STRUCTURAL INTERACTIONS BETWEEN ALPHA-SUBUNITS AND BETA-SUBUNITS OF THE GASTRIC H,K-ATPASE

Structural and functional interactions between alpha- and beta-subunit s of the H,K-ATPase were explored. The sensitivity to trypsinolysis of alpha-subunit was monitored by SDS-PAGE in control H,K-ATPase-enriche d microsomes and in microsomes in which disulfide bonds of the beta-su bunit were reduced using 2-mercaptoethanol (2-ME). Reduction of beta-s ubunit disulfide bonds increased the susceptibility of the alpha-subun it to tryptic digestion. Kinetics of trypsinolysis were also carried o ut in the presence of ligands known to bind with H,K-ATPase and favor a particular conformer state in the native enzyme. The time-course for release of tryptic peptides was monitored in protein stained gels and Western blots probed with monoclonal antibody alpha-H,K,12.18. In con trol preparations, where beta-subunit disulfides remained intact, tryp sinolysis in the presence of ATP or K+ produced distinctive patterns o f tryptic fragments, each characteristic of the conformational states induced by the respective ligand. For 2-ME-treated microsomes the alte red alpha-subunit was unable to undergo ligand-induced conformational changes. The increased susceptibility of the alpha-subunit to trypsini zation, the change in accessibility of tryptic cleavage sites and the inability of the alpha-subunit to undergo ligand-induced conformationa l changes after reduction of the beta-subunit disulfides suggest that the interactions between alpha- and beta-subunits are important for th e conformational stability of the functional holoenzyme. A model local izing the most susceptible tryptic cleavage sites in control and 2-ME- reduced states is presented.