The kinetic identity between occluded Rb+ formed in the Na+/K+ ATPase by the direct or by the physiological route

RODOLFO MARTIN GONZALEZ LEBRERO; SERGIO BENJAMÍN, KAUFMAN; JENS G, NOERBY; PATRICIO J, GARRAHAN; ROLANDO CARLOS, ROSSI

INTERNATIONAL CONGRESS SERIES

ELSEVIER

Lugar: Amsterdam; Año: 2000 p. 433 - 436

0531-5131

Occlusion of K+ or its congeners in the Na+/K+‑ATPase can occur either through the reversal of the deocclusion or after K+‑dependent dephosphorylation of the enzyme. The former is called direct occlusion and becomes apparent after mixing the enzyme with K+ or its congeners in media lacking Na+ and ATP whereas the latter is called physiological occlusion and can be measured during hydrolysis of ATP. Direct occlusion leads to thermodynamic equilibrium whereas physiological occlusion tends to a steady-state which lasts as long as the supply of ATP is adequate and there is no accumulation of Pi and ADP. It is generally assumed that the species that holds occluded K+ is the same regardless of the route used for occlusion. This may not be so since multiple enzyme states occluding K+ might exist and the distribution among them might differ according to the occlusion route. In the experiments reported here we used our rapid filtration technique to measure the kinetics of deocclusion of the K+‑congener Rb+ formed both by the direct and the physiological routes and the effects of ATP and Pi on this process. The experiments were performed using a partially purified Na+/K+‑ATPase from pig kidney and measuring the time course of deocclusion in media containing from zero to 8 mM Pi or from zero to 2.5 mM ATP. Results showed that that the occluded intermediates released Rb+ following a kinetic equation that had the same shape and the same values for its rate coefficients and that responded in the same way to the concentration of Pi and ATP regardless of the route used for obtaining occlusion. This strongly suggests that the direct and the physiological routes generate the same enzyme intermediate holding occluded Rb+.Supported by the EU and by CONICET, UBA and ANPCYT, Argentina.