Fluoride irreversibly inhibits Na,K-ATPase activity with aluminum and divalent cations

Objectives: Na,K-ATPase activity is irreversibly inhibited by fluoride in the presence of some ligands. We examined the effect of aluminum and divalent cations on irreversible inhibition of Na,K-ATPase activity by fluoride. Methods: Na,K-ATPase was preincubated in the presence of 2.5 mM NaF or KF, and with or without aluminum, divalent cations, inorganic phosphate and aluminum chelater, deferoxamine. After preincubation, the enzyme solution was diluted ten times and then Na,K-ATPase activity was measured to test the reversibility of inhibition. Results: When ATPase activity was measured with different concentrations of NaF or KF without dilution, NaF and KF inhibited Na,K-ATPase activity depending on their concentrations, and the concentration of half maximal inhibition was about 1.3 to 1.4 mM. NaF less than 2.5 mM of final concentration did not inhibit Na-ATPase activity. On the other hand, KF less than 1.5 mM increased the Na-ATPase activity, but decreased the activity with more than 2.5 mM. We also found that aluminum ion increased the inhibition of ATPase activity by fluoride depending on its concentration. After the enzyme pretreated with 2.5 mM NaF or KF was diluted, almost 50 % of activity was inhibited. The inhibition increased to 90 % when preincubation was done with fluoride plus more than 7.5 µM aluminum ion, but the inhibition by fluoride was not observed in the presence of deferoxamine. The irreversible inhibition requires Mg²⁺ depending on its concentration, and the effect of Mg²⁺ was replaced with Mn²⁺ or Ca²⁺. Inorganic phosphate did not affect the irreversible inhibition. Conclusions: These results suggest that irreversible inhibition by fluoride requires aluminum, divalent cations and potassium, and that the complex of fluoride and aluminum binds to Na,K-ATPase irreversibly in the presence of divalent cations.