scholarly journals Interaction of rat liver glucocorticoid receptor with adenosine 5'-triphosphate. Characterization of interaction by use of ATP-sepharose affinity chromatography

1980 ◽  
Vol 190 (3) ◽  
pp. 809-818 ◽  
Author(s):  
V K Moudgil ◽  
J K John
Keyword(s):  
Glucocorticoid Receptor ◽  
Triamcinolone Acetonide ◽  
Rat Liver ◽  
Sodium Molybdate ◽  
Receptor Complex ◽  
Competitive Binding Assay ◽  
Receptor Complexes ◽  
Salt Activation ◽  
Free Nucleotides

An interaction between rat liver glucocorticoid—receptor complex and immobilized ATP was identified. Rat liver cytosol preparations were incubated with [3H]triamcinolone acetonide for 4 h at 4 degrees C and partially purified by precipitation with (NH4)2SO4 before use. The resulting glucocorticoid—receptor complex could be selectively adsorbed on to columns of ATP—Sepharose. The freshly prepared cytosol [3H]triamcinolone acetonide—receptor complex had very little affinity for binding to the ATP—Sepharose column, but acquired this ability on temperature- or salt-activation. The presence of 10 mM-sodium molybdate during this salt- or temperature-dependent activation blocked the binding of the receptor complex to ATP—Sepharose. The interaction is reversible, since it can be disrupted by high-salt conditions. A competitive binding assay, using free nucleotides in samples to be chromatographed, revealed a preferential interaction between ATP and the glucocorticoid—receptor complex. Buffer containing ATP was also used to elute the glucocorticoid—receptor complex from ATP—Sepharose columns successfully. When ATP was added to the preparations containing [3H]triamcinolone acetonide—receptor complexes, the steroid specificity or sedimentation properties of the complex remained unaltered. Our results demonstrate an interaction between rat liver glucocorticoid—receptor complex and immobilized ATP and suggest a role of this nucleotide in receptor function.

scholarly journals ATP-dependent activation of glucocorticoid receptor from rat liver cytosol

1980 ◽  
Vol 190 (3) ◽  
pp. 799-808 ◽  
Author(s):  
V K Moudgil ◽  
J K John
Keyword(s):  
Glucocorticoid Receptor ◽  
Rat Liver ◽  
Sodium Molybdate ◽  
Receptor Activation ◽  
Activation Process ◽  
Receptor Complex ◽  
Liver Cytosol ◽  
Receptor Complexes ◽  
Steroid Binding ◽  
Rat Liver Cytosol

The glucocorticoid—receptor complex from freshly prepared rat liver cytosol is in a non-activated form, with very little affinity to bind to isolated nuclei. When such preparations were incubated with 5—10 mM-ATP at 4 degrees C, the receptor complex acquired the properties of an ‘activated’ transformed form, which readily bound to nuclei, ATP—Sepharose, phosphocellulose and DNA—cellulose. This transformation was comparable with the activation achieved by warming the steroid—receptor complex at 23 degrees C. The effect of ATP was specific, as it was more effective than ADP, whereas AMP had no such effect on activation. The process of receptor activation was sensitive to the presence of 10 mM-sodium molybdate; the latter blocked activation by both ATP and heat. Bivalent cations had no observable effect on the receptor activation at low temperature, but they decreased the extent of activation by ATP. The steroid-binding properties of glucocorticoid receptor remained intact under the above conditions. However, a significant increase in steroid binding occurred when ATP was preincubated with cytosol receptor before the addition of [3H]triamcinolone acetonide. ATP also stabilized the glucocorticoid—receptor complexes at 23 degrees C. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor under mild assay conditions.

scholarly journals Inactivation of rat liver glucocorticoid receptor by molybdate. Effects on both non-activated and activated receptor complexes

1981 ◽  
Vol 198 (3) ◽  
pp. 447-455 ◽  
Author(s):  
N Murakami ◽  
V K Moudgil
Keyword(s):  
Glucocorticoid Receptor ◽  
Rat Liver ◽  
Sodium Molybdate ◽  
Dependent Manner ◽  
Receptor Complex ◽  
Liver Cytosol ◽  
Isolated Nuclei ◽  
Receptor Complexes ◽  
Dose Dependent ◽  
Rat Liver Cytosol

