ISOFORM-SPECIFIC MONOCLONAL-ANTIBODIES TO NA,K-ATPASE ALPHA-SUBUNITS - EVIDENCE FOR A TISSUE-SPECIFIC POSTTRANSLATIONAL MODIFICATION OF THEALPHA-SUBUNIT

Monoclonal antibodies to isoforms of the Na,K-ATPase have become impor tant tools in the study of the enzyme's distribution, physiological ro les, and gene regulation, and when their epitopes are defined, they ar e useful in the study of enzyme structure as well. Evidence is present ed that the alpha 3-specific antibody McBX3 recognizes an unusual epit ope that is not present on alpha 3 in the heart. The epitope, which is also found in kidney oil from some species, was mapped to a site on t he large intracellular loop near the ATP binding site. DNA sequencing of reverse transcribed-PCR products encompassing the corresponding reg ions from alpha 3 from brain (where McBX3 recognizes alpha 3) and hear t demonstrated that the tissue difference in epitope is not due to alt ernative splicing of the mRNA. Instead, hydroxylamine sensitivity indi cated that the antibody recognizes a post-translational modification. The epitope for a new antibody for alpha 3, XVIF9-G10, was mapped to a site near the N terminus, a location analogous to the sites for the w ell-characterized antibodies McK1 (alpha 1) and McB2 (alpha 2). The an tibody XVIF9-G10 reacted with the alpha 3 of the heart as well as that of the brain; however, McBX3 and XVIF9-G10 both stained the same cell ular structures in sections of the rat retina. A new alpha 1-specific antibody, 6F, was characterized and mapped to another site near the N terminus; this antibody has broader species specificity than the other well-characterized alpha 1 antibody, McK1.