Quantitative Analysis of the High-Affinity Binding Sites for [3H]Ouabain in the Rat Vas Deferens and Their Immunological Identification as the α2 Isoform of Na+/K+-ATPase

Binding assays were performed with [H-3]ouabain to investigate the presence of, and to characterize, a Na+/K+-ATPase isoform with high affinity for cardiac glycosides in the rat vas deferens. Nonlinear regression analysis of equilibrium experiments carried out with crude preparations in a Mg-P-i medium indicated the presence of high-affinity sites characterized with good precision (individual coefficients of variation = 11-35%) by their density (B-max = 0.42 to 0.72 pmol/mg protein) and dissociation constant (K-d = 0.069 to 0.136 mu M) values. The values of the dissociation rate constant (k(-1)) and the association rate constant (k(+1)) for these sites were 0.151 to 0.267 min(-1) and 2.87 to 3.60 mu M-1.min(-1), respectively. A higher number of low-affinity sites (K-d around 15 mu M), supposed to correspond to the alpha(1) isoform, was also identified, but their K-d and B-max values were not quantified precisely in this crude preparation. Western blot assays indicated hybridization with specific anti-alpha(1) and anti-alpha(2) isoform antibodies but not with anti-alpha(3) isoform antibody. Taken together, the present results indicate the existence of a low proportion of the alpha(2) isoform of Na+/K+-ATPase in the rat vas deferens that can be quantified precisely by [H-3]ouabain binding even in a crude membrane preparation that is suitable for studies under conditions of plasticity. (C) 1998 Elsevier Science Inc.