Effect of aldosterone on abundance and phosphorylation kinetics of Na-K-ATPase of toad urinary bladder

Abstract

The possibility was explored that aldosterone-dependent modulation of either the abundance or the kinetic properties of the Na+ pump (Na-K-ATPase) is involved in the mechanism of natriferic action on toad urinary bladder. Cell membranes from the epithelium of urinary bladders of toads were prepared by a sucrose-Ficoll step gradient method, which yielded a 10-fold increase in specific activity of plasma membrane marker enzymes and only minor contamination with other subcellular fractions. Phosphorylation of Na-K-ATPase by gamma-PO4 of [gamma-32P]ATP in the presence of Mg2+ was Na+ dependent, whereas dephosphorylation was K+ dependent. Km for phosphorylation by ATP was 3 X 10(-8) M and K1/2 (half-maximal stimulation) for Na+ was 13 +/- 2 mM. Pretreatment of cell membranes with sodium deoxycholate (DOC) increased the maximal extent of phosphorylation (Nmax) about threefold without changing the Km for ATP. Aldosterone (5 X 10(-8) M) stimulated transepithelial Na+ transport two- to threefold in 5 h but had no significant effect on Km for ATP in the phosphorylation reaction (with or without activation by DOC) or on the abundance of the enzyme inferred from Nmax of phosphorylation in the absence of activation by DOC. After pretreatment with DOC, average Nmax was 13% greater in the aldosterone-treated population. Regression analysis of these responses revealed no significant correlation between increments in short-circuit currents and Nmax values of phosphorylation with or without pretreatment with DOC.(ABSTRACT TRUNCATED AT 250 WORDS)