Steroid-binding Activity Research Articles

Characterization and Functional Properties of the A and B Forms of Human Progesterone Receptors Synthesized in a Baculovirus System

Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [32Pi], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormone-dependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.

Creation of “super” glucocorticoid receptors by point mutations in the steroid binding domain.

Almost all modifications of the steroid binding domain of glucocorticoid receptors are known to cause a reduction or loss of steroid binding activity. Nonetheless, we now report that mutations of cysteine 656 of the rat receptor, which was previously suspected to be a crucial amino acid for the binding process, have produced "super" receptors. These receptors displayed an increased affinity for glucocorticoid steroids and a decreased relative affinity for cross-reacting steroids such as progesterone and aldosterone. The increased in vitro affinity of the super receptors was maintained in a whole cell bioassay. These results indicate that additional modifications of the glucocorticoid receptor, and probably the other steroid receptors, may further increase the binding affinity and/or specificity.

Structural and functional reconstitution of the glucocorticoid receptor-hsp90 complex.

Untransformed steroid receptors in cytosol preparations are associated with the 90-kDa heat shock protein hsp90, but the study of how hsp90 affects receptor function has been held back by the inability to reassociate steroid receptors with hsp90 in cell-free systems. Recently we showed (Dalman, F.C., Bresnick, E. H., Patel, P. D., Perdew, G. H., Watson, S. J., and Pratt, W. B. (1989) J. Biol. Chem. 264, 19815-19821) that glucocorticoid receptors translated in rabbit reticulocyte lysate bind to hsp90 at the termination of receptor translation. In this work we show that rabbit reticulocyte lysate promotes the temperature-dependent association of hsp90 with immunopurified mouse L cell glucocorticoid receptors. Reticulocyte lysate also promotes the temperature-dependent dissociation of hormone-free receptors from a prebound receptor-DNA complex. The glucocorticoid receptor is released from DNA in association with rabbit hsp90, and reconstitution of the receptor-hsp90 complex is accompanied by complete restitution of steroid binding activity and repression of DNA binding activity. This is the first time that transformation of a DNA-bound steroid receptor has been reversed and it raises the question of whether the same or a similar system is involved in the termination of transcriptional activation when steroid dissociates from DNA-bound receptors in intact cells.

Interaction between corticosteroid binding globulin and activated leukocytes in vitro

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.

Fluorescence investigation of the sex steroid binding protein of rabbit serum: steroid binding and subunit dissociation

The relationship between steroid binding and protein subunit interactions of rabbit sex steroid binding protein (rSBP) has been studied by steady-state and time-resolved fluorescence spectroscopy. The high-affinity (Ka approximately 10(8) M-1 at 4 degrees C), fluorescent estrogen d-1,3,5(10),6,8-estrapentaene-3,17 beta-diol [dihydroequilenin (DHE)] was used as a fluorescent probe of the steroid-binding site. Perturbation of the binding site with guanidinium chloride (Gdm.Cl) was monitored by changes in the steady-state fluorescence anisotropy of DHE as well as by changes in fluorescence quenching of DHE with acrylamide. The results of acrylamide quenching at 11 degrees C show that, while between 0 and 1 M Gdm.Cl the steroid-binding site is completely shielded from bulk solvent, there is decreased DHE binding. To study the subunit-subunit interactions, rSBP was covalently labeled with dansyl chloride in the presence of saturating 5 alpha-dihydrotestosterone (DHT), which yielded a dansyl-conjugated protein that retained full steroid-binding activity. The protein subunit perturbation was monitored by changes in the steady-state fluorescence anisotropy of the dansyl group. At 11 degrees C, the dansyl anisotropy perturbation, reflecting changes in global and segmental motions of the dimer protein, occurs at concentrations of Gdm.Cl above 1 M. The Gdm.Cl titration in the presence of steroids with equilibrium association constants less than 10(8) M-1 shows a plateau near 3 M Gdm.Cl at 11 degrees C; at this Gdm.Cl concentration, no DHE is bound. No plateau is observed at 21 degrees C. At higher Gdm.Cl concentrations, the dansyl fluorescence anisotropy decreases further and shows no steroid dependence. Recovery of steroid-binding activity (assayed by saturation binding with [3H]DHT), under renaturation conditions, is dependent on both steroid concentration and affinity. Both unlabeled and dansyl-labeled protein recovery the same amount of activity, and according to fluorescence anisotropy, dansyl-labeled rSBP re-forms a dimer upon dilution below 1 M or removal of Gdm.Cl. From the steroid requirement for recovery of steroid-binding activity, it appears that a conformational template is required for the dimeric protein to re-form a steroid-binding site with native-like properties.

