Electrogenic Partial Reactions of the Gastric H,K-ATPase

AbstractThe fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K+ and H+ ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E1 and P-E2, are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and ≤4.5 in the E1 conformation; 6.7 and ≤2 in the P-E2 conformation. The equilibrium dissociation constants for K+ binding on the cytoplasmic side were 11 and 16mM. The respective equilibrium dissociation constants on the luminal side were obtained via K+ concentration dependence of the enzyme activity and determined to be 0.11mM for both luminal binding sites