Studies on (K + + H +)-ATPase : II. Role of sulfhydryl groups in its reaction mechanism

Abstract

1. 1. Preincubation of a microsomal membrane preparation from pig gastric mucosa with 5,5′-dithiobis(2-nitrobenzoic acid) inhibits the (K + + H +)-ATPase and K +-stimulated 4-nitrophenylphosphatase activities of the preparation. 2. 2. The activating monovalent cation, K +, in the presence of CDTA, increases the rate of inactivation of (K + + H +)-ATPase by 5,5′-dithiobis(2-nitrobenzoic acid), indicating a conformational change in the presence of K +. 3. 3. In the absence of added Mg 2+ or in the presence of CDTA, the semilogarithmic plot of ATPase activity vs. reaction time is linear. Upon addition of Mg 2+, a fast and a slow phase become discernible. 4. 4. The nucleotides ATP, ADP and dATP in 0.5 mM concentration strongly protect the enzyme against inactivation by the modifying agent in the presence of CDTA. The ATP analogues AMPPNP and AMPPCP exert moderate protection, whilst other nucleotides like GTP, ITP and CTP show only minor protection. 5. 5. Analysis of the protective effect of ATP indicates that in the presence of 2 mM CDTA, 5,5′-dithiobis(2-nitrobenzoic acid) and ATP do not compete for the same site, suggesting that the essential sulfhydryl groups are not located in the ATP binding site. 6. 6. The slow inactivation phase, which occurs in the presence of Mg 2+, is completely blocked by ATP, and its non-phosphorylating analogue AMPPNP now appears to be equally potent.