Identification of lysine 134 in the steroid-binding site of the sex steroid-binding protein of human plasma.

The sex steroid-binding protein of human plasma SBP (or sex hormone-binding globulin, SHBG) was specifically inhibited with the alkylating affinity label, 17 beta-[[( 2-14C]bromoacetyl)oxy]-5 alpha-androstan-3-one. The natural ligand, 5 alpha-dihydrotestosterone, was shown to protect against inactivation and labeling. The steroid-binding activity of the protein was abolished when approximately 1 mol of label was incorporated into 1 mol of dimeric SBP. In order to identify and locate the labeled amino acid in the steroid-binding site, the steroidal portion of the bound label was first removed and the protein was digested with Achromobacter protease and subdigested with trypsin. Seven radioactive peptides were isolated, sequenced, and found to contain the common sequence QVSGPLTSXR. Residue X was identified as lysine-134 from the SBP amino acid sequence (Walsh, K. A., Titani, K., Kumar, S., Hayes, R., and Petra, P. H. (1986) Biochemistry 25, 7584-7590). The results indicate that only 1 of the 2 lysine-134 residues in the homodimer was labeled. This suggests that the steroid-binding site is constructed from an association of the two subunits in an AB to BA "sandwich" configuration with lysine-134 residue of one subunit on one surface near the D-ring and the lysine-134 of the other subunit at the opposite end of the steroid, or well away from the steroid-binding site. Although the nature of the data does not allow description of a specific role for lysine-134, its proximity to the 17 beta-OH of the steroid nucleus suggests participation in the binding process through direct or indirect hydrogen bonding.

A Role for Corticosteroid-Binding Globulin in Delivery of Cortisol to Activated Neutrophils*

In human blood, cortisol is transported by a plasma protein known as corticosteroid-binding globulin (CBG). As anticipated from primary structure comparisons of CBG and alpha 1-proteinase inhibitor (A1-PI), CBG acts as a substrate for neutrophil elastase. However, unlike A1-PI, CBG does not alter the activity of this enzyme, but is cleaved by it at a single location close to its carboxy-terminus, and this reduces its molecular size by 5 kDa with the concomitant release of more than 80% of CBG-bound cortisol. Three small molecular size fragments are detected after elastase cleavage, and carbohydrate analysis of these fragments suggests that they represent the same polypeptide fragment which has been differentially glycosylated. To assess the biological significance of these observations, CBG was incubated with either mononuclear cells or granulocytes obtained from patients with acute inflammation (sepsis) and from a normal volunteer. Only granulocytes from septic patients reduced the mol wt of CBG by about 5 kDa and destroyed its steroid-binding activity. Preincubation with A1-PI prevented this, which demonstrates that neutrophil elastase plays a key role in this event. These results suggest a physiological role for CBG in the delivery of cortisol to sites of inflammation.

Selective molybdate-directed covalent modification of sulfhydryl groups in the steroid-binding versus the DNA-binding domain of the glucocorticoid receptor.

