The effect of dithiothreitol on the kinetics of dissociation of dexamethasone from the non-transformed mammary cytosolic glucocorticoid receptor.
Sulfhydryl reducing agents such as dithiothreitol are required for maximum binding of dexamethasone to the mammary cytosolic glucocorticoid receptor, but little is known concerning the effects of dithiothreitol on the kinetics of the binding reaction. In this report we have examined the influence of dithiothreitol on the dissociation kinetics of dexamethasone from the non-transformed glucocorticoid-receptor complex at 0-4 degrees C under various experimental conditions. Without dithiothreitol, the rate of dissociation of dexamethasone remains essentially the same (t1/2 approximately 17 h) regardless of the method chosen to monitor dissociation. With dithiothreitol, however, there is a marked acceleration in the rate of dissociation of receptor-bound dexamethasone when an excess of unlabeled dexamethasone is used to study dissociation (t1/2 approximately 5 h) but not when dissociation is investigated by removal of free labeled dexamethasone by charcoal adsorption (t1/2 approximately 21 h); dithiothreitol also accelerates the observed rate of dissociation when a combination of these methods is used. An acceleration in the rate of receptor-bound dexamethasone is also observed when an excess of the synthetic progestin, R5020, is used in the dissociation assay. The possible reasons and importance underlying these findings have been discussed.
R H
RH
Buell
L O
LO
Wosu
G
G
Shyamala
D000818:Animals; D002606:Charcoal / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology*; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D011397:Promegestone / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D014316:Tritium
D011965:Receptors, Glucocorticoid; D014316:Tritium; D002606:Charcoal; D003907:Dexamethasone; D011397:Promegestone; D004229:Dithiothreitol