The overall goal of this research is to understand the cellular and molecular mechanisms of HCl secretion by the stomach. The particular research focus will define structure, function and synthetic pathway of the enzyme responsible for HCl secretion, the H, K-ATPase. These data are of immediate interest to the normal operation of the human stomach, and are directly applicable to disorders ranging from peptic ulcer to autoimmune disease. The proposal has four major projects. The first project will establish the pathway for synthesis and turnover of gastric H, K-ATPase. The enzyme 15 composed of (x- and beta-subunits whose synthesis will be followed from radiolabeled amino acid precursors to the final assembly of the mature functional enzyme in the parietal cell of intact animals. This particular cell model is an excellent one in which to compare synthetic activities of H, K-ATPase with the very closely related Na, K-ATPase a critical enzyme for all cells in the human body. The role of widely used pharmacological agents on the synthesis and turnover of H, K-ATPase will be tested. The beta-subunit of the H, K-ATPase is a glycoprotein, and the second project will study several unusual features of glycoconjugates within the beta-subunit. Initial questions wig establish structural features of beta- subunit glycoconjugates for human and several animals, asking about location, composition and functional activity. The anomeric alpha- galactosyl linkage has been identified as a 'capping' sugar in animal beta- subunit glycoconjugates, but the enzyme responsible for this linkage is normally suppressed in human. Possible deregulation of the gene for expression in conditions such as gastritis and autoimmune disease will be investigated. The third project will define the sites of interaction between the alpha- and beta- subunits of H, K-ATPase. Specific peptide regions that determine alpha/beta interaction and possible functional sites on the holoenzyme will be identified. The final project will develop an in vitro cell system for evaluating synthesis and trafficking of H, K-ATPase. Data will of course be compared with those obtained in vivo, but the cell culture model presents several experimental advantages, including genetic manipulation and identification of co-processing proteins.
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