GASTRIC H+-K+-ATPASE ACTIVITY IS INHIBITED BY REDUCTION OF DISULFIDE BONDS IN BETA-SUBUNIT

H+-K+-ATPase activity of rabbit isolated gastric microsomes was irreversibly inactivated by reducing agents, such as 2-mercaptoethanol and dithiothreitol. Similar to what has been observed for Na+-K+-ATPase, high concentrations of reagents, at moderately elevated temperatures, were required to inactivate H+-K+-ATPase, suggesting relative inaccessibility of the responsible disulfide bonds. Resistance against inactivation was conferred by monovalent cation activators of K+-stimulated ATPase and p-nitro-phenylphosphatase. The effectiveness of K+ congeners in protecting the enzyme was similar in sequence (Tl+ > K+ > Rb+) and concentration to their respective affinities for stimulating enzymatic activity, suggesting that the K+-bound form of the enzyme is more resistant to reduction than the free enzyme. Furthermore, Na+ antagonized the protective effect of K+. Labeling studies using fluorescein-maleimide indicated that 60-70% of the cysteine residues in the beta-subunit are in the oxidized form. Coupled with primary sequence data, this suggests that three disulfide bonds are present in the native beta-subunit. In contrast, <10% of the cysteine residues in the alpha-subunit are in the oxidized form. Kinetic studies showed that the 2-mercaptoethanol-induced loss of H+-K+-ATPase activity was correlated with a reduction of disulfide groups in the beta-subunit, while there was no significant change in the alpha-subunit. We conclude that reduction of disulfide bonds irreversibly inhibits H+-K+-ATPase activity, binding of K+ to the enzyme confers a resistance to disulfide bond reduction, and the responsible disulfide bonds are present in the beta-subunit. It thus appears that structural integrity of the beta-subunit is essential for holoenzyme function and that cooperative interactions between the alpha- and beta-subunits of the H+-K+-ATPase are important for stabilizing the enzyme.