Temperature-Dependent Kinetic Correlates of the Activation of the Glucocorticoid-Receptor Complex

The effects of temperature on the kinetics of activation were studied in [3H]triamcinolone acetonide[( 3H]TA)-labeled cytosol preparations from mouse whole brain. After removal of unbound [3H]TA and molybdate (which prevents activation) from the unactivated steroid-receptor complex by gel exclusion chromatography, activation was initiated by incubation at 6-30 degrees C for 0.75-24 min and then rapidly quenched at -5 degrees C with Na2MoO4 (20 mM final concentration). The loss of the 9.2S (unactivated) form of the [3H]TA-receptor complex and the concomitant formation of the 3.8S (activated) form increased dramatically with increases in the activation temperature. These hydrodynamic changes were correlated directly with rapid time- and temperature-dependent increases in the binding of [3H]TA-labeled cytosol to DNA-cellulose (DNA-C). Further analyses of these data revealed a greater than 50-fold increase in the apparent first-order rate constant for the increased binding to DNA-C as the activation temperature was increased from 6 degrees C to 30 degrees C. An Arrhenius plot of these temperature-dependent kinetic constants revealed an energy of activation of 116 kJ. These data support a proposed model for activation of the glucocorticoid-receptor complex that includes the splitting of a 297 kDa, unactivated species into a 92 kDa, activated species.     

Kinetic analyses of activation-induced changes in the hydrodynamic and surface properties of the glucocorticoid-receptor complex in mouse brain

Unactivated, molybdate-stabilized, [³H]triamcinolone acetonide-labeled, glucocorticoid receptors from mouse whole brain were activated by removal of the molybdate and incubation at 22°C for 1.5 to 24 min and then rapidly quenched at 0°C with molybdate. The loss of the 9.2 S (unactivated) form of the [³H]TA-receptor complex and the concomitant formation of the 3.8 S (activated) form displayed first-order kinetics with a half-time of less than two min. The increase in the 3.8 S form correlated nearly perfectly with an increased binding to DNA-cellulose, and with a decreased and increased adsorption to DEAE-cellulose and glass fiber filters, respectively. The changes in adsorption to these filters, which occurred at a faster rate than did the changes in binding to DNA-C, are thought to reflect an increase in the relative number of positive charges and hydrophobic groups on the surface of the activated complex.     

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex

The molybdate-stabilized rat liver glucocorticoid receptor complex was purified 9000-fold with a 46% yield by steroid-affinity chromatography and DEAE-Sephacel ion-exchange chromatography. The purified glucocorticoid receptor was identified as a 90-92-kDa protein by SDS/polyacrylamide gel electrophoresis. Raising the temperature to 25 degrees C in the absence of molybdate resulted in increased binding of the receptor complex to DNA-cellulose or nuclei, similar to the effect on the cytosolic complex. The purified complex has a sedimentation coefficient of 9-10 S before and after heat treatment in the absence of molybdate. The appearance of smaller 3-4-S species was unrelated to the extent of DNA-cellulose binding of the complex. The process termed 'transformation', i.e. increasing the affinity for DNA, is not concomitant with subunit dissociation or loss of RNA. Highly purified glucocorticoid receptor could be covalently modified with biotin to retain its steroid-binding activity but with a 50% decrease in nuclear binding capacity. The biotin-modified complex reacts with streptavidin in solution without losing its steroid.     

The effect of alkaline phosphatase on the activation of glucocorticoid-receptor complexes in rat liver cytosol

Calf intestinal alkaline phosphatase was found to stimulate the rate of in vitro activation of rat liver glucocorticoid-receptor complexes. This effect was registered both at 0 and 25 degrees C and could be prevented by sodium molybdate. The resulting change in sedimentation behaviour (shift of sedimentation coefficient from 9.6 S to 4.8 S for molybdate-stabilized and alkaline phosphatase-treated complexes, respectively) was similar to that observed after heat activation.     

