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Volume 272, Number 26, Issue of June 27, 1997 pp. 16315-16321
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

2-O-Dansyl Analogs of ATP Bind with High Affinity to the Low Affinity ATP Site of Na⁺/K⁺-ATPase and Reveal the Interaction of Two ATP Sites during Catalysis

(Received for publication, November 21, 1996, and in revised form, March 20, 1997)

From the Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen; Frankfurter Strasse 100, D-35392 Giessen, Germany

Na⁺/K⁺-transport through mammalian cell membranes by Na⁺/K⁺-ATPase (EC 3.6.1.37) needs the interaction of ATP sites with different binding affinities during catalysis: one with catalytic (high affinity site) and one with regulatory properties (low affinity site). To find affinity labels for the latter one, the effects of 2-O-dansylated ATP analogs on Na⁺/K⁺-ATPase and its partial activities were analyzed. DANS-ATP (2-O-(6-dimethylaminonaphthalenesulfonyl)adenosine 5-triphosphate) inhibited noncompetitively at low ATP concentrations and competitively at high ATP concentrations the Na⁺/K⁺-activated hydrolysis of ATP under turnover conditions. It interacted preferentially with the low affinity ATP site as shown by its protective effect against the inactivation of Na⁺/K⁺-ATPase by Co(NH₃)₄ATP and Cr(H₂O)₄ATP. DANS-N₃-ATP, however, inactivated Na⁺/K⁺-ATPase. The initial velocity of inactivation shows a sigmoid concentration dependence that was converted to a hyperbola in the presence of ATP. DANS-N₃-ATP inhibited competitively the K⁺-activated hydrolysis of p-nitrophenyl phosphate in a fluorescein isothiocyanate-blocked enzyme but did not effect Na⁺-dependent phosphoenzyme formation from [-³²P]ATP in a Co(NH₃)₄PO₄-blocked enzyme. These effects could be described by a Koshland-Némethy-Filmer model assuming two nucleotide binding sites in strong cooperation. Fitting all data to this model revealed that ATP was bound in a negative cooperative way with a K_d = 0.3-1 µM to the first site and a K_d = 100-120 µM to the second site of the enzyme containing already one ATP bound. The hydrolysis of ATP through a pathway with two ATP bound was 30 times faster than hydrolysis with one ATP bound. DANS-N₃-ATP bound in a positive cooperative way with a K_d = 500 ± 100 µM to the first site and a K_d = 2.5 ± 0.5 µM to the second site containing already one DANS-N₃-ATP bound. Therefore, DANS-N₃-ATP may be an useful affinity marker of the low affinity, regulatory ATP site.

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