Phylogenetic conservation of isoform-specific regions within alpha-subunit of Na(+)-K(+)-ATPase

Site-directed polyclonal antibodies were generated against four oligopeptides derived from the rat alpha-isoforms of Na(+)-K(+)-adenosinetriphosphatase (i.e., Na(+)-K+ pump) and were used to assess the phylogenetic conservation of the target regions. The first antibody was directed against a region near the phosphorylation site that is conserved among alpha-subunits from numerous species. The remaining antibodies were specific for the isoforms of alpha and recognized a region near the fluorescein isothiocyanate-reactive lysine of the adenine binding site. Immunoblots of protein from a number of species probed with these antibodies revealed dramatic differences in the conservation of the target sequences. The region derived from alpha 1 was found in four of the six mammals screened. In contrast, the analogous region derived from alpha 2 was found in all the screened mammals. Most conserved was the region derived from alpha 3, which was found in all the screened mammals as well as in chickens, catfish, dogfish, and sea slugs. Conservation of the region from alpha 3 suggests functional significance and implies that strong selective pressures have discouraged mutational change. Results from the immunoblots further suggest that isoforms are present in all vertebrates.