Activation of cytosol aldosterone receptors in rat kidney

Cytosolic aldosterone-protein complexes are isolated from rat kidney slices after incubation with [3H]aldosterone and dexamethasone. Activated and unactivated forms of the complex are characterized by gel electrophoresis and hydroxyapatite chromatography after incubation at 4 degrees C and 25 degrees C respectively. It is found that the activated form reaches a maximum after 30 min at 25 degrees C and can be separated as an homogeneous peak by electrophoresis. Intermediate forms can also be identified. In the presence of 10 mM ATP, activation immediately occurs at 4 degrees C and is almost complete. In the presence of 10 mM molybdate, the activation is strongly enhanced and the increase in activated form may be about fifteen-fold whether molybdate is added during kidney homogenization or just before incubation at 25 degrees C. On the other hand molybdate reduces to one third the binding of the aldosterone-receptor complexes to nuclei. In the presence of the steroid RU 26988 which is a pure glucocorticoid, experiments done on aldosterone-receptors complexes and their binding to nuclei are confirmed. This proves that aldosterone is specific for mineralocorticoid sites. The general pattern of the mineralocorticoid receptor activation is discussed and its resemblance to the case of other steroid hormones is emphasized.     

Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.     

Characterization of the effect of sodium molybdate and diisopropylfluorophosphate on the human myometrial estrogen and progesterone receptors

The human myometrial estrogen receptor (ER) and progesterone receptor (PR) are stabilized in the presence of 25 mM molybdate. The stabilizing effect is very palpable for the PR; even after 15 min of incubation at 37°C only 15% of the binding activity is lost in the presence of molybdate, whereas during the same period of time 75% of the binding activity is lost in the absence of molybdate. In the presence of molybdate the ER yield is increased by 27.7 ± 16.3% (mean ± SD) and the yield of PR is increased by 34.3 ± 18.3%. Also in the presence of 2 mM diisopropylfluorophospate (DFP) the PR yield is increased by 25.5 ± 16.7% whereas the ER recovery is decreased by 22.7 ± 12.8%. The DNA-binding activities of ER and PR are greatly enhanced in the presence of DFP whereas this activity is blocked by molybdate. No shifts in the sedimentation values of either ER or PR was observed in the presence of molybdate as compared to the sedimentation values obtained in the presence of DFP. Also the equilibrium dissociation constants of ER and PR bindings were not affected by molybdate. DFP however caused a decrease in both the affinity and the binding capacity of the ER whereas the binding characteristics of the PR remained unchanged.     

Changes in the sedimentation properties of cytosolic androgen receptors associated with activation in vitro and in vivo

In cell-free systems androgen receptor (AR) labeled with (3H)DHT at 0 degrees C in the presence of 50mM molybdate remains unactivated (less than 3% binding to nuclei) and untransformed (7-8S on sucrose density gradients containing 0.4M KCl and 50mM molybdate). In the absence of molybdate, however, these complexes undergo activation and transformation even at 0 degrees C, albeit, very slowly. Incubation of unactivated, untransformed AR complexes at 18 degrees C, or at 0 degrees C in the presence of 0.4M KCl, greatly accelerated both activation and transformation. Activation and transformation are also associated with formation of high affinity (3H)DHT-receptor complexes as indicated by decreased rates of (3H)DHT dissociation from the receptor. Cytosolic AR complexes labeled with (3H)DHT in tissue slices at 37 degrees C, or in vivo, undergo rapid activation, transformation and nuclear translocation. The data suggest that activation and transformation of cytosolic AR in cell-free systems is associated with changes in the physicochemical properties of AR similar to those occurring upon hormone binding in intact cells and in vivo.     

Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.     

The effect of nucleotides and molybdate ions on the activation of glucocorticoid-receptor complexes

The effects of various nucleotides and sodium molybdate on the activation of glucocorticoid-receptor complexes (GRC) isolated from tissue cytosol of 6- and 25-month-old rats was studied. It was shown that nucleoside triphosphates activate GRC in the livers of 6-month-old rats, the activating effect being decreased in the following order: UTP greater than or equal to ATP greater than GTP greater than or equal to CTP. Nucleoside di- and monophosphates exert a far lesser stimulating effect. These effects of nucleotides decrease with ageing. Molybdate ions exert a 3-fold effect on the activation of GRC from various rat tissues, i.e., stimulating, inhibiting and zero effects.     

Tyrosine phosphorylation of the receptor for insulin-like growth factor II is inhibited in plasma membranes from insulin-treated rat adipocytes.

