Physicochemical parameters affecting the charcoal adsorption assay for quantitative retinoid-binding measurement.

The different parameters affecting the accuracy and reliability of the dextran-coated charcoal adsorption assay for characterization of retinoic acid receptors ligand binding activity were investigated. Using dextran-coated charcoal (DCC) at a final 10 mg/ml concentration, an efficient adsorption of free [3H]retinoic acid was observed with a yield in the range 99.2 to 99.

Hydrodynamic characterization and DNA-binding properties of the liganded and unliganded forms of the retinoic-acid receptor alpha from human HL-60 cells.

The hydrodynamic parameters of the retinoic-acid receptor from human myeloblastic leukemia HL-60 cells were accurately investigated. The ligand-bound retinoic-acid receptor (RAR) has a Stokes radius of 3.5 nm when analyzed by size-exclusion chromatography.

A protein kinase C-dependent activity modulates retinoic acid-induced transcription.

The retinoic acid receptors (RARs) and retinoid X receptors, which are members of the nuclear receptor family, mediate the effects of vitamin A derivatives on cellular growth and differentiation. The protein kinase C isozyme family also controls these processes in response to extracellular stimuli. We have investigated the relationship between these two signal transducing pathways using gene transfer techniques.

Purification and functional characterization of the ligand-binding domain from the retinoic acid receptor alpha: evidence that sulfhydryl groups are involved in ligand-receptor interactions.

The pGEX-2T expression vector was used to produce the ligand-binding domain from the human retinoic acid receptor alpha (hRAR alpha LBD) in Escherichia coli. The resulting fusion protein, containing the glutathione S-transferase separated from the truncated receptor (hRAR alpha 186-462) by a thrombin cleavage site, was purified with use of affinity chromatography on immobilized glutathione. A 90% homogeneity was obtained, with a specific activity of 100 pmol/mg and an overall 10% yield.

Biochemical and immunological evidence that an acidic domain of hsp 90 is involved in the stabilization of untransformed glucocorticoid receptor complexes.

Polyclonal antibodies (AS 232-266) have been raised against the 232-266 amino acid sequence of the mouse hsp 84. This sequence possesses 54% acidic residues. AS 232-266 react with both the denatured and the free native murine hsp 84, but not with the bound hsp 84 present in the untransformed glucocorticoid receptor complexes (GR).

Binding of RU486 and deacylcortivazol to the glucocorticoid receptor is insensitive to sulfhydryl-modifying agents.

The differential sensitivity of the rat liver glucocorticoid receptor (GR) to sulfhydryl group modifying agents when bound to various agonist and antagonist ligands was studied. [3H]Triamcinolone acetonide (TA) binding was completely abolished by previous treatment of the unbound receptor with various N-alkylmaleimides. On the contrary, [3H]RU486 binding was only slightly affected by treatment with N-ethylmaleimide (NEM) and more significantly decreased with maleimides bearing bulky substituents.

Steroid receptor exchange assay in the presence of acetonitrile: application to the study of glucocorticoid- and anti-glucocorticoid-receptor complexes.

Acetonitrile was used to modify the binding parameters of glucocorticoid-receptor complexes. Acetonitrile (8%) caused a striking increase of the rate constant of dissociation of non-transformed [3H]triamcinolone and [3H]RU 486 receptor complexes. The latter complexes appeared significantly less sensitive to acetonitrile than the former.

Improvement in glucocorticoid receptor binding affinity concomitant to shift from antagonist to agonist activity in a series of 17 beta-carboxamide derivatives of dexamethasone.

Modification of the 17 beta-side chain of the synthetic glucocorticoid agonist dexamethasone by periodic oxidation and subsequent coupling to various primary amines yield secondary 17 beta-carboxamide derivatives displaying antiglucocorticoid activity in vitro, but not in vivo. To obtain more potent antiglucocorticoids, new secondary and tertiary 17 beta-carboxamide derivatives were synthesized. Although they displayed an improved affinity for the glucocorticoid receptor in rat thymus cytosol and antiglucocorticoid activity in rat hepatoma (HTC) cells, these new compounds were again devoid of in vivo antiglucocorticoid activity in the rat.

