A model for the interaction between Rb+, ATP and Na+/K+-ATPase

SPIAGGI AJ; MONTES MR; GONZÁLEZ-LEBRERO RM; ROSSI RC

Salta

Congreso; SAB 2010-Workshop CeBEM-Protein Society Meeting; 2010

Occlusion of K+ (or its congeners Rb+ or Tl+) in the Na+/K+-ATPase occurs afterK+-dependent dephosphorylation (“physiological route”) or through a “direct route” notrelated to ATPase activity.Our results indicate that: i) occlusion of Rb+ through the direct route, as well asits release from the occluded states, are at least two-steps processes that can be describedby the sum of two exponential functions of time, for every [Rb+] and [ATP] tested; ii) thefraction of Rb+ that is occluded or released in the faster phase is enlarged by ATP; iii) theinitial rate of occlusion is directly proportional to free [Rb+] up to 250 μM in the absenceof ATP, whereas it decreases with [ATP] and becomes an increasing sigmoidal function offree [Rb+] at [ATP] = 2000 μM; iv) the nucleotide markedly increases the initial rate andthe rate coefficients of both phases of Rb+ deocclusion; v) at equilibrium, the direct routeresults in enzyme states that hold either one or two occluded Rb+ ions even in enzymeswith ATP bound; vi) equilibrium between bound and occluded Rb+ seems to be almostcompletely poised towards the latter in spite of the mentioned effect of ATP on occlusionand deocclusion rates. Part of these results had been previously observed in our lab.The kinetics of Rb+ occlusion and deocclusion through the direct route in thepresence of ATP are consistent with a model that assumes that: 1) these processes canfollow two alternative and interrelated pathways associated with the conformers E1 andE2; 2) only E2 can occlude Rb+; 3) uptake or release of each cation involves an ordered-sequential process; and 4) ATP can bind to E1 and E2, being its affinity for E1 much higher.