Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.     

The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor

Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor complex that accompanies transformation to the DNA-binding state. When steroid-receptor complexes that are prebound to DNA-cellulose are exposed to MMTS, the steroid rapidly dissociates, but the receptor remains bound to DNA. Thus, the presence of steroid is not required for the receptor to remain bound to DNA in a high affinity manner. Treatment of cytosol containing transformed glucocorticoid-receptor complexes at 0 degrees C with 20 mM hydrogen peroxide also inactivates the DNA-binding activity of the receptor. The peroxide-induced inactivation is reversed by DTT. Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25 degrees C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. The presence of peroxide during heating of the cytosol also prevents dissociation of the receptor complex as assayed both by reduction in sedimentation value of the receptor and by dissociation of the 90-kDa heat shock protein from the steroid-binding protein. These results strongly suggest that critical sulfur moieties in the receptor complex must be in a reduced form for the temperature-mediated dissociation of the receptor to occur.     

Activation of the human glucocorticoid receptor: evidence for a two-step model

The relationship between glucocorticoid receptor subunit dissociation and activation was investigated by DEAE-cellulose and DNA-cellulose chromatography of monomeric and multimeric [3H]triamcinolone acetonide ([3H]TA)-labeled IM-9 cell glucocorticoid receptors. Multimeric (7-8 nm) and monomeric (5-6 nm) complexes were isolated by Sephacryl S-300 chromatography. Multimeric complexes did not bind to DNA-cellulose and eluted from DEAE-cellulose at a salt concentration (0.2 M KCl) characteristic of unactivated steroid-receptor complexes. Monomeric [3H]TA-receptor complexes eluted from DEAE-cellulose at a salt concentration (20 mM KCl) characteristic of activated steroid-receptor complexes. However, only half of these complexes bound to DNA-cellulose. This proportion could not be increased by heat treatment, addition of bovine serum albumin, or incubation with RNase A. Incubation of monomeric complexes with heat inactivated cytosol resulted in a 2-fold increase in DNA-cellulose binding. Unlike receptor dissociation, this increase was not inhibited by the presence of sodium molybdate. Fractionation of heat inactivated cytosol by Sephadex G-25 chromatography demonstrated that the activity responsible for the increased DNA binding of monomeric [3H]TA-receptor complexes was macromolecular. These results are consistent with a two-step model for glucocorticoid receptor activation, in which subunit dissociation is a necessary but insufficient condition for complete activation. They also indicate that conversion of the steroid-receptor complex to the low-salt eluting form is a reflection of receptor dissociation but not necessarily acquisition of DNA-binding activity.     

Activated steroid-receptor complex. Comparison of assays using DNA-cellulose or homologous nuclei.

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [3H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the later only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.     

The "activated" hepatic glucocorticoid-receptor complex. Its generation and properties.

The glucocorticoid receptor-glucocorticoid complex of the hepatic cytosol need undergo an "activation" to enable its binding to nuclei, chromatin, or stripped DNA. The conditions of this activation have been studied using native calf thymus DNA absorbed to cellulose. At low ionic strength, activation is very slow at 0 degrees, but, takes place rapidly at 25 degrees, reaching completion at 1 hour. Addition of 10 mm CaCl2 or 150 mm NaCl increases the rate of activation of the receptor at 0 degrees. Neither magnesium nor manganese ions can replace calcium with respect to enabling activation of the steroid-receptor complex to occur at low temperatures. Isofocusing studies reveal that the major component of the unactivated steroid-receptor complex has an isoelectric point of 7.1. Incubation of the steroid-receptor complex at 25 degrees for 30 min leads to its conversion to a form with an isoelectric point of 6.1 concurrent with the development of its ability to bind to DNA-cellulose. Sucrose density gradient analysis reveals that no detectable alteration in the sedimentation coefficient of the steroid-receptor complex occurs during its activation. MnCl2 (20mm) effeciently precipitates the unactivated hormone-receptor complex and to a lesser degree, precipitates the activated hormone-receptor complex.     

Evidence that the hormone-binding domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a major portion of receptors that are "misfolded" after removal of hsp90.

After dissociation of cytosolic heteromeric glucocorticoid receptor complexes by steroid, salt, and other methods, only 35-60% of the dissociated receptors can bind to DNA-cellulose. The DNA-binding and non-DNA-binding forms of the dissociated receptors have the same Mr and are phosphorylated to the same extent (Tienrungroj, W., Sanchez, E. R., Housley, P. R., Harrison, R. W., and Pratt, W. B. (1987) J. Biol. Chem. 262, 17347-17349). The basis for the different DNA-binding activities is unknown, but the DNA-binding fraction of the receptor has a more basic pI than the non-DNA-binding fraction (Smith, A. C., Elsasser, M. S., and Harmon, J. M. (1986) J. Biol. Chem. 261, 13285-13292). We have separated the non-DNA-binding state of the receptor from the DNA-binding state and then cleaved it with trypsin and chymotrypsin. We find that the 15-kDa tryptic fragment derived from the non-DNA-binding state of the dissociated receptor is fully competent in binding DNA, whereas the 42-kDa chymotryptic fragment containing both the hormone-binding and DNA-binding domains does not bind DNA. Trypsin cleavage of the molybdate-stabilized untransformed receptor also yields a 15-kDa fragment that is fully competent in binding DNA. Reducing agents do not restore DNA-binding to the non-DNA-binding fraction of the receptor and the hormone-binding domain can be separated from the DNA-binding domain on nonreducing gel electrophoresis. These results argue that the two domains are not linked by disulfide bridges, and they are consistent with the proposal that there are two least energy states of folding after dissociation of hsp90. A significant portion of the receptors is "misfolded" in such a manner that the steroid binding domain is directly preventing DNA-binding activity.     

