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 Cu(2+)-phenanthroline cross-linking. The Cu(2+)-phenanthroline reagent reacted with the H+, K(2+)-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, then reduction and fluorescent tryptic peptide of about 6.5 kDa that had been cross-linked. Since its N-terminal amino acid is Val561, the peptide probably ends at Arg616 or Arg621 and Cys565 and/or Cys615 are probably within the region of closest contact between the two alpha-subunits.