Cellular Biochemical Studies on Reulatory Mechanism of Gastric Acid Secretion

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[1] We prepared the site-specific polyclonal antibodies recognizing amino-terminal 70 residues of the gastric proton pump alpha subunit. The antibodies for beta subunit was also prepared. The expression plasmids for the alpha and beta subunit cDNAs were constructed and introduced into Cos1 cells. The subnits of proton pump were detected by the anitibodies. It is now possible to study the effects of phosphorylation of amino-terminal Tyr residues of the alpha subunit on the cellular localization of the proton pump.[2] The above expression plasmids were introduced into CHO-K1 cells and the cell lines which permanently express proton pump were isolated. They showed positive growth in the presence of ouabain, suggesting that the structurally related proton pump (H^+/K^+-ATPase) may surrogate Na^+-pump (Na^+/K^+-ATPase). These cell lines are useful to screen proton pump inhibitors.[3] The 5'-upstream regions of rat proton pummp alpha and beta subunit genes were ligated to the reporter gene (luciferase). The resulting plasmids were introduced into HeLa cells. The transcription of the reporter gene was enhanced in the presence of the expression plasmid for gastric GATA proteins. Site-directed mutagenesis demonstrated that the GATA protein binding site proximal to the TATA-box of the upstream region is important for the transcriptional activation of alpha and beta subunit genes.