Detection of occluded Rb+ intermediates during ATP hydrolysis catalyzed by Na+/K+-ATPase in nominally Na+-free media

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

Rosario, Argentina

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

Sociedad Argentina de Biofísica

The membrane protein Na+/K+-ATPase couples the hydrolysis of 1 ATP molecule to the exchange of 3 cytoplasmic Na+ for 2 extracellular K+. Mg2+ is an essential activator of the catalysis. The catalytic mechanism remains unclear. It is known that during the catalytic cycle the enzyme becomes phosphorylated and that the transported cations become occluded (trapped within the enzyme). K+-occluded intermediates of the Na+/K+-ATPase can be obtained either by the direct route, without formation of phosphoenzyme, or by the physiological route, after K+-stimulated dephosphorylation of the phosphoenzyme. Several cations can replace K+, including Rb+ (1-3). Previous studies (4) show that as [Na+] is increased at a fixed [Rb+], occluded Rb+ and ouabain-sensitive ATPase activity increase along a sigmoidal curve with an initial slope equal to zero, as expected if 3 Na+ were needed for ATP phosphorylation of the pump. However, a small but significant ATPase activity is measured when no Na+ is added to the reaction media. Moreover, under these conditions, there is an increase and then a decrease of ATPase activity as [Rb+] increases. The inhibition of ATPase activity at high [Rb+] is explained by the inhibition of enzyme phosphorylation due to formation of occluded Rb+ intermediates through the direct route. On the other hand, the increase in ATPase activity observed at low [Rb+] can be explained by assuming that Rb+ enhances dephosphorylation of the same phosphointermediate that is formed through Na+‑activated phosphorylation. Because such dephosphorylation should lead to Rb+ occlusion, we have measured steady-state levels of occluded Rb+ at 25 °C using a purified preparation of pig kidney Na+/K+-ATPase in nominally free Na+ media (with less than 10 mM Na+) containing 2.5mM ATP, imidazole-HCl 25mM, pH=7.4, and different concentrations of [86Rb+]RbCl and CholineCl (an inert salt added in order to keep constant the ionic strength at 170mM), with 0.5mM free Mg2+ or without Mg2+. A small but significant increase of occluded Rb+ in the presence of Mg2+ is observed, suggesting that Na+ activated phosphorylation leads to the same Rb+-sensitive phosphointermediate that is formed in the assayed conditions. Since only H+ and Li+ have been reported as possible Na+ analogs (5,6), it remains an open question how enzyme is phosphorylated. Supported with grants from: UBACYT, ANPCYT, and CONICET. References [1]  Robinson JD, Flashner MS, Biochim, Biophys. Acta, 1979, 549, 145 - 176. [2]  Forbush B, J. Biol. Chem., 1987, 262, 11104 - 11115. [3]  Rossi RC, González-Lebrero RM, Kauffman SB, Garrahan PJ, J. Gen. Physiol., 2005, 126, 29a-30a. [4]  Monti JLE, Schvartz PG, González-Lebrero RM, Kauffman SB, Rossi RC,Garrahan PJ, J. Gen. Physiol., 2005, 126, 28a-29a. [5]  Beaugé L, Biochim, Biophys. Acta, 1978, 527, 472 - 484. [6]  Hara Y, Nakao M, J. Biol. Chem., 1986, 261, 12655 - 12658.