The H+/ATP transport ratio of the (K+ + H+)-ATPase of pig gastric membrane vesicles.

Various values have been reported for the H+/ATP transport ratio of the (K+ + H+)-ATPase of the gastric parietal cell: 4, 2 and 1. We have, therefore, reinvestigated this matter with a vesicle preparation isolated from pig gastric mucosa. The vesicles are suspended in glycylglycine buffer (pH 6.11) at 22 degrees C, and incubated until equalization of the K+ concentration inside and outside (75 mM). After addition of ATP, the initial rates of H+ uptake and ATP hydrolysis are then measured. Proton uptake is inhibited in the absence of K+ or in the presence of nigericin. The K0.5 value for proton transport is 154 microM and the Km value for ATP hydrolysis is 61 microM. The Lineweaver-Burk plot for ATP hydrolysis vs. ATP concentration is linear with a Vmax of 5.5 nmol/mg protein per s, but that for H+ uptake is not. Thus with increasing ATP concentration (6.7 to 1670 microM) the transport ratio increases from 0.3 to 1.8. Extrapolation to infinite ATP concentration gives a value of 1.89. (S.E. 0.13, N = 5) and a Hill coefficient of n = 1.21 (S.E. 0.06, N = 5) implying that the true transport ratio is 2 H+/ATP with positive cooperativity between the protons.