Identification of antigenic sites on the Na+/K(+)-ATPase beta-subunit: their sequences and the effects of thiol reduction upon their structure.

In contrast to the catalytic (alpha) subunit of the Na+/K(+)-ATPase holoenzyme, the glycoprotein (beta) subunit has proven to be a poor antigen for monoclonal antibody (Mab) production. However, in this work six Mabs directed against the beta-subunit of the lamb kidney holoenzyme have been isolated. These Mabs all recognize the holoenzyme, but their 'in solution' binding affinities for deglycosylated enzyme or isolated beta are generally at least 10-fold higher. Species specificity mapping, antibody patterns of binding to beta-fragments and competition binding studies indicated that there were only three distinct epitopes, with two antibodies binding in the NH2-terminal half (epitopes I and II) and 4 Mabs binding at the same or overlapping site (III) in the -COOH terminal half of beta. DNA sequence analysis of isolated collections of bacteriophage M13 that contain a 15 amino-acid 'epitope library' insert in the pIII protein, which enables them to bind to the antibodies, revealed the residues KYRDS (amino acids 111-115) and LETYP (amino acids 197-201) to be the deduced sequences for the epitopes of Mabs M19-P7-E5 (II) and M17-P5-F11 (III), respectively. The epitope I site was not, however, identified. Further studies showed that antibody binding to these three determinant sites had no affect on the Na+/K(+)-ATPase and K(+)-stimulated p-nitrophenylphosphatase (pNPPase) activities of either holoenzyme or deglycosylated enzyme, nor any affect on the cation- (Na+, K+ or Mg2+) and ouabain-induced conformational changes monitored with FITC-labeled deglycosylated enzyme. Interestingly, anti-beta Mab access to the three epitopes was increased following beta-mercaptoethanol inactivation of the holoenzyme, but this thiol reduction abolished the binding of two conformation-sensitive anti-alpha Mabs to the enzyme. These results are consistent with the previous suggestion of Kirley ((1990) J. Biol. Chem. 265, 4227-4232) that the beta-disulfide linkages not only maintain beta-structure but they are critical for maintaining alpha-conformation and holoenzyme activity.