Hydrogen peroxide produces all of the effects on glucocorticoid receptors that are produced by molybdate, including stabilization of the receptor 90-kDa heat shock protein (hsp90) complex (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). When the glucocorticoid receptor is exposed simultaneously to molybdate and peroxide at concentrations that are optimal for receptor stabilization if each agent is present alone, there is an irreversible loss of steroid binding activity. The effect is accompanied by a covalent modification of the receptor, which is demonstrated by an increase in its apparent Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of the receptor with the sulfhydryl-modifying reagents methyl methanethiosulfonate or N-ethylmaleimide prevents covalent modification, suggesting that cysteine moieties are the site of attack. The covalently modified receptor can still bind to DNA. Molybdate-peroxide treatment does not covalently modify the 15-kDa tryptic fragment containing the DNA-binding domain and 11 of the 20 cysteine moieties in the receptor. However, the 27-kDa tryptic fragment, which contains the steroid-binding domain and 5 cysteines, is covalently modified. The 27-kDa tryptic fragment is covalently modified by the molybdate-peroxide combination when [3H]dexamethasone 21-mesylate is covalently bound to Cys-644. This leaves some combination of 4 cysteines in the steroid-binding domain (628, 649, 671, and 742) as the modified groups. These modifications occur in a region of the receptor that is known to contain its sites of interaction with both hsp90 and molybdate, with the latter having a well-established avidity for sulfur. These observations raise the possibility that the covalent modification caused by the molybdate-peroxide combination represents a modification of sulfur ligands involved in molybdate stabilization of the receptor.

Effect of atp on the regulation of the steroid binding activity of the oestradiol receptor

We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity ( K a 3.3 × 10 8M −1) and lower sedimentation coefficient (4S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [γ 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [ 3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [ 3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [γ 32P]ATP and subsequently affinity labelled with [ 3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [ 3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed.

Developmental Changes in Testicular Inhibin and Androgen-Binding Protein during Sexual Maturation in the Cynomolgus Monkey, Macaca fascicularis*

Inhibin was measured by RIA in testicular extracts and plasma of cynomolgus monkeys during four stages of sexual maturation. Immunoactive inhibin levels were compared to those of another Sertoli cell secreted protein, androgen-binding protein (ABP). ABP steroid-binding (bioactive) activity was measured in testes and epididymal segments using the radiolabeled ligand [3H]dihydrotestosterone (DHT). Testicular immunoreactive inhibin concentrations were maximal in late prepubertal monkeys, 2.5-3.5 yr old, while the total testicular content of inhibin progressively increased with age into adulthood. Bioactive testicular ABP concentrations were maximal during the pubertal period of the cynomolgus monkey (3.5-4.0 yr old), while the total ABP content of the testes also increased with sexual maturation. Mean (+/- SE) plasma concentrations of inhibin and testosterone (T) in adults, 6-8 yr old (17.72 +/- 3.5 microliters inhibin equivalents/ml and 7.07 +/- 2.45 ng/ml T, respectively), were significantly higher (P less than 0.05 and P less than 0.001, respectively) than those in early prepubertal, juvenile monkeys, aged 1.5-2.5 yr (5.85 +/- 2.1 microliters inhibin equivalents/ml and 0.27 +/- 0.02 ng/ml T). The increased plasma levels of inhibin and T in adults were associated, respectively, with the increased inhibin and androgen contents of the testes in these same animals. The developmental changes in testicular steady state mRNA concentrations for the inhibin alpha-, beta A-, and beta B-subunits as well as ABP were examined during sexual maturation by Northern blot analysis using heterologous human cDNA probes. Densitometric analysis of the autoradiograms revealed that the inhibin alpha-subunit mRNA concentrations were higher than those of inhibin-beta A and -beta B and ABP mRNA during all stages of pubertal development. Although the relative concentrations of each inhibin subunit mRNA were decreased in the adult animals relative to those in the juvenile monkeys, the total amount of steady state mRNA for the subunits was greater than that in the immature animals. A similar situation existed for the ABP mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

The Methyltrienolone Binding Protein of Human Placenta Requires Nicotinamide Adenine Dinucleotide Cofactor(s) for Steroid Binding*

The methyltrienolone binding protein (MTBP) found in human placental cytosol was found to require a low molecular weight modulator for steroid binding activity. Purification and characterization of the modulating activity showed that NAD+ is the endogenous substance responsible for activating MTBP to a form capable of steroid binding. The hierarchy of potency of exogenously added nucleotides is NADH greater than NAD+ = NADPH = NADP+. An investigation of the tissue distribution of human MTBP demonstrated that MTBP binding activity was present in placenta and chorion but absent from amnion and umbilical cord. Preliminary studies showed that rat, mouse, and rabbit placenta do not contain MTBP and suggest that MTBP may be a species-specific protein.

