Phosphorylation of the plasma membrane calcium pump at high ATP concentration. On the mechanism of ATP hydrolysis

M. M. ECHARTE; R. C. ROSSI; J. P. F. C. ROSSI

BIOCHEMISTRY

American Chemical Society

Año: 2007 vol. 46 p. 1034 - 1041

0006-2960

The Plasma Membrane Calcium ATPase (PMCA) reacts with ATP to form acid-stable phosphorylated intermediates (EP) that can be measured using (gamma32P)ATP. However, the steady-state level of EP at [ATP] higher than 100 µM has not yet been studied due to methodological problems. Using a microscale method and a purified preparation of PMCA from human red blood cells, we measured the steady-state concentration of EP as a function of [ATP] up to 2 mM at different concentrations of Mg2+, both at 4 and 25ºC. We have measured the Ca2+-ATPase activity (v), under the same conditions as those used for phosphorylation experiments. While the curves of ATPase activity vs. [ATP] were well described by the Michaelis-Menten equation, the corresponding curves of EP required more complex fitting equations, exhibiting at least a high- and a low-affinity component. Mg2+ increases the apparent affinity for ATP of this latter component but it shows no significant effect on its high-affinity one or on the Ca2+-ATPase activity. We calculated the turnover of EP (kpEP) as the ratio v/EP. At 1 mM Mg2+, kpEP increases hyperbolically with [ATP] while at 8 mM Mg2+ it exhibits a behavior that cannot be explained by the currently accepted mechanism for ATP hydrolysis. These results, together with measurements of the rate of dephosphorylation at 4ºC, suggest that ATP is acting in additional steps involving the interconversion of phosphorylated intermediates during the hydrolysis of the nucleotide.