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  • Title: The effect of calcium on the hepatic glucocorticoid receptor.
    Author: Kalimi M, Hubbard J, Ray A.
    Journal: J Steroid Biochem; 1983 Jun; 18(6):665-71. PubMed ID: 6865410.
    Abstract:
    The unbound glucocorticoid receptor of rat hepatic cytosol was very unstable at 25 degrees C. This receptor instability was increased by the addition of 1-10 mM Ca2+ such that the unbound glucocorticoid receptor was completely inactivated within 30 min at 25 degrees C in the presence of 5 mM Ca2+. Interestingly, Ca2+ inactivation was blocked approximately 80% by simultaneous addition of either 10 mM molybdate or 10 mM molybdate plus 5 mM dithiothreitol. Ten millimolar leupeptin or 10 mM alpha 2-macroglobulin did not inhibit Ca2+ inactivation of the receptor. In fact, leupeptin alone slightly inactivated the receptor. Prior treatment of cytosol with 10 mM molybdate plus 5 mM DTT at 25 degrees C for 30 min offered a complete protection against the subsequent addition of 5 mM Ca2+. The effects of Ca2+ were found to be irreversible since addition of molybdate or molybdate plus dithiothreitol after 5 mM Ca2+ treatment (20 min for 25 degrees C) did not reactivate partially inactivated receptors but did stabilize the remaining receptor binding at the time of molybdate or molybdate plus dithiothreitol addition. Addition of 1-5 mM Ca2+ to preformed [3H]-dexamethasone--receptor complexes slightly inhibited steroid--receptor complex transformation into a nuclear binding form at 25 degrees C. In addition, 10 mM Ca2+ altered the agarose gel filtration profile of the complex. Control cytosol or cytosol treated with 10 mM EDTA or 10 mM leupeptin showed three distinct macromolecular bound radioactivity peaks having Stokes radii of 65, 40 and 20 A. On the other hand, Ca2+-treated cytosol showed a single large aggregated component which was excluded in the void volume of the column. Trypsin (0.5 microgram/ml) generated a small steroid--receptor fragment of 20 A when added alone, but interestingly when added with 10 mM Ca2+ the [3H]-dexamethasone--receptor complex eluted in the agarose gel exclusion volume. These results show that Ca2+ has a profound effect on rat hepatic glucocorticoid receptors and that molybdate can block Ca2+-dependent receptor inactivation.
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