Precocious induction of tryptophan dioxygenase by glucocorticoid in suckling rats and correlation with change in glucocorticoid receptor

When young rats (less than 14 days old) were treated once a day for 2 days with 100 micrograms/100 g body weight of dexamethasone, their liver cytosol showed a sharp new peak of glucocorticoid binding protein (peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography. When the young rats were given a single injection of the hormone, the chromatogram showed a dominant peak of binding protein (peak B), eluted with 0.07 M NaCl, which was similar to that in untreated rats. The appearance of peak C on two treatments of young rats with hormone was confirmed by both in vivo and in vitro labeling and also studies on the nuclear fraction. Peaks B and C were specific hormone-binding proteins as shown with excess unlabeled hormone. The appearance of peak C was concomitant with the precocious induction of tryptophan dioxygenase in the liver of the young rats, and pretreatment with two injections of dexamethasone were necessary for maximal enzyme induction. On the other hand, in adult rats a single injection of dexamethasone (of 20 micrograms/100 g body weight or more) was enough to cause the appearance of peak C and induce tryptophan dioxygenase activity maximally; an additional injection of the hormone did not change the chromatographic pattern of the specific binding or the enzyme activity. For this effect in young rats, dexamethasone could not be replaced by other hormones such as glucagon, growth hormone, thyroid hormones, insulin, sex steroids or short-acting glucocorticoid.     

Physicochemical characterization of a new glucocorticoid receptor

A new glucocorticoid-binding protein (Peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography was identified previously in the rats subjected to stress or treated with glucocorticoid (100 micrograms/100 g body wt.), while the 'classic' glucocorticoid receptor (Peak B) eluted with 0.07 M NaCl was found predominantly in untreated rats. The new glucocorticoid-binding protein, Peak C, was characterized by Scatchard analysis and competition with other steroids as a glucocorticoid receptor. The saturation curve of Peak C for dexamethasone was sigmoidal, whereas that of Peak B was hyperbolic. The Hill coefficient was 1.0 for Peak B and 3.1 for Peak C. These results show that Peak C has multiple binding sites. Peak C bound specifically to only natural or synthetic glucocorticoids, whereas Peak B bound not only to glucocorticoids but also to progesterone and aldosterone. Peak C was far more labile than Peak B, its binding activity decreasing 80% when it was incubated for 30 min at 25 degrees C. The molecular sizes of these two peaks (B and C) were similar, being about 90 000-100 000 as determined by Sepharose 6B column chromatography at high ionic strength (0.34 M KCl). The hormone-receptor complex of Peak C bound to rat liver chromatin specifically, but did not bind to calf thymus DNA. The complex of Peak B bound to not only the chromatin but also calf thymus DNA. Peak B reacted well with antiserum to the 'classic' glucocorticoid receptor, but Peak C did not react with this antiserum. These results indicate that Peak C is a different glucocorticoid receptor protein from Peak B, or classic glucocorticoid receptor, and plays physiologically important roles as a glucocorticoid receptor mediating the action of the hormone at a high level.     

