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

Abstract

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°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 K 0.5 value for proton transport is 154 μM and the K m value for ATP hydrolysis is 61 μM. The Lineweaver-Burk plot for ATP hydrolysis vs. ATP concentration is linear with a V max of 5.5 nmol/mg protein per s, but that for H + uptake is not. Thus with increasing ATP concentration (6.7 to 1670 μM) 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.