QUANTITATIVE-ANALYSIS OF THE HGH-AFFINITY BINDING-SITES FOR [H-3]OUABAIN IN THE RAT VAS-DEFERENS AND THEIR IMMUNOLOGICAL IDENTIFICATION AS THE ALPHA(2) ISOFORM OF NA+ K+-ATPASE/

Binding assays were performed with [H-3]ouabain to investigate the pre sence of, and to characterize, a Na+/K+-ATPase isoform with high affin ity for cardiac glycosides in the rat vas deferens. Nonlinear regressi on analysis of equilibrium experiments carried out with crude preparat ions in a Mg-P-i medium indicated the presence of high-affinity sites characterized with good precision (individual coefficients of variatio n = 11-35%) by their density (B-max = 0.42 to 0.72 pmol/mg protein) an d 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 co nstant (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 si tes (K-d around 15 mu M), supposed to correspond to the alpha(1) isofo rm, was also identified, but their K-d and B-max values were not quant ified precisely in this crude preparation. Western blot assays indicat ed hybridization with specific anti-alpha(1) and anti-alpha(2) isoform antibodies but not with anti-alpha(3) isoform antibody. Taken togethe r, the present results indicate the existence of a low proportion of t he 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 membra ne preparation that is suitable for studies under conditions of plasti city. (C) 1998 Elsevier Science Inc.