Study of the heteromeric structure of the untransformed glucocorticoid receptor using chemical cross-linking and monoclonal antibodies against the 90K heat-shock protein.

The untransformed rat glucocorticoid receptor is assumed to be a hetero-oligomeric complex, containing a non-steroid binding component, the 90K heat-shock protein (HSP 90). Direct measurement of its molecular weight by chemical cross-linking provides new evidence for a trimeric structure with a Mr of ca. 270,000.

Association of the glucocorticoid receptor binding subunit with the 90K nonsteroid-binding component is stabilized by both steroidal and nonsteroidal antiglucocorticoids in intact cells.

The interaction of various antiglucocorticoids with the glucocorticoid receptor from intact rat thymocytes was investigated. Reversible antiglucocorticoids (RU 486, cortexolone, progesterone) underwent more limited nuclear transfer than potent glucocorticoids (dexamethasone, triamcinolone acetonide, progesterone). This behavior was correlated with an impeded dissociation of cytosolic antiglucocorticoid receptor complexes preformed in intact cells, as assayed by high-performance size exclusion chromatography in physiological conditions (i.

Inhibition of glucocorticoid receptor transformation, subunit dissociation, and temperature-dependent inactivation by various N-substituted maleimides.

A series of N-substituted maleimides were synthesized, and their effect on the activation to the DNA binding state of the rat liver glucocorticoid receptor was studied. Unactivated (preincubated at 0 degrees C) cytosolic [3H]triamcinolone acetonide-receptor complexes were pretreated with various N-alkylmaleimides at 0 degrees C and then heated at 25 degrees C and assayed for DNA-cellulose binding. No inhibition of the DNA binding activity was observed with either N-ethylmaleimide or N-substituted maleimides bearing an ionizable substituent, like N-(omega-carboxyalkyl)maleimides and N-[2-(trimethylammonio) ethyl]maleimide.

RNA binding to the untransformed glucocorticoid receptor. Sensitivity to substrate-specific ribonucleases and characterization of a ribonucleic acid associated with the purified receptor.

The cytosolic untransformed molybdate-stabilized glucocorticoid-receptor complex from rat liver was eluted as a heterogenous peak containing two components with Stokes radii (Rs) of 8.3 nm and 7.1 nm when analyzed by size-exclusion HPLC even in the absence of molybdate.

RU 486 stabilizes the glucocorticoid receptor in a non-transformed high molecular weight form in intact thymus cells under physiological conditions.

When incubated at 37 degrees C for 1 h with intact rat thymocytes [3H]RU 486 underwent only partial nuclear transfer since more than 65% of the receptor bound radioactivity was still cytosolic (versus less than 10% for [3H]triamcinolone acetonide). Moreover when prepared and assayed in physiological buffers, i.e.

Improvements in the purification of an antisteroid antiserum resistant to conventional immunoadsorption chromatography.

Antibodies against dexamethasone, a synthetic steroid, have been induced in rabbits immunized with a 3-carboxymethyloxime dexamethasone derivative conjugated to bovine serum albumin. The antiserum displaying the highest affinity for dexamethasone (KD = 0.5 nM) appeared to be resistant to purification on an agarose matrix bearing the same 3-carboxy-methyloxime dexamethasone derivative.

RU 486 stabilizes a high molecular weight form of the glucocorticoid receptor containing the 90K non-steroid binding protein in intact thymus cells.

The interaction with the glucocorticoid receptor of RU 486, a recently described antiglucocorticoid, was investigated in intact cells. When incubated at 37 degrees C with intact rat thymocytes [3H] RU 486 underwent negligible nuclear transfer. Moreover when assayed in physiological buffers, i.

