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Early Events in Glucocorticoid Action

ACTIVATION OF THE STEROID RECEPTOR AND ITS SUBSEQUENT SPECIFIC NUCLEAR BINDING STUDIED IN A CELL-FREE SYSTEM
  • Stephen J. Higgins
    Affiliations
    From the Departments of Biochemistry and Biophysics and Medicine, and the Metabolic Research Unit, University of California at San Francisco, San Francisco, California 94122
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  • Guy G. Rousseau
    Affiliations
    From the Departments of Biochemistry and Biophysics and Medicine, and the Metabolic Research Unit, University of California at San Francisco, San Francisco, California 94122
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  • John D. Baxter
    Affiliations
    From the Departments of Biochemistry and Biophysics and Medicine, and the Metabolic Research Unit, University of California at San Francisco, San Francisco, California 94122
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  • Gordon M. Tomkins
    Affiliations
    From the Departments of Biochemistry and Biophysics and Medicine, and the Metabolic Research Unit, University of California at San Francisco, San Francisco, California 94122
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Open AccessPublished:August 25, 1973DOI:https://doi.org/10.1016/S0021-9258(19)43583-7
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      Nuclear binding of receptor-glucocorticoid complexes can be observed at 20° in a cell-free system from cultured hepatoma cells. This association requires two steps: “activation” of the complex to a form which will bind and nuclear binding. These processes have been studied separately and an assay for the active form of the receptor is described. In cytoplasmic extracts, activation is favored by dilution and by increasing the ionic strength or the temperature. Using activated cytosol, receptor-glucocorticoid complexes bind to nuclei at 0° and at low ionic strength. This binding is specific in that it is of high affinity (Kd = 2.1 x 10-10m) to a fixed number of sites (15,000 to 16,000 per nucleus). A Scatchard analysis suggests that a single class of nuclear acceptor sites is involved in the binding. Further, activated receptors bound with nonradioactive steroid competitively inhibit nuclear binding of tritiated complexes. The cell-free nuclear binding reaction is similar in a number of ways to that which occurs in the intact cell, including the number of sites, the high affinity of the interaction, the necessity for activation, and the fact that the receptor must be bound with steroid. These data suggest that the cell-free system described here adequately represents some of the early steps in glucocorticoid action.

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