The activated hepatic glucocorticoid-receptor complex: A simple one-step method for its separation from nonactivated complex
✍ Scribed by Mohammed Kalimi
- Book ID
- 102985264
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 560 KB
- Volume
- 86
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Protamine sulfate was found to precipitate completely the nonactivated [3H]dexamethasone-receptor complex of rat liver. This observation was then used as the basis of a method to separate activated from nonactivated complex. Thus, addition of 10 mg/ml of protamine sulfate to the rat hepatic cytosol [3H]dexamethasone-receptor complex, incubated at 0-4°C for 2 hr, resulted in the complete precipitation of [3H]dexamethasone-receptor complex. The remaining supernatant obtained on centrifugation at 800g was unable to bind either to nuclei or to DNA-cellulose. An increase in temperature to 25°C or the addition of 10 mM CaCl, to the cytosol resulted in the appearance of activated [3H]dexamethasonereceptor complex in the supematant obtained by addition of protamine sulfate. This was determined by characteristic binding to nuclei or DNA cellulose and by PI. F'rotamine sulfate could not affect the separation of activated rH]dexamethasone-receptor complex at salt concentrations above 100 mM NaCl. This procedure therefore had to be carried out under conditions of relatively low ionic strength. Finally, a one-step rapid method is described for the separation of activated [3H]dexamethasone-receptor complex from nonactivated receptor complex. The homogeneous population of activated complex thus obtained should have considerable applicability in studies of the mechanisms of in vitro glucocorticoidreceptor activation.