Characterization of the fourth alpha isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, alpha and beta, and tissue- specific isoforms exist for each of these, alpha 1, alpha 2 and alpha 3 and beta 1, beta 2 and beta 3. We have proposed that an additional alpha isofo rm, alpha 4, exists based on genomic and cDNA cloning. The mRNA for this ge ne is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the cur rent study, the putative alpha 4 isoform has been functionally characterize d as a novel isoform of the Na,K-ATPase in both rat testis and in alpha 4 i soform cDNA transfected 3T3 cells. Using an alpha 4 isoform-specific polycl onal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding com petition assays reveal the presence of high affinity ouabain receptors in b oth rat testis and in transfected cell lines that have identical K-D values . Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPas e in testis.