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Biological half-life and transfer of maternal corticosteroid-binding globulin to amniotic fluid in the rabbit.

Rabbit corticosteroid-binding globulin (CBG) from the serum of pregnant and nonpregnant females differs in terms of charge microheterogeneity, and both forms were, therefore, radiolabeled and injected iv into 23- to 27-day pregnant rabbits (n = 6) to assess their biological half-lives and possible transfer to the fetal compartment. After an initially rapid distribution phase, the serum half-lives of both forms of [125I]CBG were essentially identical (approximately 13 h) and did not vary at different gestational ages. There was also no difference in the transfer of either form of [125I]CBG from maternal to fetal compartments in any of the animals studied. Moreover, [125I]CBG showed no sign of degradation and retained its steroid-binding activity in fetal urine and amniotic fluid. Twenty-two hours after administration of [125I]CBG to rabbits (n = 2) at 23 days gestation, its mean level in fetal urine (7 cpm/microliter) and amniotic fluid (2.5 cpm/microliter) was much higher than that in fetal blood (0.6 cpm/microliter). More importantly, the specific activities of [125I]CBG in fetal urine and amniotic fluid were comparable to that in maternal serum, and approximately 2 orders of magnitude higher than that in fetal serum. Taken together, these results suggest that CBG in fetal urine and amniotic fluid is largely of maternal origin, and that maternal CBG crosses the fetal kidney preferentially.

Steroid Binding Activity Is Retained in a 16-kDa Fragment of the Steroid Binding Domain of Rat Glucocorticoid Receptors

The steroid binding domain of the rat glucocorticoid receptor is considered as extending from amino acids 550 to 795. However, such a synthetic protein (i.e. amino acids 547-795; Mr approximately 31,000) has been reported to show very little affinity for the potent synthetic glucocorticoid dexamethasone. We now disclose that digestion of steroid-free rat glucocorticoid receptors with low concentrations of trypsin yields a single species, of Mr = 16,000, that is specifically labeled by dexamethasone 21-mesylate. This 16-kDa fragment retains high affinity binding for [3H]dexamethasone that is only approximately 23-fold lower than that seen with the intact 98-kDa receptor. Analysis of the protease digestion patterns obtained both with trypsin and with lysylendopeptidase C allowed us to deduce the proteolytic cleavage maps of the receptor with these enzymes. From these protease maps, the sequence of the 16-kDa fragment was identified as being threonine 537 to arginine 673. These results show that glucocorticoid receptor fragments smaller than 34 kDa do bind steroids and that the amino acids Thr537-Arg673 constitute a core sequence for ligand binding within the larger steroid binding domain. The much slower kinetics in generating the 16-kDa fragment from affinity-labeled receptors suggests that steroid binding causes a conformation change in the receptor near the cleavage sites.

Glucocorticoid receptor expression in receptorless mutants isolated from the human leukemic cell line CEM-C7.

The molecular basis for the loss of steroid binding activity in receptorless (r-) glucocorticoid-resistant (dexr) mutants isolated from the glucocorticoid-sensitive (dexs) cell line CEM-C7 was investigated. Although there was little binding of the reversibly associating ligand [3H]dexamethasone in r- mutants, labeling with the covalent affinity ligand [3H] dexamethasone 21-mesylate revealed significant amounts of a 92 kilodalton human glucocorticoid receptor (hGR) protein. Immunoblots of hGR protein in r- and normal cells showed that r- mutants expressed approximately half the amount of immunoreactive hGR protein seen in dexs cells. Comparison of the genomic organization of the hGR genes in normal and mutant cells revealed no discernable differences in the structure, or dosage, indicating that the r- phenotype was not the result of gross deletion or rearrangement of the hGR genes. In addition, r- cells expressed the same 7 kilobase mRNA as normal cells. More importantly, the amount of hGR mRNA expressed in r- cells was never significantly less, and in some cases was greater than, that seen in normal cells, indicating that the decrease in immunoreactive hGR protein seen in r- cells is not the result of loss of hGR mRNA expression. Taken together with the known mutation rate of the hGR gene(s) in these cells, these results suggest that the hGR genes in dexs CEM-C7 cells are allelic and that dexs cells express both a normal hGR protein and one with an altered steroid binding site. Furthermore, they suggest that the r- phenotype is acquired as the result of mutation within the coding region of the originally functional allele, leading to loss of ligand binding and expression of immunoreactive product.

