Phosphorylation of the plasma membrane calcium pump at high ATP concentration. On the mechanism of ATP hydrolysis.
The plasma membrane calcium ATPase (PMCA) reacts with ATP to form acid-stable phosphorylated intermediates (EP) that can be measured using (gamma-32P)ATP. However, the steady-state level of EP at [ATP] higher than 100 microM 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 25 degrees C. 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 (k(pEP)) as the ratio v/EP. At 1 mM Mg2+, k(pEP) increases hyperbolically with [ATP], while at 8 microM 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 4 degrees C, suggest that ATP is acting in additional steps involving the interconversion of phosphorylated intermediates during the hydrolysis of the nucleotide.
Echarte
María M
MM
Departamento de Química Biológica, IQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
Rossi
Rolando C
RC
Rossi
Juan Pablo F C
JP
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000097:blood / Q000737:chemistry / Q000378:metabolism*
D000255:Adenosine Triphosphate; D053499:Plasma Membrane Calcium-Transporting ATPases; D008274:Magnesium