Kinetics of transient pump currents generated by the (H,K)-ATPase after an ATP concentration jump.
(H,K)-ATPase containing membranes from hog stomach were attached to black lipid membranes. Currents induced by an ATP concentration jump were recorded and analyzed. A sum of three exponentials (tau 1(-1) approximately 400 sec-1, tau 2(-1) approximately 100 sec-1, tau 3(-1) approximately 10 sec-1; T = 300 K, pH 6, MgCl2 3 mM, no K+) was fitted to the transient signal. The dependence of the resulting time constants and the peak current on electrolyte composition, ATP conversion rate, temperature, and membrane conductivity was recorded. The results are consistent with a reaction scheme similar to that proposed by Albers and Post for the NaK-ATPase. Based on this model the following assignments were made: tau 2 corresponds to ATP binding and exchange with caged ATP. tau 1 describes the phosphorylation reaction E1 x ATP-->E1P. The third, slowest time constant tau 3 is tentatively assigned to the E1P-->E2P transition. This is the first electrogenic step and is accelerated at high pH and by ATP via a low affinity binding site. The second electrogenic step is the transition from E2K to E1H. The E2K<==>E1H equilibrium is influenced by potassium with an apparent K0.5 of 3 mM and by the pH. Low pH and low potassium concentration stabilize the E1 conformation.
Max-Planck-Institut für Biophysik, Frankfurt am Main, FRG.
M
M
Stengelin
K
K
Fendler
E
E
Bamberg
D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D017506:H(+)-K(+)-Exchanging ATPase / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D011188:Potassium / Q000494:pharmacology; D013270:Stomach / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure; D013552:Swine; D013997:Time Factors
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium