The plasma membrane Ca⁺² pump (PMCA) is responsible for the fine regulation of the intracellular Ca⁺² and is thus involved in the control of several cellular processes. This enzyme belongs to the P-type ATPase family, as well as the sarcoplasmic reticulum Ca⁺²-ATPase (SERCA) and Na⁺/K⁺ ATPase. It is described that AlF_x, MgF_x and BeF_x inhibit P-type ATPases, possibly by interacting with the phosphate-binding site of the enzymes. To date there is no study that completely explored the mechanism of interactions between beryllium fluoride and PMCA. Our group is studying the effects of treatment of PMCA with the BeF_x complex on the enzymatic activity. Binding of BeF_x to ghosts was done in medium containing 30 mM Tris-Cl (pH 7.4), 30 mM CaCl₂, 5 mM MgCl₂, 100 mM KCl, 0.2 mg/ml ghost membranes, and 0.5 mM ouabain. When necessary, the BeF_x complex was pre-formed by previous mixing of 50 m M BeSO₄ with 5 mM NaF for 10 min in water. Experiments of Ca²⁺-dependent ATPase activity of ghost membranes were done with or without CaM, and in presence or absence of ADP, at different BeSO₄ concentrations, with a fixed concentration of NaF (1mM). In the absence of BeSO₄, NaF by itself inhibited the hydrolytic activity of ghosts by about 50%. This inhibition was greater in the presence of BeSO₄, leading to complete inhibition with 10-20 m M BeSO₄. In presence of CaM and 0.5 µM BeSO₄, increasing concentrations of NaF completely inhibited the Ca²⁺-dependent hydrolytic activity of ghosts, with a k_0.5 of 400 µM, either in presence or absence of ADP. In absence of CaM and BeSO₄, the k_0.5 for NaF was shifted to almost 1 mM, evidencing that the BeF_x complex acts by formation of a tighter adduct than fluoride alone with the enzyme.Time course dependence experiments showed that this inhibition was fast. At 5 min of preincubation BeF_x complex inhibited the hydrolytic activity of ghosts. These data suggest that the BeF_x complex can bind to the E₁Ca conformation of PMCA and drive it to an E_?BeF_x inactive complex that remains stable after addition of ATP. It is possible that the BeF_x forms a dead-end complex in the E₂BeF_x conformation as described for SERCA.
This work is supported by CAPES, CNPq, FAPERJ. Mail to: jmignaco@bioqmed.ufrj.br