Stabilization of glucocorticoid receptors in vitro using divalent cations plus cation chelators

Abstract

The specific glucocorticoid receptor binding of rat liver cytosol was very unstable in vitro at 25 and 4°C. However, 5 mM CaCl 2 added with 5 mM EDTA to cytosol prior to incubation markedly stabilized unbound glucocorticoid receptors at both temperatures. Optimal effectiveness was achieved using equimolar (5 mM) amounts of CaCl 2 and EDTA. On the other hand, 5 mM CaCl 2 (added alone) further destabilized the unbound glucocorticoid receptor, while 5 mM EDTA (added alone) had no effect at 25°C. EGTA (in lieu of EDTA) added with CaCl 2 stabilized hepatic receptor binding at 25°C. On the other hand, citrate added with calcium was ineffective in stabilizing the hepatic glucocorticoid receptor. MgCl 2 effectively replaced CaCl 2 as a stabilizing agent at 25°C if added with 5 mM EDTA. When added alone, MgCl 2 slightly destabilized the unbound receptor. Sucrose density gradient analysis (in low salt) revealed that CaCl 2 plus EDTA enhanced the steroid-receptor complex sedimentation coefficient from 2 S to about 10 S. Unlike molybdate, CaCl 2 plus EDTA had no apparent effect on steroid-receptor complex thermal transformation into a nuclear binding form, while MgCl 2 plus EDTA partially reduced transformation. These results suggest a novel means to chemically stabilize unbound hepatic glucocorticoid receptors in vitro which may be of particular importance for receptor purification studies.