Structural interactions between a- and β-subunits of the gastric H,K-ATPase

AbstractStructural and functional interactions between a- and β-subunits of the H,K-ATPase were explored. The sensitivity to trypsinolysis of α-subunit was monitored by SDS-PAGE in control H,K-ATPase-enriched microsomes and in microsomes in which disulfide bonds of the β-subunit were reduced using 2-mercaptoethanol (2-ME). Reduction of /3-subunit disulfide bonds increased the susceptibility of the α-subunit to tryptic digestion. Kinetics of trypsinolysis were also carried out 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 α-H,K,12.18 In control preparations, where β-subunit disulfides remained intact, trypsinolysis in the presence of ATP or K+ produced distinctive patterns of tryptic fragments, each characteristic of the conformational states induced by the respective ligand. For 2-ME-treated microsomes the altered a-subunit was unable to undergo ligand-induced conformational changes. The increased susceptibility of the a-subunit to trypsinization, the change in accessibility of tryptic cleavage sites and the inability of the a-subunit to undergo ligand-induced conformational changes after reduction of the β-subunit disulfides suggest that the interactions between a- and β-subunits are important for the conformational stability of the functional holoenzyme. A model localizing the most susceptible tryptic cleavage sites in control and 2-ME-reduced states is presented