Activated steroid-receptor complex comparison of assays using DNA-cellulose or homologous nuclei

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [³H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the latter only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.     

Transformation of the chick oviduct progesterone receptor in the absence of hormone

Chicken oviduct progesterone receptor in cytosol was found to be transformed from the 8S to 4S form by incubation at 25 degrees C as well as by 0.3 M KCl in the absence of hormone. Heat transformation of ligand-free receptor took place at a much slower rate than that of ligand-bound receptor. The eventual percentage of transformation, however, was almost the same. The 4S form of the receptor transformed by KCl in the absence of hormone could bind to DNA-cellulose, but not to nuclei. However, upon exposure it acquired the ability to bind to nuclei. It was shown that the transformed ligand-free receptor could bind to progesterone to form the normal activated steroid-receptor complex. Conversely, when activated 4S progesterone-receptor complex was treated with DCC to peel off the hormone, a resulting ligand-free receptor was formed which behaved just like the KCl-transformed receptor in the absence of hormone.     

Studies on the translocation inhibitor of 3H-dexamethasone-receptor complex.

Recent reports on the binding of glucocorticoid-receptor complexes to rat liver nuclei suggested the presence of components which inhibited the binding. The inhibitory component(s) of the receptor translocation was observed not only in the cytosol of the liver but also in cytosols of the kidney, the spleen and the thymus. The cytoplasmic levels of the inhibitor in these tissues were not modified by the administration of Dexamthasone (DEX). The liver inhibitor was macromolecular and clearly separated from the DEX-receptor complex on DEAE-cellulose chromatography. The mechanism of the inhibition seemed to be an interaction between the inhibitor and the steroid-receptor complex. In addition, the inhibition seemed to be less specific for the bindings of different steroid-receptor complexes to nuclei. The bindings of hepatic 3H-DEX-receptor complex by nuclei derived from livers of adrenalectomized and DEX-treated rats, in the presence or absence of the translocation inhibitor, were similar.     

Purification and comparative study of the properties of glucocorticoid- and estrogen-receptor complexes in the rat liver

Parallel purification of glucocorticoid- and estrogen-receptor complexes from rat liver cytosol has been accomplished. Some properties of purified steroid-receptor complexes (SRC) were determined. The procedure developed earlier when the two-step treatment of cytosol with DNA-cellulose alternated with SRC ammonium sulphate precipitation, was shown to be universally applicable for purification of various SRC. Certain modifications have been devised allowing some increase in the degree of receptor purification. The amount of estrogen-receptor complexes (ERC) isolated from male liver cytosol was 20-40 times less than that of glucocorticoid-receptor complexes (GRC) isolated simultaneously from the equal volume of the same cytosol. Both SRC types bind intensively to homologous DNA but not to poly(A). The elution of GRC from DNA cellulose was mainly achieved at 0.4 M NaCl. With this, GRC and ERC showed small, but reliable differences in the salt resistance of their associates with DNA: the ERC-DNA link was stable toward NaCl up to 0.1 M, whereas an appreciable amount of GRC was eluted from DNA-cellulose at 0.1 M NaCl. The stability of purified ERC exceeded that of purified GRC, which apparently reflects the differences in the hormone-receptor binding constants. The receptor stability under various environmental conditions is discussed and some recommendations on the improvement of the SRC stability and its control are given.     

Sensitivity of progesterone receptors to aurintricarboxylic acid: Inhibition of nuclear uptake,ATP and DNA binding

When hen oviduct cytosol samples containing progesterone receptor complexed to [³H]progesterone were included with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose or ATP-Sepharose was abolished under simmilar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that auintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest the use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Reversible precipitation of the glucocorticoid receptor from rat liver by La3+ ions

It was shown that La3+ ions are capable of precipitating the glucocorticoid receptor from rat liver. Treatment of cytosol containing either free receptor or the hormonereceptor complex with 0,005 M La(NO3)3 results in recptor precipitation. The pellet is readily dissolved in buffer with EDTA. Transcortin and transcortin-like proteins are not affected by La3+ ions. The lanthanium-treated receptor does not lose its ability to bind DNA and chromatin in vitro. It is suggested that precipitation by La3+ ions can be used for separation of the receptor from transcortin and transcortin-like proteins as well as for evaluation of binding parameters for steroid-receptor complexes in rat liver cytosol.     

