The long term goal of this poject is to determine the mechanisms responsible for H+ and Cl- secretion in the stomach. It is known that H+ secretion is dependent upon H+/K+ exchange activity of the (H+,K+) ATPase and that in the non-secreting state this activity is limited by K+ accessibility at a K+ dependent catalytic site located within the tubulovesicles of the parietal cell. Following the morophological changes accompanying the transition from resting to secreting state this ATPase is localized within the intracellular canaliculus. We will investigate the pump complex isolated from either resting or stimulated tissue to determine what changes this morphological transformation exerts on the basic mode of pump operation and ancillary routes of K+ and Cl- permeability. In the short term, this proposal addresses the solubilization, purification and reconstitution of the gastric proton transport system. The detergent, n-octylglucoside, will be utilized to extract the active solubilized (H+,K+) ATPase from both resting and stimulated mucosa. Immunoaffinity chromatography using the monoclonal antibody H,K III will then be used to purify the solubilized, active enzyme. This purified protein will then be inserted into artificial phospholipid vesicles and studied by optical probes of pH and membrane potential. A possible significance of this work is in the treatment of ulcer disease. Inhibition of this enzyme by the substituted benzimidazoles has been correlated with inhibition of acid secretion in humans. A detailed knowledge of this ATPase is necessary to design inhibitors of acid secretion which are selective for this enzyme.
| Rabon, E C; Smillie, K; Seru, V et al. (1993) Rubidium occlusion within tryptic peptides of the H,K-ATPase. J Biol Chem 268:8012-8 |
| Rabon, E; Sachs, G; Bassilian, S et al. (1991) A K(+)-competitive fluorescent inhibitor of the H,K-ATPase. J Biol Chem 266:12395-401 |
| Hall, K; Perez, G; Sachs, G et al. (1991) Identification of H+/K(+)-ATPase alpha,beta-heterodimers. Biochim Biophys Acta 1077:173-9 |
| Rabon, E C; Bassilian, S; Sachs, G et al. (1990) Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons. J Biol Chem 265:19594-9 |
| Rabon, E C; Bassilian, S; Jakobsen, L J (1990) Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase. Biochim Biophys Acta 1039:277-89 |
| Rabon, E C; Reuben, M A (1990) The mechanism and structure of the gastric H,K-ATPase. Annu Rev Physiol 52:321-44 |
| Rabon, E C; Gunther, R D; Bassilian, S et al. (1988) Radiation inactivation analysis of oligomeric structure of the H,K-ATPase. J Biol Chem 263:16189-94 |
| Gunther, R D; Bassilian, S; Rabon, E C (1987) Cation transport in vesicles from secreting rabbit stomach. J Biol Chem 262:13966-72 |
| Rabon, E; Wilke, M; Sachs, G et al. (1986) Crystallization of the gastric H,K-ATPase. J Biol Chem 261:1434-9 |