Physicochemical characterization of a new glucocorticoid receptor

A new glucocorticoid-binding protein (Peak C) eluted with 0.14 M NaCl on DEAE-cellulose chromatography was identified previously in the rats subjected to stress or treated with glucocorticoid (100 μg/100 g body wt.), while the ‘classic’ glucocorticoid receptor (Peak B) eluted with 0.07 M NaCl was found predominantly in untreated rats. The new glucocorticoid-binding protein, Peak C, was characterized by Scatchard analysis and competition with other steroids as a glucocorticoid receptor. The saturation curve of Peak C for dexamethasone was sigmoidal, whereas that of Peak B was hyperbolic. The Hill coefficient was 1.0 for Peak B and 3.1 for Peak C. These results show that Peak C has multiple binding sites. Peak C bound specificially to only natural or synthetic glucocorticoids, whereas Peak B bound not only to glucocorticoids but also to progesterone and aldosterone. Peak C was far more labile than Peak B, its binding activity decreasing 80% when it was incubated for 30 min at 25°C. The molecular sizes of these two peaks (B and C) were similar, being about 90 000–100 000 as determined by Sepharose 6B column chromatography at high ionic strength (0.35 M KCl). The hormone-receptor complex of Peak C bound to rat liver chromatin specifically, but did not bind to calf thymus DNA. The complex of Peak B bound to not only the chromatin but also calf thymus DNA. Peak B reacted well with antiserum to the ‘classic’ glucocorticoid receptor, but Peak C did not react with this antiserum. These results indicate that Peak C is a different glucocorticoid receptor protein from Peak B, or classic glucocorticoid receptor, and plays physiologically important roles as a glucocorticoid receptor mediating the action of the hormone at a high level.     

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.     

Application of high-performance ion-exchange chromatography to the analysis of cytosolic glucocorticoid receptor

The use of high-performance ion-exchange chromatography (HPIEC) on a Mono Q column was investigated for the analysis of glucocorticoid receptor. In the presence of 10 mM sodium molybdate, both liganded and unliganded glucocorticoid receptor were eluted as a single and sharp peak (0.32 M NaCl). In the absence of molybdate and after exposure to heat and salt, another peak of specifically bound radioactivity was eluted with 0.08 M NaCl. When HPIEC was performed in the absence of molybdate, two molecular forms of the liganded receptor were detected which eluted with 0.08 M NaCl (Stokes' radius Rs = 5.1 nm, s20,w = 4.6 S, calculated mol. wt Mr approximately 100,000) and 0.32 M NaCl (Rs = 7.3 nm, S20,w = 9.0 S, calculated Mr approximately 280,000). Analysis of both forms with mini-columns of DNA-Ultrogel, DEAE-Trisacryl and hydroxylapatite (HA-Ultrogel) confirmed the identity of the two peaks with transformed and non-transformed glucocorticoid-receptor complexes. These results suggest that HPIEC may provide a useful tool for the rapid resolution and quantification of receptor molecular forms.     

Characterization of testosterone binding protein of rat liver cytosol

Testosterone binding protein from rat liver cytosol, which had been incubated with [3H]testosterone followed by treatment with dextran-coated charcoal, was analyzed by DEAE-cellulose and phosphocellulose chromatography. On DEAE-cellulose chromatography, two distinct peaks of radioactivity were eluted at 0.07 M and 0.19 M KCl, both sedimented in 4 S regions. Phosphocellulose chromatography resulted in a broad peak at 0.08 M KCl, with a shoulder at 0.04 M KCl, both sedimented at 4 S. These findings indicated that testosterone binding protein consists of two types of components each with 4 S.     

Activity of cAMP-dependent protein kinases and cAMP-binding proteins from rat renal cytosol upon dehydration

The activity of cAMP-dependent protein kinases, cAMP binding and the spectrum of cAMP-binding proteins in renal papillary cytosol of intact rats and of rats kept on a water-deprived diet for 24 hours were investigated. It was found that the stimulation of protein kinases by 10(-6) M cAMP in the experimental group was significantly higher than in the control one. On DEAE-cellulose chromatography, the position of peaks of the specific cAMP binding corresponded to those of the regulatory cAMP-dependent protein kinases type I and II. Under these conditions, more than 80% of the binding activity in intact animals was localized in peak II, whereas in rats kept on a water-deprived diet over 60% of the binding activity was localized in peak I. The total binding activity of cytosol in experimental animals remained unchanged is compared to intact rats. It is suggested that in renal papilla dehydration is accompanied by the induction of synthesis of regulatory subunits of cAMP-dependent protein kinase type I.     

