Characterization of ouabain-sensitive phosphatase activity in the absence of potassium ion in purified pig kidney Na,K-ATPase.
The ouabain-sensitive phosphatase activity of purified pig kidney Na,K-ATPase preparation in the absence of potassium ion ((-K)phosphatase) was examined precisely. During the preparation procedures, the (-K)3-O-methylfluoresceinphosphatase ((-K)3-OMFPase) activity or the (-K)p-nitrophenylphosphatase ((-K)pNPPase) activity appeared to be purified in parallel with the Na,K-ATPase activity. The (-K)phosphatase activity was competitively inhibited by ATP and by ADP, with the K1 values of 0.25 microM and 1.4 microM, respectively. These values are consistent with their Kd values for the high-affinity ATP binding site of the Na,K-ATPase (Hegyvary, C. & Post, R.L. (1971) J. Biol. Chem. 246, 5234-5240). The substrate, pNPP, apparently competed with covalently bound fluorescein-5'-isothiocyanate (FITC), which is known to bind in the neighborhood of the high-affinity ATP binding site of the Na,K-ATPase, in both the (-K)phosphatase and the (+K)phosphatase reactions. The FITC-fluorescence intensity of FITC-labeled enzyme at the maximal steady-state activity of the (-K)phosphatase reaction was at a similar level to that of the E2 species. However, the FITC-labeled enzyme in the presence of only magnesium ion or only pNPP gave a fluorescence level similar to that of the E1 species. Oligomycin inhibited the (-K)phosphatase activity by at most 46%. On the basis of these results, it is strongly suggested that the (-K)phosphatase reaction is catalyzed at the high-affinity ATP binding site of Na,K-ATPase, and the (-K)phosphatase reaction proceeds in a cyclic manner (E1----E2----E1).
D000818:Animals; D002384:Catalysis; D004573:Electrolytes / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008024:Ligands / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine; D013861:Thiocyanates
D004573:Electrolytes; D005452:Fluoresceins; D008024:Ligands; D009840:Oligomycins; D013861:Thiocyanates; D010042:Ouabain; D010744:Phosphoric Monoester Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium