Inhibition of the Na,K-ATPase by fluoride. Parallels with its inhibition of the sarcoplasmic reticulum CaATPase.
Addition of lithium fluoride to a suspension of Na,K-ATPase undergoing turnover produced a slow (minutes) complete loss of ouabain-sensitive ATPase activity. Persistence of the effect in the presence of deferoxamine showed that fluoride inhibits independent of aluminum. The time course of onset of inhibition was adequately fit by a function corresponding to a monophasic transformation with a pseudo first-order rate constant (k(obs)). This constant varied hyperbolically with [Mg2+] (half-maximal effect at 9 mM Mg2+), whereas it increased with no sign of approaching saturation as the square of [F-], implying that inhibition requires binding of two fluorides/ATPase. The value of k(obs) was found to be increased by greater than 10-fold in the presence of potassium ([K+]1/2 = 0.6 mM) or ouabain. Sodium, ATP, and ADP, which favor the E1 form of the enzyme, had a protective effect. These results implicate the potassium-occluded MgE2(K2) complex as the main fluoride-susceptible form. Protection by Pi and orthovanadate suggests that fluoride exerts its effect at the phosphorylation site. Inhibition was reversible, although slowly, with t1/2 = 7 h at 37 degrees C. Sodium greatly accelerated reversal (t1/2 = 3 min with 150 mM Na+ present), and potassium antagonized this acceleration. The value of k(obs) for reactivation increased steeply with [Na+], with the sodium dependence being about the same at pH 8.0 as at pH 7.4. All of these effects have parallels to effects of fluoride on the sarcoplasmic reticulum CaATPase (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235).
Murphy
A J
AJ
Department of Biochemistry, School of Dentistry, University of the Pacific, San Francisco, California 94115.
Hoover
J C
JC
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D004285:Dogs; D005459:Fluorides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D012984:Software
D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D005459:Fluorides