Improved Stokes radius measurement of the glucocorticoid receptor using TSK G4000SW and TSK G3000SW high-performance size-exclusion columns. Analytical and preparative applications.

The Stokes radius of the rat liver glucocorticoid receptor was determined using TSK G3000SW and TSK G4000SW high-performance size-exclusion columns. The accuracy of the calibration graph for proteins larger than 6 nm on the TSK G4000SW column allowed the resolution of a heterogeneous structure for the cytosolic untransformed receptor, giving two forms with Rs values of 8.3 and 7.

Evidence for an increased glycation of IgG in diabetic patients.

The levels of non-enzymatically glycated total plasma proteins, albumin and IgG were determined in diabetic and non-diabetic patients using affinity chromatography on boronate-agarose gels. A significant increase in both glycated albumin and IgG, and in total glycated plasma proteins was demonstrated. A good correlation between glycated albumin and glycated hemoglobin was observed, while the correlation between glycated IgG and glycated albumin (or hemoglobin) was lower.

Microheterogeneity of agonist and antagonist glucocorticoid receptor complexes detected by isoelectric focusing and modifications induced by receptor activation.

Rat-liver glucocorticoid receptor was incubated with either [3H]triamcinolone acetonide or [3H]RU 486, a well known antiglucocorticoid. Once formed, the steroid-receptor complexes were analyzed by isoelectric focusing in agarose gel slabs. A careful slicing of the receptor tracks revealed the presence of three distinct radioactive peaks focused at the following pI values: 5.

[Preparation of an anti-idiotypic antibody directed against anti-dexamethasone antibody and demonstration of its ability to acknowledge the receptor of glucocorticoids].

By using a 3-carboxymethyloxime dexamethasone derivative coupled to bovine serum albumin we have prepared specific anti-dexamethasone antibodies in rabbits. These antibodies were then purified by affinity chromatography and administered to a second set of rabbits. One of them produced anti-idiotypic antibodies able to impede the [3H] dexamethasone binding of the initial anti-dexamethasone antibodies and to displace the [3H] dexamethasone-antibodies complexes towards high molecular weight species in gel filtration experiments.

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography.

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera.

Physical characterization of the activated and non-activated forms of the glucocorticoid-receptor complex bound to the steroid antagonist [3H]RU 486.

We have compared the physicochemical characteristics of the non-activated (molybdate stabilized) glucocorticoid-receptor complex bound either to [3H]triamcinolone acetonide or to the antagonist [3H]RU 486 with those of the activated (25 degrees C preheated) complexes. The level of activation was measured by a DNA cellulose assay. The physicochemical features of the steroid-receptor complexes were analyzed by various techniques including high-performance size exclusion chromatography, sucrose gradient sedimentation and high-performance DEAE ion exchange chromatography.

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex.

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.

Covalent chromatography by thiol-disulfide interchange of the highly-purified non-transformed rat liver glucocorticoid-receptor.

Highly-purified non-transformed rat liver [3H]triamcinolone acetonide-receptor complex was shown to be covalently adsorbed on activated thiol sepharose 4B, a reactive sulfhydryl matrice. Elution by mercaptoethanol in excess and inhibition of binding by previous treatment of the complex with N-ethylmaleimide clearly demonstrated the specificity of the binding by thiol disulfide interchange. The transformed [3H]triamcinolone acetonide-receptor complex, partially purified by DNA-cellulose chromatography, was also retained on activated thiol sepharose 4B.

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures.

A series of N-alkylmaleimides was shown to inactivate effectively the rat liver glucocorticoid receptor at neutral pH. A partial purification of the unbound cytosolic receptor by protamine sulfate precipitation and a careful stabilization of the essential thiol by dithiothreitol and sodium molybdate before the alkylation step appeared essential to obtain pseudo-first-order kinetics. Moreover, performing the experiment at -12 degrees C in buffer containing 40% glycerol as antifreeze agent resulted in increased receptor stabilization and a slowing-down of the inactivation process, which could then be more accurately studied.