Purification, characterization, and activation of the glucocorticoid-receptor complex from rat kidney cortex

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Abstract
The unactivated molybdate-stabilized glucocorticoid receptor (GcR) was purified from rat kidney cortex cytosol (RKcC) by using a modification of the procedure for rat hepatic receptor. The purification includes affinity chromatography, gel filtration and ion-exchange chromatography. The final preparation (.apprx. 1000-fold pure as determined from specific radioactivity) was used in subsequent physicochemical and functional analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single heavily Coomassie-stained band at 90 kilodaltons. Density gradient ultracentrifugation indicated a sedimentation coefficient of 10.5 .+-. 0.05 S (n = 2). Chromatography on an analytical gel filtration column produced a Stokes radius (Rs) of 6.4 .+-. 0.07 nm (n = 5). The Rs was unchanged when the molybdate-stabilized GcR was analyzed in the presence of 400 mM KCl or when analyzed in the unpurified (cytosolic) state. The hepatic GcR existed as a larger form in cytosol (7.7 .+-. 0.2 nm). Following purification, or upon gel filtration analysis under hypertonic conditions, the Rs was similar to that of the unpurified RKcC GcR. Following removal of molybdate from RKcC GcR and thermal activation (25.degree. C/30 min), DNA-cellulose binding increased 1.5- to 2-fold over the unheated control. Addition of RKcC or hepatic cytosol (endogenous receptors thermally denatured at 90.degree. C/30 min or presaturated with 10-7 M radioinert ligand) during thermal activation increased DNA-cellulose binding an additional 2- to 6-fold beyond the heated control. Although the purified unactivated kidney cortex and hepatic forms of the GcR are similar, the presumably more native cytosolic, unactivated forms may differ in Rs which may relate this difference to the concept of receptor polymorphism.