We have identified a factor from rat liver cytosol that enhances the DNA-cellulose-binding ability of the glucocorticoid receptor and lowers the sedimentation value from 9-10 S to 4-5 S. Cytosol is prepared in the presence of molybdate, and unactivated receptor is isolated by chromatography on DEAE-cellulose in the presence of molybdate. This receptor sediments at 9-10 S and has little affinity for DNA. If the molybdate is removed and the receptor is incubated at 25 degrees C with the low-salt wash of the DEAE-cellulose column, DNA binding is enhanced by 50-600% relative to controls incubated with buffer only. In addition, the factor present in the low-salt wash converts the 9-10 S receptor into a mixture of 5 S and 4 S forms. The factor must be present during the incubation in order to exert its maximal effect. Factor added after the incubation has only marginal effects on the DNA-binding ability of the receptor, indicating that the factor does not increase the DNA-binding ability of activated receptor. Moreover, the factor is significantly less effective on receptor that has been activated before incubation with the factor. These results suggest that the factor acts as an activation enhancer. Preliminary characterization indicates that the activation enhancer is a trypsin-sensitive protein of approx. 70,000 Da, whose activation-enhancing properties are inhibited by ATP. RNAase A, which has effects similar to those described above on the 7-8 S receptor, does not mimic the effects of the activation enhancer on the 9-10 S receptor.     

Fractionation of serum transcobalamins on charged cellulose filters.

A simple and rapid fractionation procedure of the three transcobalamins, TCI, TCII, and TCII, of human serum was achieved by filtration through a stack of charged cellulose filters composed of one cellulose-nitrate and three DEAE-cellulose (DE-81) disks. A reaction mixture containing microliter amounts of serum was incubated with excess of 57Co B12 of high specific activity, diluted with 0.1 M sodium borate buffer (pH 8.5), and passed through the filter stack by applying vacuum. Under these conditions TCII is selectively and quantitatively adsorbed to the cellulose-nitrate filter while both TCI and TCIII adsorb to the DE-81 filters. In the second step TCIII is selectively desorbed from the latter filters by a 0.05 M monopotassium phosphate solution of pH 4.6. Using sera of different distribution of transcobalamins the data obtained were comparable to those determined by the more laborious methods employing DE-52 column chromatography combined with procedures to remove TCII.     

Purification, characterization, and activation of the glucocorticoid-receptor complex from rat kidney cortex

The unactivated molybdate-stabilized glucocorticoid receptor (GcR) was purified from rat kidney cortex cytosol (RKcC) by using a modification of the procedure previously described by this laboratory for rat hepatic receptor. The purification includes affinity chromatography, gel filtration, and ion-exchange chromatography. The final preparation (approximately 1000-fold pure as determined from specific radioactivity) was used in subsequent physicochemical and functional analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single heavily Coomassie-stained band at 90 kilodaltons. Density gradient ultracentrifugation indicated a sedimentation coefficient of 10.5 +/- 0.05 S (n = 2). Chromatography on an analytical gel filtration column produced a Stokes radius (Rs) of 6.4 +/- 0.07 nm (n = 5). The Rs was unchanged when the molybdate-stabilized GcR was analyzed in the presence of 400 mM KCl or when analyzed in the unpurified (cytosolic) state. In contrast, the hepatic GcR was observed to exist as a larger form in cytosol (7.7 +/- 0.2 nm). Following purification, or upon gel filtration analysis under hypertonic conditions, the Rs was similar to that of the unpurified RKcC GcR. Following removal of molybdate from RKcC GcR and thermal activation (25 degrees C/30 min), DNA-cellulose binding increased 1.5-2-fold over the unheated control. Addition of RKcC or hepatic cytosol (endogenous receptors thermally denatured at 90 degrees C/30 min or presaturated with 10(-7) M radioinert ligand) during thermal activation increased DNA-cellulose binding an additional 2-6-fold beyond the heated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adsorption of dissolved organic matter to the inorganic filter substrate and its implications for C uptake measurements

Inorganic carbon uptake rates for glass fiber-filtered samples are higher than those for membrane-filtered samples because of adsorption of dissolved organic matter to the filter substrate. Experimentally derived values for adsorption onto filters were as follows (relative units): GF/F filter, 1, quartz filter, 1.1, GF/C filter, 0.6; GN-6 Gelman filter, 0.1; Nuclepore and Poretics filter, 0.0; Anodisc filter, 0.4 to 1.9.     

