Ouabain binding during plasma membrane biogenesis in duck salt gland

Abstract

The conditions necessary for optimal ouabain binding in the avian salt gland were examined. Binding was enhanced by ATP and Mg²⁺ and was decreased by K⁺, but was unaffected by added Na⁺. Both maximal binding and complete inhibition of Na,K-ATPase activity were obtained at 1 × 10⁻⁶ M ouabain. Half maximal binding and half maximal inhibition of Na,K-ATPase activity were obtained at 1·7×10⁻⁷ M ouabain. Ouabain binding increased in parallel with increasing specific activity of the Na,K-ATPase during salt-induced salt gland specialization. The ratio of Na,K-ATPase activity to ouabain-binding sites remained constant during the salt stress as well as after removal of the salt diet. Autoradiography indicated binding to partially and fully differentiated secretory cells of the salt gland. The ouabain binding assay appeared to be a more useful indicator of membrane amplification than Na,K-ATPase activity since it is rapid, essentially irreversible, less sensitive to tissue fixatives, and quantitatively measured the number of enzyme molecules.