Evidence That the 90-kDa Heat Shock Protein Is Necessary for the Steroid Binding Conformation of the L Cell Glucocorticoid Receptor

Using L cell glucocorticoid receptors that have been immunopurified by adsorption to protein A Sepharose with a monoclonal antireceptor antibody, we have developed an assay to study the requirements for maintenance of steroid-binding capacity. After rapid purification by immunoadsorption, heteromeric receptor complexes retain the ability to bind glucocorticoid hormone. When the receptor complexes are warmed at 20 degrees C, steroid-binding capacity is lost, and the 90-kDa heat shock protein (hsp90) dissociates from the receptor. The rates of both temperature- and salt-dependent dissociation of hsp90 parallel the rates of loss of hormone-binding activity. Molybdate and hydrogen peroxide stabilize the hsp90-receptor complex against temperature-dependent dissociation. Molybdate, however, is much more effective in stabilizing steroid-binding capacity than peroxide. Receptors that have been inactivated in the absence of molybdate or peroxide cannot be reactivated. Inactivation of steroid-binding capacity occurs in the presence or absence of reducing agent, and inactivation is not accompanied by receptor cleavage or dephosphorylation. Under no conditions does an hsp90-free receptor bind steroid. Receptor bound to hsp90 can be cleaved to the 27-kDa meroreceptor in the presence of molybdate with retention of both hsp90 and steroid-binding activity. These observations lead us to propose that hsp90 is necessary but not sufficient for maintaining a competent high affinity glucocorticoid-binding site. Although the 27-kDa meroreceptor fragment is not itself sufficient for a competent binding site, it is sufficient when it is associated with hsp90.

Characterization of the human sex hormone binding globulin (SHBG) gene and demonstration of two transcripts in both liver and testis.

A genomic cosmid clone for human sex hormone binding globulin (SHBG), a liver-secreted plasma glycoprotein that binds sex steroids, was isolated with a previously characterized liver cDNA as probe. Southern blot analysis of genomic DNA indicated that only one SHBG gene is present in the human haploid genome. A 3.8 Kb Xba I-fragment of the clone containing the entire coding region of SHBG was sequenced. The SHBG gene has 8 exons. The 5'-end preceding the translation start site had no TATA box or CAAT box promoter elements. Screening of a human testis cDNA library resulted in the isolation of two distinct cDNA forms. One cDNA was identical with the previously characterized liver SHBG cDNA, thus suggesting that human SHBG and the androgen binding protein (ABP) produced by Sertoli cells are coded for by the same gene. The second cDNA differed from the first by having exon I exchanged with a completely different sequence and exon VII deleted. An exon coding for the 5'-end of this cDNA was found in the cosmid clone 1.5 kb upstream of the first SHBG exon. Primer extension experiments showed the alternatively spliced transcript corresponding to the second cDNA to be present in both liver and testis. From the primary structure of this putative SHBG-gene-related protein, it may be deduced that it is a protein very different from SHBG and probably without steroid binding activity.

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. On the contrary, treatment with long-chain alkylmaleimides like N-heptylmaleimide resulted in significant inhibition. The highest inhibition was obtained with N-benzylmaleimide and, to a lesser extent, N-(ethylphenyl)-maleimide, whereas N-benzylsuccinimide was ineffective. Treatment of cytosol containing unactivated glucocorticoid complexes at 3 degrees C with N-benzymaleimide also prevents the temperature-mediated conversion of 8S receptor to 4S. Moreover, N-benzylmaleimide was able to inhibit the inactivation of the receptor steroid-binding activity caused by heat. N-Benzylmaleimide shares with molybdate ions the ability to inhibit glucocorticoid receptor activation, dissociation, and inactivation. However, their respective mechanisms of action are probably distinct, since their effects on receptor inactivation appear additive. It is suggested from the comparison of the various maleimides tested that the sulfhydryl groups essential for receptor activation and dissociation lie in a rather nonpolar environment including aromatic amino acid(s).