Interaction of sodium molybdate with the thyroid hormone receptor

The 3,5,3'-triiodothyronine (T3) binding activity of solubilized nuclear proteins from rat liver was decreased when molybdate (10 mM) was present in the incubation medium in the absence of thiol reagents. The equilibrium affinity constant was reduced by 40%. The rate of degradation of T3-receptor complexes at 37 degrees C remained unchanged, but when the extracts were further reincubated in the presence of beta-mercaptoethanol, molybdate had a protective effect after 5 h incubation at 37 degrees C. In contrast, the thyroxine (T4) binding activity was not affected by heating at 37 degrees C or by molybdate. Ion-exchange chromatography confirmed the existence of a molybdate-receptor interaction: the T3-receptor complexes shifted from elution at 0.22 to 0.20 M NaCl with the progressive appearance of a small leader peak, whereas the T4-receptor complexes eluted in a large and split peak (0.22-0.4 M NaCl). The destabilizing effect on T3 binding induced by exogenous dephosphorylation is more efficiently reversed by beta-mercaptoethanol when the extracts were pretreated by molybdate. In controls, the loss of saturable T3 binding activity was recovered by 50% at a 10 mM concentration of beta-mercaptoethanol, but in the presence of molybdate, the loss of T3 binding activity was recovered by 50% at a 5 mM concentration of beta-mercaptoethanol. This molybdate-receptor interaction is similar to that with nuclear receptor models in term of (i) stabilization of hormone binding, (ii) dependency on a thiol, and (iii) reversibility of the destabilizing effect by exogenous dephosphorylation.     

Adsorption of Dissolved Organic Matter to the Inorganic Filter Substrate and Its Implications for 14C Uptake Measurements

Inorganic carbon uptake rates for glass fiber-filtered samples are higher than those for membrane-filtered samples because of adsorption of dissolved organic matter to the filter substrate. Experimentally derived values for adsorption onto filters were as follows (relative units): GF/F filter, 1, quartz filter, 1.1, GF/C filter, 0.6; GN-6 Gelman filter, 0.1; Nuclepore and Poretics filter, 0.0; Anodisc filter, 0.4 to 1.9.     

ATP-dependent activation of L cell glucocorticoid receptors to the steroid binding form.

The specific glucocorticoid binding capacity in cytosols prepared from L929 mouse fibroblasts (L cells) is inactivated with a half-life of approximately 2 h at 25 degrees C. As previously published, this inactivation can be prevented with 10 mM molybdate and markedly slowed by addition of other phosphatase inhibitors such as glucose 1-phosphate and fluoride. We have now found that ATP (5 to 10 mM) also slows the rate of this inactivation. After extensively inactivating the receptor by preincubating cytosol at 25 degrees C for 4 and preventing further inactivation by addition of molybdate, addition of ATP results in reactivation of the steroid binding capacity. Maximal reactivation of 40 to 70% is achieved with 5 to 10 mM ATP. The activation is temperature-dependent and specific for ATP. ADP, GTP, CTP, and UTP do not cause activation and preliminary results indicate no effect of cyclic nucleotides in this system. If activation is prevented by addition of 10 mM EDTA to the cytosol, addition of 3 to 10 mM magnesium permits ATP-dependent activation of the binding capacity. The level of reactivation can be enhanced by addition of a heat-stable factor prepared from the same L cell supernatant. These results support the proposal that L cell glucocorticoid receptors can be activated to the glucocorticoid binding state by an ATP-dependent phosphorylation mechanism.     

Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form

In this report we examine the DNA-cellulose binding and sedimentation properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors from rat intestine and cultured human mammary cancer cells (MCF-7) extracted in nonactivating (low salt) buffers. Receptors prepared in hypotonic buffer had low DNA binding (13%) compared to receptors extracted with 0.3 M KCl (50%). Treatment of low salt receptor preparations with KCl significantly increased (approximately 3-fold) DNA-binding (activation), demonstrating that receptors can be "activated" in vitro. Activated receptors eluted from DNA-cellulose at 0.18 M KCl. Sedimentation analysis followed by DNA-cellulose binding indicated that activated receptors are approximately 3.2 S and unactivated receptors 5.5 S in size. These results suggest that dissociation of an aggregated moiety may lead to receptor activation. Treatment of unactivated receptor with RNase did not alter DNA binding or sedimentation properties of the aggregated receptor. Treatment of unactivated receptor complexes with heat did not increase DNA binding, and molybdate did not block subsequent salt activation. In summary these results suggest that 1,25(OH)2D3 receptors undergo a salt-induced activation step similar to that described for other steroid receptor systems. However, 1,25(OH)2D3 receptors differ from other steroid receptors in not exhibiting heat activation nor having salt activation blocked by molybdate.     

