The Na K-ATPase is one of the P-type ATPase family of primary active cation pumps. and subsequent reactivation by high Na+ after treatment of shark Na K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the LKB1 Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway which in Na K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain but not its aglycone ouabagenin prevented reactivation of this metal fluoride form by high Na+ demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway. were prepared by homogenization followed by washing and isolation by centrifugation in 30 mm histidine 1 mm EDTA 0. 25 m sucrose pH 6.8. The purified microsomes were activated by a moderate DOC treatment (～0.15% DOC) to extract extrinsic proteins and to open sealed vesicles. After washing and resuspension the purified membrane preparation was obtained by differential centrifugation essential as previously explained (17). The preparation was suspended in histidine/EDTA buffer with 25% glycerol and kept at ?20 °C. The preparation had a specific hydrolytic activity of ～30 models/mg at 37 °C and contained the α1- β1-subunits together with the FXYD10 regulatory subunit (18). Protein concentrations ranging from 3-5 mg/ml were decided using Peterson’s modification (19) GW 5074 of the Lowry method (20) using bovine serum albumin as a standard. Na K-ATPase Activity The specific enzyme activity was measured using either the Fiske and SubbaRow method (21) with Amidol as the reducing agent or the more sensitive method of Baginsky (22). The activity was measured at 23 °C in a test medium made up of 130 mm NaCl 20 mm KCl 4 mm MgCl2 3 mm Tris-ATP and 0.33 mg/ml bovine serum albumin. Histidine or imidazole (30 mm) was used as buffer depending on pH. Inhibition of Na K-ATPase by Fluorides and Cardiotonic Steroids The inhibition of Na K-ATPase activity by MgFx by equilibrium binding was characterized by combining 5 mm MgCl2 and increasing concentrations of NaF (1-100 mm) in imidazole 30 mm pH 6.5 7.5 or 8.5 followed by addition GW 5074 of Na K-ATPase and preincubation for 10 min. at 23 °C. The inhibition by BeFx and GW 5074 AlFx was performed by mixing 5 mm NaF and increasing concentrations of BeSO4 or AlCl3 (1-200 μm) followed by preincubation with the enzyme as explained above. The reaction with AlFx·ADP was produced by including 1 mm ADP in the AlFx reaction media. Following preincubation the Na K- ATPase activity at 23 °C was decided at optimal turnover circumstances in 130 mm Na+ 20 mm GW 5074 K+ 4 mm Mg2+ 3 mm ATP and 30 mm imidazole (pH 7.5). Enough time span of inhibition at several concentrations of fluorides was dependant on varying enough time of preincubation with fluorides (15-180 s) as defined above accompanied by addition from the enzyme activity check moderate. After 1 min the response was ended by 50% TCA and liberated Pi was motivated. The speed of inhibition induced by binding of CTS to non-phosphorylated or MgPi-phosphorylated Na K-ATPase was dependant on preincubation of enzyme in 30 mm imidazole (pH 7.5) with either 5 mm Mg2+ or 5 mm Mg2+ + 1 mm Pi with 1 μm ouabain GW 5074 ouabagenin or anthroyl ouabain for differing time periods accompanied by measurement of hydrolytic activity. Reactivation of Na K-ATPase after Fluoride Treatment The enzyme was initially reacted with steel fluoride complexes by incubating in 5 mm NaF (or KF) 30 mm imidazole pH 7.5 and either 5 mm MgCl2 5 μm BeSO4 200 μm AlCl3 or 200 μm AlCl3 plus 1 mm ADP for 10 min. at 23 °C to acquire maximum inhibition. 150 mm NaCl was added Then.