The gastric H/K ATPase (E.C. 1.2.1.36) is a Mg2+-dependent, K+- dependent, and H+ transporting enzyme which is responsible for HCI secretion by the parietal cell. A class of natural and synthetic polypeptides which are novel, specific, and potent Inhibitors of the H/K ATPase have been Identified. These are amphipathic helical polypeptides, and include the bee venom toxin, melittin and synthetic polypeptides of similar 3-dimensional structure and physiochemical properties. Significantly, physiologically relevant cations, including K+, protect against inhibition. We have also Identified an endogenous parietal cell protein which exhibits determinants In common with melittin, and this protein appears to exhibit stimulus-dependent association with the H/K ATPase. The broad, long term objectives are to define the mechanisms which underlie regulated gastric HCI secretion. Therapeutic control of gastric ulcer disease relies upon interventions which control HCI secretion.
The specific aims of this proposal are directed toward understanding the structure of this polypeptide binding site on the H/K ATPase; the mechanism whereby occupation of this site causes inhibition; how protection by cations is achieved; and the physiological significance of these findings.
The specific aims are: 1) To compare and contrast the suitability of using p-benzoylphenylalanine-containing synthetic polypeptide photoaffinity probes with [125-I]azidosalicylyl melittin for the purposes of sequence determination of the polypeptide receptor on the H/K ATPase. Purified fragments will be used for 2) identification of the residues of the H/K ATPase which interact with polypeptides by NH2 terminal analysis and determination of amino acid composition. These sequences will be mapped ana the photolabeled amino acids will be identified on the known primary structure of the H/K ATPase. 3) We will examine the functional consequences of occupation of the amphipathic polypeptide binding site by melittin, and define the mechanisms whereby physiologically relevant cations protect the enzyme from inhibition. The results of transport and ATP hydrolysis studies will be correlated with fluorescent studies of conformation of the H/K ATPase. Structural and kinetic models will be developed and tested. 4) The endogenous polypeptide with melittin-like determinants will be purified by immunoaffinity purification by HPLC. Binding studies, photoaffinity labeling, conformational studies, and the functional consequences of interaction of this protein with purified H/K ATPase will be investigated. It is our hypothesis that the alpha subunit of the H/K ATPase contains a polypeptide binding site to which melittin and this endogenous protein bind. The melittin-like protein may play a physiological role in regulation of HCI secretion. These studies represent an important new direction for the study of structure/function relationships which underlie regulated function of the gastric H/K ATPase.