Characterization of the in vitro stability of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The in vitro stability of the Ah receptor from rat hepatic cytosol was evaluated by [3H]TCDD binding studies, gel filtration, and sucrose density gradient ultracentrifugation. Thermal inactivation of unoccupied receptor followed first-order kinetics between 5 and 40 degrees C, with an estimated Ea for inactivation of approximately 35 kcal/mol. Protease inhibitors did not reduce and dilution slightly increased the inactivation rate at 20 degrees C. Recovery and 20 degrees C stability decreased with increasing ionic strength. The TCDD-receptor complex was less susceptible to degradation at 20 degrees C, even in the presence of 0.4 M KCl. Specific binding was markedly pH dependent, with maximum recovery at 7.6. Analysis of the pH curve suggested that cysteine sulfhydryl groups may be involved in TCDD binding. Dithiothreitol (1 mM) maximized recovery and 20 degrees C stability, and addition of the thiol largely reactivated binding sites lost from cytosol prepared without it. Removal of low molecular weight components of cytosol by gel filtration resulted in a rapid 20 degrees C inactivation rate that could not be lessened by dithiothreitol. Glycerol (10% v/v) and EDTA (1.5 mM) maximized recovery of specific binding, but both decreased 20 degrees C stability in a concentration-dependent manner. Calcium chloride (4 mM) increased stability at 20 degrees C by approximately 20%, and retarded the characteristic shift in sedimentation coefficient from approximately 9 to approximately 6 S in high-salt sucrose gradients. The fact that sodium molybdate (20 mM) decreased recovery and 20 degrees C stability when dithiothreitol was present but slightly increased stability in its absence suggested an antagonism between the two compounds. Molybdate mitigated the inactivation induced by 0.4 M KCl, an effect which may be related to the observation of dual peaks in molybdate-containing high-salt sucrose gradients. These data indicate that thermal inactivation of the unoccupied rat hepatic Ah receptor primarily may be due to physical rather than enzymatic processes; (ii) sulfhydryl oxidation, removal of low molecular weight cytosolic components, and high ionic strength result in rapid rates of inactivation at 20 degrees C; and (iii) the large degree of protection conferred by TCDD binding implies a very tight ligand-receptor interaction, and as such accords with TCDDs extraordinary potency and persistence in producing its toxic and biochemical effects.     

Heterogeneity of the glucocorticoid receptors: molecular transformations during activation, detected by electrofocusing

Isoelectric focusing (IEF) of glucocorticoid receptor (GR) of the neural retina of the 14-day chick embryo was conducted under conditions that yielded quantitative recovery of binding activity. IEF of the cytosol, equilibrated with [3H]triamcinolone acetonide (TA) at 0-2 degrees C yielded three major TA-GR components with apparent isoelectric points (pI') of 5.4 +/- 0.3, 6.5 +/- 0.2, and 7.6 +/- 0.3, designated as I, II, and III, respectively. During temperature-induced activation (incubation at 30 degrees C for 60 min, in the presence of free [3H]TA and 0.15 M KCl), approximately 25% of the specifically bound TA was irreversibly lost. IEF reveals that this loss is accounted for by the complete loss of binding from I. During activation, II also decreases but correspondingly III increases, i.e., the sum of II and III remains unchanged. Only the bound TA of I is sensitive to the addition of KCl (a promoter of activation). This sensitivity of I is temperature dependent. Molybdate (an inhibitor of activation) protects the bound TA of I and suppresses the formation of III. These two effects of molybdate diminish simultaneously when the temperature is increased to 30 degrees C. III preferentially exhibits binding activity to nuclei. The data suggest that (i) the glucocorticoid-free cytosol contains two GRs, I and II, with possibly two different functions; (ii) activation involves the loss of bound TA from I and the transformation of II to III with increased pI; (iii) these two molecular events in GR activation are interdependent.