Dimerization of the gastric H+,K+-ATPase

When the gastric H+,K+-ATPase was solubilized by n-dodecyl beta-D-maltoside and electrophoresed in blue native-polyacrylamide gels (BN-PAGE), one major band at about 360 kDa was observed. Since this band was recognized by both monoclonal antibodies 1218 (anti-alpha) and wheat germ agglutinin (anti-beta), the H+,K+-ATPase in its native state exists in a dimeric (alpha beta)(2) form. The site of interaction between the heterodimers was determined using Cu2+-phenanthroline cross-linking. The Cu2+-phenanthroline reagent reacted with the H+,K+-ATPase to produce an alpha,alpha-dimer and inhibited H+,K+-ATPase activity. This cross-linking and enzyme inhibition were prevented by ATP. Cross-linking followed by N-ethylmaleimide blockade of maleimide-reactive SH groups, then reduction and fluorescein 5-maleimide labeling, defined a single fluorescent tryptic peptide of about 6.5 kDa that had been cross-linked. Since its N-terminal amino acid is Val(561), the peptide probably ends at Arg(616) or Arg(621) and Cys(565) and/or Cys(615) are probably within the region of closest contact between the two alpha-subunits.