A continuous flow technique for analysis of the stoichiometry of the gastric H,K-ATPase

A continuous flow method was developed for determining the stoichiometry of the gastric proton pump H,K-ATPase in its hydrolysis of ATP, translocation of H+ and the K+ congener 86Rb+. H,K-ATPase-containing vesicles which had been isolated from pig gastric mucosa were incubated at 37 degrees C for 2 h in 150 mM 86RbCl, 0.5 mM EGTA and 3 mM Mes-buffer adjusted to pH 6.1 with Tris, and then applied to a 0.45 micron pore size filter. The immobilized vesicles were superfused with 0.15 mM Mes/Tris buffer, pH 6.1, containing 150 mM choline-Cl and 0.2 mM MgCl2. After the medium was changed to one containing 0.1 mM ATP, the amounts and rates of H+ uptake, 86Rb+ efflux, and ATP hydrolysis were measured in fractions collected after the filter. The initial ratio of transported Rb+ to hydrolysed ATP gave values of 0.96 +/- 0.26 (mean +/- SD, n = 28). The initial ratio of ATP-dependent Rb+ efflux to H+ uptake gave values of 0.92 +/- 0.28 (mean +/- SD, n = 28). The MgATPase activity was measured in vesicles which had been incubated with choline-Cl instead of RbCl. In the initial fractions used for calculation of the stoichiometry, the MgATPase activity was 15.8% +/- 8.7 (mean +/- S.D.) of the maximal ATPase activity obtained with Rb(+)-loaded vesicles. The MgATPase may be an intrinsic activity of the H,K-ATPase. However, whether corrections were made for the MgATPase or not, it had only marginal effects on the calculations of the stoichiometry of the pump.(ABSTRACT TRUNCATED AT 250 WORDS)