Leupeptin inhibits the transformation of glucocorticoid receptor

The effect of leupeptin upon the transformation of the glucocorticoid receptor was tested. When the labeled receptor was treated with heat or high salt in the presence of leupeptin, the binding to DNA-cellulose decreased in a dose-dependent manner. We observed 50% inhibition with about 40 mM leupeptin. The addition of leupeptin after the transformation procedures did not inhibit the binding to DNA-cellulose. In gradient centrifugation, 40 mM leupeptin retained approximately 10S, untransformed form. Elution profiles from DEAE-cellulose showed the preservation of the peak eluted with 0.2 M KCl, corresponding to the untransformed form. These results indicate that leupeptin might have the similar effects to molybdate in regard to blocking the transformation of rat liver glucocorticoid receptor, though the effects with leupeptin were not as great as those seen with molybdate.     

The physico-chemical properties of the AtT-20 cell's glucocorticoid receptor during depletion

Glucocorticoid agonists decrease the number of glucocorticoid receptors in the cloned AtT-20 mouse pituitary tumor cell. To investigate whether the structure of the receptor is altered during this process, we monitored the physico-chemical properties of the nuclear and cytosolic receptors undergoing depletion. Agarose chromatography, DEAE-cellulose chromatography and sucrose gradient ultracentrifugation were employed. Cells were sampled after 2, 24, 48 and 96 h incubation with 10 nM tritiated triamcinolone acetonide. Agarose chromatography yielded, in each case, a single receptor-containing peak that had a Stokes radius of 5.8 nm. Nuclear and cytosolic glucocorticoid receptors from each preparation eluted from DEAE-cellulose as a single, symmetric peak at a KCl concentration of 0.075 M. Sucrose gradient ultracentrifugation of all samples also yielded only a single peak. For each technique the amount of receptor recovered was inversely related to the length of intact cell incubation. Thus, depletion of the glucocorticoid receptor is not accompanied by observable changes in its size, surface charge or hydrodynamic properties. These results suggest that the first step of agonist-induced glucocorticoid receptor depletion in the AtT-20 cell involves the loss or alteration of the receptor's steroid-binding site.     

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.     

An isozyme of acid alpha-glucosidase with reduced catalytic activity for glycogen

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0.08, 0.12, and 0.17 M NaCl, whereas the two peaks of the variant, with 0.01 and 0.06 M NaCl. The pH optimum and thermal denaturation at 57 degrees C were the same in all enzyme peaks of both isozymes. Rabbit antiacid alpha-glucosidase antibodies produced against the common isozyme were found to cross-react with both peaks of the variant isozyme. The two isozymes shared antigenic identity and had similar Km's with maltose as substrate. Normal substrate saturation kinetics were observed with the common isozyme when glycogen was the substrate, but the variant produced an S-shaped saturation curve indicating a phase of negative and positive cooperativity at low and high glycogen concentrations, respectively. The activity of the variant was only 8.6% and 19.2% of the common isozyme when assayed with nonsaturating and saturating concentrations of glycogen, respectively. A similar rate of hydrolysis of isomaltose by both isozymes was found indicating that the reduced catalytic activity of the variant isozyme toward glycogen is not the result of a reduced ability of this enzyme to cleave the alpha-1,6 linkages of glycogen.     

Comparison of pyridoxal phosphate and 0.4 M KCl-extracted nuclear glucocorticoid receptors in HeLa S3 cells

