Isoform-specific monoclonal antibodies to Na,K-ATPase alpha subunits. Evidence for a tissue-specific post-translational modification of the alpha subunit

Abstract

Monoclonal antibodies to isoforms of the Na,K-ATPase have become important tools in the study of the enzyme's distribution, physiological roles, and gene regulation, and when their epitopes are defined, they are useful in the study of enzyme structure as well. Evidence is presented that the alpha3-specific antibody McBX3 recognizes an unusual epitope that is not present on alpha3 in the heart. The epitope, which is also found in kidney alpha1 from some species, was mapped to a site on the large intracellular loop near the ATP binding site. DNA sequencing of reverse transcribed-PCR products encompassing the corresponding regions from alpha3 from brain (where McBX3 recognizes alpha3) and heart demonstrated that the tissue difference in epitope is not due to alternative splicing of the mRNA. Instead, hydroxylamine sensitivity indicated that the antibody recognizes a post-translational modification. The epitope for a new antibody for alpha3, XVIF9-G10, was mapped to a site near the N terminus, a location analogous to the sites for the well-characterized antibodies McK1 (alpha1) and McB2 (alpha2). The antibody XVIF9-G10 reacted with the alpha3 of the heart as well as that of the brain; however, McBX3 and XVIF9-G10 both stained the same cellular structures in sections of the rat retina. A new alpha1-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 alpha1 antibody, McK1.