Aldosterone receptor assay in rat kidney cytosol

Abstract

A method has been developed to measure aldosterone receptors using rat kidney cytosol preparations. Addition of 0.1 M Na molybdate to homogenization buffer markedly diminished receptor inactivation, allowing for more accurate assessment of affinity and total number of receptors. Statistical analysis of Scatchard plots was used to resolve curvilinear plots into high affinity (type I) and a low affinity (type II) components. Aldosterone and deoxycorticosterone compete most effectively for binding to type I sites, whereas dexamethasone competes most effectively for binding to type II sites. Molybdate does not alter the location of the 8.0S peak on density gradient analysis. Assay of type I sites revealed 47.7 +/- 1.7fmol/ml cytosol protein in male adrenalectomized rats. Constant of dissociation (KD) was measured at 5.08 x 10-10 M. Non-adrenalectomized male rats had 24.4 +/- 3.0 fmol/mg protein type I sites. Addition of molybdate to homogenization buffer combined with statistical analysis of curvilinear Scatchard plots allows for accurate and reproducible measurement of high affinity aldosterone receptors in rat kidney cytosol.