A comparison of the physicochemical properties between pyridoxal 5'-phosphate- and 0.4 M KCl-extracted nuclear glucocorticoid receptors has been made utilizing HeLa S3 cells as a source of receptor. Both pyridoxal 5'-phosphate/NaBH4-reduced and 0.4 M KCl-extracted receptors sedimented as approximately 3.5-4.5 S species in 5-20% sucrose gradients containing 0, 0.15, and 0.4 M KCl. Under low-ionic-strength buffer conditions, pyridoxal 5'-phosphate-extracted receptor elutes close to the void volume of a Sephacryl S-300 gel-exclusion column. Increasing the [KCl] of the column to 0.4 M resulted in the elution of receptor with a Stokes radius of 58 A and calculated Mr = 96,000. Nuclear receptors extracted with 0.4 M KCl also formed a large-molecular-weight complex which eluted close to the void volume of the gel-exclusion column. Increasing the [KCl] to 0.4 M had the effect of shifting this receptor form to a species which had a Stokes radius of 62 A and calculated Mr = 89,700. Ion-exchange analysis of nuclear-extracted receptors revealed that 0.4 M KCl-extracted receptors exhibited considerable charge heterogeneity, whereas pyridoxal 5'-phosphate-extracted receptors did not. Pyridoxal 5'-phosphate-extracted receptors (approximately 86%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M; approximately 14% of the receptors had little affinity for DEAE-cellulose. Pyridoxal phosphate-treated receptors had little affinity for hydroxylapatite, phosphocellulose, and DNA-cellulose. The predominant form of 0.4 M KCl-extracted nuclear receptors (approximately 78%) eluted from DEAE-cellulose between 0.05 and 0.15 M KCl, a position coincident with "activated" glucocorticoid receptors. The remaining receptor fraction (approximately 22%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M, a position coincident with "unactivated" glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Purification of rat adipose tissue lipoprotein lipase by affinity chromatography.

Lipoprotein lipase (EC 3.1.1.3) from rat adipose tissue was purified by affinity chromatography with heparin-Sepharose. Elution was carried out with buffered solutions of increasing NaCl molarity. Proteins without affinity for heparin were eluted with 0.5 M NaCl, while lipoprotein lipase activity was eluted as two peaks with 1.16 M NaCl (In earlier work on human adipose tissue (Etienne et al. (1974) C.R. Acad. Sc. Paris 279, 1487-1490) two fractions with lipoprotein lipase activity were also obtained). Phospholipase activity was detected in the fraction eluted with buffered 0.5 M NaCl and containing proteins without affinity for heparin. On feeding the fasting rats with fresh cream or glucose two peaks were also obtained, but the first peak had clearly increased while the second one had remained virtually unchanged.     

Ammonium sulfate precipitation of the glucocorticoid receptor from rat liver

The transformed glucocorticoid receptor (GR) from rat liver precipitated at 30% saturation of ammonium sulfate and sedimented at 4.3 S on glycerol gradient centrifugation, whereas the nontransformed GR precipitated at higher concentrations of ammonium sulfate (40-50% saturation) and sedimented at 8.6 S on a gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that heat shock protein 90 (hsp 90) precipitated at 40-50% saturation of ammonium sulfate. Moreover, hsp 90 and the nontransformed GR were eluted from DEAE high performance ion-exchange chromatography at similar salt concentrations (0.22-0.23 M NaCl), whereas the transformed GR was eluted at 0.1 M NaCl. Therefore, hsp 90 seems to be responsible for the surface charge characteristics of the nontransformed GR.     

Solubilization, separation, and partial characterization of histamine H1 and H2 receptors from calf thymocyte membranes.

Histamine membrane receptors are defined as either H1 (blocked by diphenhydramine-like antagonists) or H2 (blocked by cimetidine-like agents). We now report the solubilization, separation, and partial characterization of specific H1 and H2 membrane receptors from calf thymocytes. Membrane fragments were incubated with [3H]histamine either alone or with unlabeled histamine, diphenhydramine, or cimetidine. Maximal specific binding occurred with incubation at 37 degrees C for 2 h at a concentration of 5 x 10(-6) M [3H]histamine. Labeled receptors were solubilized from membranes with 0.3 M KCl and 1% Nonidet 40. Chromatography of the solubilized labeled receptors on ion exchange columns revealed two classes of receptor. One class bound to DEAE-cellulose and eluted as a sharp peak at 0.15 M NaCl/Pi. The other bound to phosphocellulose and eluted as a sharp peak at 0.55 M NaCl/Pi. Initial incubation of the membranes in the presence of the H1 receptor antagonist diphenhydramine virtually abolished the DEAE-cellulose peak, while incubation with cimetidine, the H2 receptor antagonist, blocked the phosphocellulose peak. We conclude that H1 and H2 histamine receptors are physically separable and can be defined by their ability to bind to either DEAE-cellulose or phosphocellulose.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

Identification and properties of renal mineralocorticoid receptors in relation to glucocorticoid binders in rat liver and kidney.

