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.
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 [gamma-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 Kd = 0.3-1 microM to the first site and a Kd = 100-120 microM 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 Kd = 500 +/- 100 microM to the first site and a Kd = 2.5 +/- 0.5 microM 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.
Thoenges
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Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen; Frankfurter Strasse 100, D-35392 Giessen, Germany.
Schoner
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D003619:Dansyl Compounds / Q000378:metabolism*; D006868:Hydrolysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D003619:Dansyl Compounds; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase