Antigenic determinant of a monoclonal antibody: extracellular domain at the M3–M4 junction of the α-subunit of Na,K-ATPase

The binding site of a monoclonal antibody, M45-80, against the α-subunit of horse Na,K-ATPase was determined. Various sizes of DNA fragments derived from rat Na,K-ATPase α1-subunit cDNA were cloned into pUC19 expression vector and some fragments of horse genomic DNA were cloned into pUC18. Escherichia coli JM83 cells harboring the plasmids were grown and the cell lysates were used as antigens. An enzyme-linked immunosorbent assay revealed that M45-80 recognizes the hexapeptide Glu-Tyr-Thr-Trp-Leu-Glu (which is identical to the rat and horse α1-subunits) at the M3–M4 junction located on the extracellular side. The ouabain-binding site is discussed in relation to the recognition site of M45-80.Key words: Na,K-ATPase α-subunit, monoclonal antibody, pUC19 expression vector, antibody-binding site, ouabain-binding site.