Heterogeneity of Na+/K+-ATPase from rectal gland of Squalus acanthias is not due to alpha 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 alpha peptide. To avoid enzyme inactivation [3H]ouabain equilibrium binding was carried out at 20 degrees C. The heterogeneity of Na+/K+-ATPase isolated from shark rectal gland was similar in [3H]ouabain binding as previously seen in hydrolytic studies. The binding isotherms were compatible with the existence of a high-affinity (Kdis 0.69 nM) and a low-affinity (Kdis 42 nM) component of 1.46 and 0.79 nmol.(mg protein)-1, respectively. In Western blots the alpha peptide of the enzyme hybridized with an isoform-specific polyclonal antibody raised to an alpha3-specific region of the large intracellular domain of rat Na+/K+-ATPase, but not with the supposed alpha3-specific monoclonal antibody MA3-915 with its epitope near the N-terminus. Semi-quantitative analysis of the reaction of the alpha3-specific polyclonal antibody with the alpha peptide from the shark enzyme compared to the reaction with alpha 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 alpha1- or alpha2-specific polyclonal antibodies, or by the alpha1-specific monoclonal antibodies 3B and F6. The large intracellular domain of Na+/K+-ATPase from shark rectal gland thus seems to be alpha3-like and no alpha isoform heterogeneity seems able to account for the heterogeneity seen in ouabain binding.