Membrane enzyme systems responsible for the Ca2+-dependent phosphorylationof Ser(27), the independent phosphorylation of Tyr(10) and Tyr(7), and thedephosphorylation of these phosphorylated residues in the alpha-chain of H/K-ATPase

H/K-ATPase preparations (the G1 membrane) from pig stomach contain both kin ases and phosphatases and show reversible phosphorylation of Tyr(7), Tyr(10 ), anti Ser(27) residues of the alpha-chain of H/K-ATPase. The Tyr-kinase i s sensitive to genistein and quercetin and recognized by anti-c-Src antibod y. The Ser-kinase is dependent on Ca2+ (K-0.5 = 0.9 mu M), sensitive to a P KC inhibitor, and recognized by antibodies against PBC alpha and PKC beta I I. The addition of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfoni c acid (CHAPS) caused a dramatic increase in the phosphorylation of added s ynthetic copolymer substrates and permitted the phosphorylation of maltose- binding proteins fused with the N-terminal domain of alpha-chains. The phos photyrosine phosphatase was inhibited by vanadate. The phosphoserine phosph atase was inhibited by okadaic acid and by inhibitor-2. The presence of pro tein phosphatase-1 was immunologically detected. Column chromatographic sep aration of CHAPS-solubilized G1 membrane and others indicate the apparent m olecular weight of the Src-kinase to be similar to 60 kDa, the PKC alpha an d/or PKC beta II to be similar to 80 kDa, the Tyr-phosphatase to be 200 kDa , and PP-1 to be similar to 35 kDa. These data show that these membrane-bou nd enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr(7), Tyr(10) and Ser(27) of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role o f which is unknown.