Understanding the mechanism of acid secretion by the parietal cell depends on the convergence of knowledge about the structure of the H+ transporting, K+ - activated ATPase with the detailed characterization of its function at the molecular level. The proposed investigation is planned to obtain new information relative to four major molecular aspects of the Gastric ATPase: Active - site structure; Molecular weight and subunit structure; Principal conformations and their relation to ATP hydrolysis and ion (H+, K+) translocation; Organization in the membrane. 1. The nature of substrate binding and phosphorylation sites and their structure will be characterized by using fluorescence and affinity labelling techniques. 2. Studies on the subunit structure will center on the determination of the molecular dimension of the native and crystal lattice - induced enzyme preparation by electron microscopy and computer - assisted image analysis. 3. The conformational states of the enzyme induced by specific ligands and the various rate constants associated with these transitions, will be investigated by using fluorescence and radioisotopic techniques. 4. We will determine the hydrophylic and hydrophobic peptide topology of the enzyme in the membrane by proteolytic digestion techniques coupled with site - specific reagents and lipophilic photo affinity labels. The topics selected for investigation are central issues in the areas of membrane transport process. Health - related significance of these studies will derive from the expansion of our information about basic molecular mechanisms for HCI secretion.