The binding of the natural mineralocorticoid aldosterone and the glucocorticoid corticosterone to macromolecules in rat liver and kidney cytoplasmic fractions was compared by various chromatographic procedures. Equilibration of kidney cytosol with 10nM-aldosterone, either alone or in the presence of a competing steroid, was ideal for ionexchange chromatography of DEAE-cellulose DE-52, and revealed the presence of four sorts of binding components. One of these, eluted in the 0.001M-phosphate pre-wash, and another, less abundant, forming a peak at 0.006M-phosphate, did not bind corticosterone at equimolar concentrations, and appear to constitute the mineralocorticoid-specific 'MR' receptor in rat kidney. They could not be detected in the liver. Radioactivity eluted in the 0.02 and 0.06M-phosphate regions on DEAE-cellulose DE-52 appears to be due to [3H]aldosterone binding to glucocorticoid-specific 'GR' receptors and to transcortin respectively, since labelling was greater with corticosterone even at 10 nM than with the mineralocorticoid at 100nM and since [14C]corticosterone bound to blood serum transcortin was always co-chromatographed in the 0.06M-phosphate region. These two components appear to be identical with those in the liver and could be labelled maximally only by 100nM-corticosterone. The separation between specific mineralo- and glucocorticoid-binding species was less clear when chromatography was attempted on DEAE-Sephadex A-50 columns, possibly because of disaggregation into subunits in the presence of the high KC1 concentrations required for elution. Competitive binding followed by filtration through Sephadex G-200 gel indicated that cellular MR binders, unlike GR receptors, exist mostly as high-molecular-weight aggregates, although both appear to exhibit a comparable monomeric molecular weight of approx. 67000.     

Developmental changes of glucocorticoid receptors in the rat pancreas

Glucocorticoids are known to play a role in the maturation of the exocrine pancreas. The exact mechanism of glucocorticoid action in pancreatic ontogeny is, however, not clear. The present study characterized and quantitated the binding of [3H]dexamethasone to cytosol fractions from pancreata of rats at various ages. Trunk blood samples from these rats were also checked for levels of free and bound corticosterone. Specific and saturable bindings for dexamethasone were found in pancreatic cytosol fractions from newborn suckling and adult rats. Competition studies showed a preference for steroids with glucocorticoid activity. Specific binding was relatively low in pancreatic cytosol from newly born and 1-day old pups. A significant rise was seen after day 15. Cytosolic binding capacities were greatest from pancreata obtained from pups at weaning (3rd to 5th weeks). Values then declined toward the adult level. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 7.3 (+/- 1.1) X 10(-8) M and number of binding sites equalled to 1.29 (+/- 0.18) X 10(-13) mole/mg of cytosolic protein in adult rat pancreas. Pancreata from 25- and 15-day old rats had Kds of 3.4 (+/- 0.8) X 10(-8) M and 2.7 (+/- 0.7) X 10(-8) M with the number of binding sites equal to 1.77 (+/- 0.21) X 10(-13) mole/mg protein and 1.31 (+/- 0.16) X 10(-13) mole/mg protein respectively. Total plasma corticosterone concentration was low before day 10. It rose significantly by day 15, peaked at day 25, and then declined after weaning. About 5-15% of corticosterone during weaning and about 20-30% before and after weaning were in the free form. The peak level of dexamethasone binding corresponded to an increase in the plasma corticosterone level during weaning. This suggests a close relationship between plasma corticosterone levels and pancreatic glucocorticoid receptors. Both may, therefore, play a role in pancreatic development in the rat.     

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.