REVERSIBLE PHOSPHORYLATION OF BOTH TYR(7) AND TYR(10) IN THE ALPHA-CHAIN OF PIG STOMACH H-ATPASE BY A MEMBRANE-BOUND KINASE AND A PHOSPHATASE(,K+)

When pig stomach membrane H+,K+-ATPase preparations were incubated wit h [gamma-P-32]ATP and Mg2+ with vanadate, P-32 was incorporated into t he oc chain of H+,K+-ATPase to a steady-state level of approximately 0 .7 mol of phosphotyrosine (Tyr(P))/mol of phosphoenzyme intermediates. The addition of a membrane H+,K+ ATPase preparation with Mg2+ acceler ated the liberation of P-32 from Tyr(P) residues in the alpha-chain, M ild tosylphenylalanyl chloromethyl ketone-trypsin treatment solubilize d P-32-containing peptides from the ct-chain almost completely. A reve rse-phase column chromatography of the supernatant gave two peaks of P -32-peptide with similar total radioactivities. The amino acid sequenc e of both peaks was shown to be Gly Lys Ala-Glu-Asn-Tyr-Glu-Leu-Tyr-Gl n-, which is consistent with the amino-terminal sequence of the alpha- chain of H+,K+-ATPase deduced from cDNA from pig stomach except that t he initial Met was absent. The comparison of the recovery of amino aci d from each Edman cycle showed that the phosphorylation of Tyr(10) occ urred preceding the phosphorylation of Tyr(7). These data and others s uggested the presence of a novel membrane-bound enzyme system to parti cipate in reversible phosphorylation of both Tyr residues in the alpha -chain of H+,K+-ATPase.