| Autor: |
Thoenges, Detlef, Schoner, Wilhelm |
| Zdroj: |
Journal of Biological Chemistry; June 1997, Vol. 272 Issue: 26 p16315-16321, 7p |
| Abstrakt: |
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 [γ-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 μmto the first site and a Kd= 100–120 μmto 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 aKd= 500 ± 100 μmto the first site and a Kd= 2.5 ± 0.5 μmto 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. |
| Databáze: |
Supplemental Index |
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