Abstract
ATP and its analogues act on the minimal functional unit of Na, K-ATPase, the alpha beta protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K+-phosphatase activity of soluble FITC-modified alphabeta protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N3-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N3-[alpha-32P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 microM TNP-8N3-[alpha-32P]ADP, and ATP or FITC treatment prevents labeling of the alpha chain. At 25 microM, however, TNP-8N3-[alpha-32P]ADP can be incorporated in the FITC-modified alpha chain, concurrently with the inactivation of the K+-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of 32P-probe per mol of alpha chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na+ or Mg2+, but not K+. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N3-[alpha-32P]ADP. Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na+ site. Partial trypsinolysis reveals that the point of TNP-8N3-[alpha-32P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified alpha chain. The affinity labeling of the fluorescein enzyme by TNP-8N3-[alpha-32P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each alpha subunit of Na,K-ATPase.
Highlights
ATP and its analogues act on the minimal functional unit of Na,K-ATPase, the ␣ protomer, with high and low affinity effects
We found that when Fluorescein isothiocyanate (FITC)-modified Na,K-ATPase was solubilized with C12E8, the soluble protomers retained their Kϩphosphatase activity but did not recover their ability to hydrolyze ATP [14]; it follows that FITC must effectively block the high affinity ATP site of every ␣ protomer and not just one half of an (␣)2 membrane dimer
TNP-8N3-ADP Photolabeling of the Catalytic Site—The problem associated with low affinity binding calls for a ligand with an intrinsically higher avidity for the site in question (e.g. Ref. 39)
Summary
Na,K-ATPase, sodium- and potassiumdependent adenosine triphosphatase (EC 3.6.1.37); FITC, fluorescein 5Ј-isothiocyanate; pNPP, p-nitrophenyl phosphate; pNPPase, p-nitrophenyl phosphatase; TNP-ADP, 2Ј(3Ј)-O-(2,4,6-trinitrophenyl)adenosine 5Ј-diphosphate; TNP-8N3-ADP, 2Ј(3Ј)-O-(2,4,6-trinitropheyl)8azidoadenosine 5Ј-diphosphate; E1, Naϩ form of enzyme; E2, Kϩ form of enzyme. It has been proposed that an enzyme with a single site, which would alternate between high and low-affinity conformations, could account for the complex behavior with respect to ATP [13] This seemed a plausible alternative when the C12E8 (octaethylene glycol dodecyl monoether)-solubilized protomers were shown to present dual responses to ATP in the absence of oligomerization [6]. The simultaneous covalent binding of FITC and TNP nucleotide to the same soluble protomer suggested that high and low affinity ATP binding occurred at two distinct sites on the protomeric enzyme unit. Photoaffinity Labeling of 2 Nucleotide Sites on Na,K-ATPase an affinity label for the high-affinity (catalytic) ATP site; we go on to show directly that the FITC-modified ␣ subunit can covalently bind TNP-8N3-[␣-32P]ADP. That the site of primary TNP-8N3-[␣-32P]ADP attachment to the FITC-modified Na,K-ATPase is within a regulatory nucleotide pocket distinct from the catalytic ATP binding site
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