Heterogeneity of Na+/K+-ATPase from rectal gland of Squalus acanthias is not due to α isoform diversity

Purified Na+/K+-ATPase (EC 3.6.1.37) isolated from the rectal gland of Squalus acanthias was characterized in ouabain-binding studies and with respect to isoform(s) of the α peptide. To avoid enzyme inactivation [3H]ouabain equilibrium binding was carried out at 20°C. The heterogeneity of Na+/K+-ATPase isolated from shark rectal gland was similar in [3H]ouabain binding as previously seen in hydrolyric studies. The binding isotherms were compatible with the existence of a high-affinity (K(dis) 0.69 nM) and a low-affinity (K(dis) 42 nM) component of 1.46 and 0.79 nmol·(mg protein)-1, respectively. In Western blots the α peptide of the enzyme hybridized with an isoform-specific polyclonal antibody raised to an α3- specific region of the large intracellular domain of rat Na+/K+-ATPase, but not with the supposed α3-specific monoclonal antibody MA3-915 with its epitope near the N-terminus. Semi-quantitative analysis of the reaction of the α3-specific polyclonal antibody with the α peptide from the shark enzyme compared to the reaction with α peptide from rat brain enzyme indicated that this region is not exactly the same in the two species. The α peptide of shark enzyme was not recognized by α1- or α2-specific polyclonal antibodies, or by the α1-specific monoclonal antibodies 3B and F6. The large intracellular domain of Na+/K+-ATPase from shark rectal gland thus seems to be α3-like and no α isoform heterogeneity seems able to account for the heterogeneity seen in ouabain binding.