Translation of glucocorticoid receptor mRNA in vitro yields a nonactivated protein

The glucocorticoid receptor is present in cytosol prepared from cell extracts of nonhormone-treated cells as a large nonactivated (i.e. non-DNA binding) 9 S heteromeric complex which contains the Mr approximately 90,000 heat shock protein, hsp90. hsp90 is expressed under physiological conditions in mammalian cells and is also present in reticulocyte lysate, as assessed by Western immunoblotting using specific anti-hsp90 antibodies. We have translated glucocorticoid receptor mRNA in reticulocyte lysates. The receptor synthesized under cell-free conditions also interacts with hsp90 both in the presence and absence of ligand, as determined by sucrose gradient centrifugation. The in vitro synthesized glucocorticoid receptor does not bind to DNA-cellulose but can be converted to a DNA binding form following labeling with dexamethasone and heat treatment. Thus, the glucocorticoid receptor is synthesized in a nonactivated form under cell-free conditions. These data indicate that the 9 S glucocorticoid receptor complex found in cytosol does not represent an artifact due to cell homogenization and supports the existence in vivo of the glucocorticoid receptor-hsp90 complex.     

Studies on the nuclear binding of steroid hormone-receptor complex; binding of liver and thymus dexamethasone-receptor complex and prostate dihydrotestosterone-receptor complex to nuclei from various tissues.

To examine the binding specificity of steroid hormone-cytoplasmic receptor complexes to nuclei, binding of 3H-dexamethasone (Dex)-liver, 3H-Dex-thymus and 3H-dihydrotestosterone (DHT)-prostate receptor complexes to nuclei from liver, prostate, thymus, spleen and kidney was studied. It was observed that a significant amount of steroid-receptor complexes was bound to any nuclei used in the present study and the extent of the binding of receptor complexes to nuclei from homologous tissues was not always greater than that to nuclei from heterogenous tissues. However, a significant portion of the 3H-Dex-liver and 3H-DHT-prostate receptor complexes was not absorbed by nuclei from kidney, spleem, and thymus, and the unabsorbed complexes were efficiently bound to liver and prostate nuclei. The results obtained indicate that two types of receptor complex with regard to nuclear binding were present in cytosols of liver and prostate; one binds to nuclei from kidney, spleen, thymus, liver and prostate and the other does not bind to nuclei from kidney, spleen and thymus but does bind to nuclei of liver and prostate. The latter type of receptor complex was not observed in the cytosol from the thymus.     

The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).     

Functions of calcium in the interaction of progesterone-receptor complex with nuclear components

Chick oviduct cytosol [³H]progesterone-receptor complex treated with 30 mm Ca²⁺ at 0 °C demonstrated a twofold greater binding to isolated chick oviduct nuclei or DNA-cellulose than such complexes activated thermally (25 °C). Divalent ions such as Mg²⁺ and Mn²⁺ were unable to mimic the effect of Ca²⁺ under identical conditions. The capacity of the Ca²⁺-treated progesterone-receptor complex to bind to nuclei or DNA-cellulose reached a peak within 45 min of Ca²⁺ treatment of the complex at 0 °C. This binding gradually declined as a function of incubation time and after 24 h at 0 °C no significant binding was observed. The Ca²⁺- and heat-treated chick oviduct [³H]progesterone-receptor complex was also characterized by DEAE-cellulose and agarose gel nitration chromatography. While heat-activated receptor could be resolved into A and B subunits on DEAE-cellulose, the receptor exposed to Ca²⁺ for 45 min at low temperature yielded the “A” subunit and a broad peak with poor affinity for the anion exchanger. The peak corresponding to “B” subunit was not discernible. The broad peak which eluted before the A peak was subsequently resolved by agarose gel filtration into receptor forms IV and V as described previously by Sherman et al. (M. Sherman, S. Atienza, J. Shansky, and L. Hoffman, 1974, J. Biol. Chem., 249, 5351–5363; M. Sherman, L. Pickering, F. Rollwagen and L. Miller, 1978, Fed. Proc., 37, 167–173). Again DEAE-cellulose chromatography of the progesterone-receptor complex treated as long as 24 h at 0 °C with Ca²⁺ revealed a poorly bound peak which on agarose gel filtration corresponded exclusively to form V. A correlation was apparent between an increase in form V and a gradual decrease in the binding capacity of the Ca²⁺-treated steroid-receptor complex to nuclei, DNA-cellulose, or DEAE-cellulose filters.Based on these findings, I postulate that Ca²⁺ has a functional role in the mechanism of progesterone action in chick oviduct. Firstly, it enhances a low temperature, time dependent binding of the progesterone-receptor complex to chick oviduct nuclear components, and subsequently promotes, by possible activation of endogenous protease(s) the cleavage of the receptor subunits.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.