When freshly prepared glucocorticoid-receptor complex from rat liver cytosol was incubated at 23 degrees C in the presence of sodium molybdate, its subsequent binding to isolated nuclei, DNA-cellulose and ATP-Sepharose was blocked. In addition, binding to these acceptors by cytosol receptor complex fractionated with (NH4)2SO4 was also blocked by incubation of the complexes with 50 mM-sodium molybdate. However, molybdate had no effect on the binding of activated receptor complexes to ATP-Sepharose. Molybdate was also effective in extracting the nuclear- and DNA-cellulose-bound glucocorticoid-receptor complexes in a dose-dependent manner. Molybdate appears to exert its effects directly on the receptor by interacting with both non-activated and activated receptor forms.

Glucocorticoid receptor activation in isolated perfused rat hearts

1989 ◽  
Vol 256 (2) ◽  
pp. C219-C225 ◽  
Author(s):  
S. M. Czerwinski ◽  
E. E. McKee ◽  
R. C. Hickson
Keyword(s):  
Glucocorticoid Receptor ◽  
Triamcinolone Acetonide ◽  
Deae Cellulose ◽  
Potassium Phosphate ◽  
Receptor Activation ◽  
Receptor Complexes ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Cellulose Binding ◽  
Isolated Perfused Rat Hearts

The formation of unactivated and activated glucocorticoid receptor complexes was studied in intact, isolated, perfused rat hearts in the presence of [3H]triamcinolone acetonide. Receptor activation, as quantified by the DNA-cellulose-binding assay, began to increase within 30 s of perfusion and reached a final steady-state level (t 1/2 = 4.6 min) with 46% of the steroid-receptor complexes bound to DNA-cellulose. With the use of a linear potassium phosphate (KP) gradient (5-400 mM), unactivated receptors eluted from DEAE-cellulose anion exchange columns at approximately 250 mM KP. Two activated receptor forms appeared, which eluted either in the wash fraction (binder IB) or between 50 and 100 mM KP (binder II) and occurred with half times of 1.3 and 2.7 min, respectively. Postperfusion cytosol preparation did not markedly influence the results as receptor binding was reduced by 10% or less when a 100-fold excess of unlabeled triamcinolone acetonide was included in the homogenizing buffer. We conclude from these results that glucocorticoids are able to exert a direct effect on the heart through binding to their own receptor in the absence of endogenous hormones. The time dependency of receptor activation supports a physiological role for this process. However, activation rates, determined from conformational changes associated with altered DEAE-cellulose elution profiles and appearance of activated receptor forms, occur earlier and may not be coordinated with the rate of activation as quantified by DNA-cellulose binding.

scholarly journals Interaction of rat liver glucocorticoid receptor with sodium tungstate

1982 ◽  
Vol 204 (3) ◽  
pp. 777-786 ◽  
Author(s):  
N Murakami ◽  
S P Healy ◽  
V K Moudgil
Keyword(s):  
Glucocorticoid Receptor ◽  
Rat Liver ◽  
Sodium Tungstate ◽  
Binding Capacity ◽  
Receptor Complex ◽  
Heat Activation ◽  
Ph Dependent ◽  
Steroid Binding ◽  
Cellulose Binding ◽  
Rat Liver Cytosol

Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10-20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3-4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor.

scholarly journals Binding of the glucocorticoid receptor to the rat liver nuclear matrix. The role of disulfide bond formation.

1986 ◽  
Vol 261 (26) ◽  
pp. 11962-11967 ◽  
Author(s):  
S H Kaufmann ◽  
S Okret ◽  
A C Wikström ◽  
J A Gustafsson ◽  
J H Shaper
Keyword(s):  
Glucocorticoid Receptor ◽  
Rat Liver ◽  
Disulfide Bond ◽  
Nuclear Matrix ◽  
Bond Formation ◽  
Disulfide Bond Formation
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