-Lebrero R.M., Kaufman S.B., Garrahan P.J. and Rossi R.C.,  2005,   J Gen Physiol, 126, 30a-31a. [3] Schvartz P.G., Monti J. L. E., González-Lebrero R. M., Kaufman S. B., Garrahan P. J., and Rossi R. C., 2004, XXXIII Reunion de la SAB, Mar del Plata [4]  Rossi, R.C., Kaufman, S.B., González-Lebrero, R.M., Norby, J. G., and Garrahan P.J., 1999, Anal. Biochem., 270, 276?285.   [Supported by CONICET, Agencia Nacional de Promoción Científica y Tecnológica and Universidad de Buenos Aires]