GONZALEZ LEBRERO Rodolfo Martin
The sidedness of the direct route of occlusion of K+ in the Na+/K+ ATPase.
RODOLFO MARTIN GONZALEZ LEBRERO; SERGIO BENJAMÍN, KAUFMAN; PATRICIO J, GARRAHAN; ROLANDO CARLOS, ROSSI
ANNALS OF THE NEW YORK ACADEMY OF SCIENCES.
Lugar: Oxford; Año: 2003 vol. 986 p. 301 - 303
Forbush (1988, Prog. Clin. Biol. Res., 268A, 229) found evidence that deocclusion of K+ stimulated by Mg2+ and orthophosphate (MgPi) occurs through extracellular sites of the Na+/K+‑ATPase. It is also believed that occlusion that takes place after mixing the enzyme with K+ or its congeners (e.g. Rb+) in the absence of other pump ligands (direct route) occurs through intracellular sites, although there is no clear cut evidence to support this view. Direct route leads to the occlusion and deocclusion of two Rb+ through an ordered process (González-Lebrero et al., 2002, J. Biol. Chem., 277, 5910) with a fast and a slow components of the same size, thus resembling the kinetics for Rb+ deocclusion in the presence of MgPi (Glynn et al., 1985, J. Physiol., 368, 453; Forbush, 1987, J. Biol. Chem., 262, 11116). In experiments at 25ºC, we investigated the sidedness of K+ occlusion through the direct route using 86Rb+. Enzyme was first equilibrated in 100 µM Rb+ and then incubated in 500 µM Rb+ for 48 s before measuring 86Rb+ loss in media with 8 mM Pi, 1 mM Mg2+ and 10 mM (unlabelled) Rb+. 86Rb+ was present during either the first or the 48 s incubation periods, to label the slow- or fast-exchange pools, respectively. Results show that the 86Rb+ that remained occluded decreased with time with rate coefficients of 0.5 s-1 or 15 s-1, depending on whether 86Rb+ had been present during the first or the 48 s incubation periods, respectively. This strongly suggests that Rb+ was released through the same sites as those from which the cation entered the enzyme and that these sites are the external ones, since 86Rb+ deocclusion was performed in media with MgPi.With grants from ANPCyT, CONICET and UBA.Keywords: Sidedness, occlusion, direct route