Structural interactions between alpha- and beta-subunits of the gastric H,K-ATPase.
Structural and functional interactions between alpha- and beta-subunits of the H,K-ATPase were explored. The sensitivity to trypsinolysis of alpha-subunit was monitored by SDS-PAGE in control H,K-ATPase-enriched microsomes and in microsomes in which disulfide bonds of the beta-subunit were reduced using 2-mercaptoethanol (2-ME). Reduction of beta-subunit disulfide bonds increased the susceptibility of the alpha-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 alpha-H,K,12.18. In control preparations, where beta-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 alpha-subunit was unable to undergo ligand-induced conformational changes. The increased susceptibility of the alpha-subunit to trypsinization, the change in accessibility of tryptic cleavage sites and the inability of the alpha-subunit to undergo ligand-induced conformational changes after reduction of the beta-subunit disulfides suggest that the interactions between alpha- and beta-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.
Tyagarajan
K
K
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Chow
D C
DC
Smolka
A
A
Forte
J G
JG
D000818:Animals; D004220:Disulfides / Q000737:chemistry; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry*; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011817:Rabbits; D013270:Stomach / Q000201:enzymology*; D014357:Trypsin
D004220:Disulfides; D008623:Mercaptoethanol; D014357:Trypsin; D017506:H(+)-K(+)-Exchanging ATPase