1-anilino-8-naphtalene sulfonate probes a gastric HK-ATPase potassium site whose access requires ionophores.
1-anilino-8-naphtalenesulfonate (ANS) is a hydrophobic dipole previously used to demonstrate that the proton for potassium exchange by the gastric HK-ATPase is electroneutral. In this paper, we demonstrate that ANS binds to gastric membranes and probes conformational changes of the HK-ATPase independently of any active H for K exchange. Conformational changes require the presence of potassium-valinomycin and are not triggered by sodium. Potassium effect is enhanced by ATP, in the presence and in the absence of magnesium and, by ADP, in the presence of magnesium. Labeling of the pig HK-ATPase K518 by fluorescein-5-isothiocyanate inhibits the enzyme activity and knocks out the ATP effect on ANS fluorescence. Scherring 28080 and the monoclonal antibody 95-111, two competitive inhibitors of K-activated ATPase dephosphorylation, do not modify K-effect on ANS fluorescence but inhibit ATP effects. This supports that ANS does not probe K-site between the H1-H2 loop. Treatment of gastric membranes with trypsin does not inhibit the ANS response to potassium but does inhibit the response to ATP. This suggests that the ATP site inducing the ANS response is cytoplasmic and the potassium site is intramembranous. Titration reveals that one mole of ANS interacts with one mole of ATPase. We suggest that ANS probes a hydrophobic potassium site of gastric ATPase and that addition of ATP and ADP-Mg embed that site.
Festy
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F
INSERM U10, Hôpital Bichat, Paris, France.
Lins
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L
Gallet
X
X
Robert
J C
JC
Thomas-Soumarmon
A
A
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D005456:Fluorescent Dyes; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology*; D008958:Models, Molecular; D011188:Potassium / Q000378:metabolism* / Q000494:pharmacology; D017510:Protein Folding; D013270:Stomach / Q000201:enzymology; D014634:Valinomycin / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007476:Ionophores; D014634:Valinomycin; D000244:Adenosine Diphosphate; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium