Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase.
A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [14C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [14C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump.
Keeling
D J
DJ
Smith Kline & French (Research) Ltd., Welwyn, Herts, United Kingdom.
Fallowfield
C
C
Lawrie
K M
KM
Saunders
D
D
Richardson
S
S
Ife
R J
RJ
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000494:pharmacology*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D005753:Gastric Mucosa / Q000201:enzymology*; D007093:Imidazoles / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D011188:Potassium / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D002250:Carbon Radioisotopes; D007093:Imidazoles; D007202:Indicators and Reagents; C035235:Sch 28080; C059036:8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazol(1,2-a)pyridinium; D011188:Potassium