Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H+K(+)-ATPase.
Membrane fragments containing the H+K(+)-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na+K(+)-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985. EMBO J. 4:3079-3085). Since H+K(+)-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K+. By separation of the H+ and K+ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K+ or at low concentrations of K+. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was concluded that the K+ transport during dephosphorylation is electrogenic, too, in contrast to the Na+K(+)-ATPase where the K+ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H+K(+)-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.
van der Hijden
H T
HT
Department of Biochemistry, University of Nijmegen, The Netherlands.
Grell
E
E
de Pont
J J
JJ
Bamberg
E
E
D000244:Adenosine Diphosphate / Q000502:physiology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D004553:Electric Conductivity / Q000502:physiology; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D009853:Omeprazole / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011522:Protons; D013552:Swine
D010710:Phosphates; D011522:Protons; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D008274:Magnesium; D009853:Omeprazole