Heterogeneity of Na+/K+-ATPase from rectal gland of Squalus acanthias is not due to αisoform 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 alpha peptide. To avoid enzyme inactivation [H-3]ouabain equilibrium binding was carried out at 20 degrees C. The heterogeneity of Na+/K+-ATPase isolated from shark rectal gland was similar in [H-3]ouabain binding as previously seen in hydrolytic 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 a peptide of the enzyme hybridized with an isoform-specific polyclonal antibody raised to an alpha(3)-specific region of the large intracellular domain of rat Na+/K+-ATPase, but not with the supposed alpha(3)-specific monoclonal antibody MA3-915 with its epitope near the N-terminus. Semi-quantitative analysis of the reaction of the alpha(3)-specific polyclonal antibody with the a peptide from the shark enzyme compared to the reaction with a peptide from rat brain enzyme indicated that this region is not exactly the same in the two species. The alpha peptide of shark enzyme was not recognized by alpha(1)- or alpha(2)-specific polyclonal antibodies, or by the alpha(1)-specific monoclonal antibodies 3B and F6. The large intracellular domain of Na+/K+-ATPase from shark rectal gland thus seems to be alpha(3)-like and no alpha isoform heterogeneity seems able to account for the heterogeneity seen in ouabain binding.