The studies on the loss and recovery of the steroid binding ability of rat liver glucocorticoid receptor

We examined the mechanism of loss and recovery of steroid binding activity (inactivation and reactivation) of the rat liver glucocorticoid receptor (GR) in the cell free condition. Male Sprague-Dawley rats were adrenectomized 2 days before the hepatectomy for the experiments, and were fed with normal diet and saline. The cytosol was prepared in 10mM phosphate buffer (pH 7.4) containing 10% glycerol (PG buffer). The nuclei were isolated by centrifuging at 800 x g for 20 min at 4 degrees C in 10mM phosphate buffer (pH 7.4) containing 0.32M sucrose, 3mM MgCl2, then centrifuged again at 40,000 x g for 20 min at 4 degrees C in 10mM phosphate buffer (pH 7.4) containing 1.8M sucrose, 3mM MgCl2. The glucocorticoid receptor was partially purified by affinity chromatography with DOC-Sepharose 6B, and column chromatography with Sephacryl S-300 and DEAE-cellulose in the presence of molybdate throughout the procedure. The steroid binding activity of the cytosol prepared in PG buffer was inactivated by following procedures; either 1) treatment with 1% activated charcoal, 2) dialysis against 500 volumes of PG buffer, 3) incubation at 37 degrees C for 30 min, or 4) incubation in high salt condition (0.4M KCl) at 0 degrees C for 16h. When the steroid binding activity was inactivated by incubation at 0 degrees C for 16hr and charcoal treatment, dithiothreitol (DTT) and NADPH were effective on reactivation, while glutathione (reduced form) was ineffective. Antipain increased the reactivated steroid binding activity in these cases. The steroid binding activity, which was inactivated by dialysis, was slightly recovered by DTT or NADPH, but antipain was ineffective. We could not reactivate steroid binding activity which was inactivated by incubation at 37 degrees C for 30 min or high salt (0.4M KCl) treatment with any reagent we tested. Leupeptin, however, partially prevented the high salt inactivation. When the cytosol prepared in PG buffer was labeled with 3H-dexamethasone (Dex) and then incubated at 25 degrees C for 30 min, 3H-Dex labeled steroid receptors was transformed and it also lost its steroid binding activity partially. DTT or NADPH increased steroid binding activity, but did not increase nuclear binding activity. Interestingly, by dialysis, the cytosol glucocorticoid receptors was not transformed in the PG buffer. We also examined the reactivation of steroid receptor using the partially purified receptor. To remove molybdate, the partially purified receptor was dialyzed against PG buffer. Large part of steroid binding activity was lost during the dialysis. DTT, however, reactivated steroid binding activity without molybdate, and also S-value of reactivated receptor was undistinguishable from the untransformed (molybdate stabilized) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Sex hormone binding globulin in saliva.

Measurement of salivary steroid hormone concentrations has frequently been advocated as a convenient alternative to plasma measurements. This is partly due to the belief that salivary steroid concentrations are a reliable reflection of the plasma free hormone level, a belief reinforced by earlier inability to demonstrate the presence of significant quantities of steroid binding proteins. Recent reports confirm that such quantities of these proteins are present in saliva and that they retain their steroid binding activity. We have measured sex hormone binding globulin (SHBG) and albumin in saliva from 14 men, 21 non-pregnant women and 36 pregnant women. No differences in the concentration of salivary albumin was evident in any of the groups studied whereas a significant difference in the concentration of SHBG was evident between men and non-pregnant women, and non-pregnant and pregnant individuals. Although much lower, salivary SHBG and albumin concentrations broadly reflect those found in plasma. Furthermore, a highly significant correlation existed between salivary SHBG and albumin concentrations in all groups studied. It now seems generally accepted that the albumin present in saliva arises from contamination by either traces of blood or gingival fluids. The close relationship between the concentrations of albumin and SHBG in saliva suggests that they both gain entry by a similar route. Furthermore, their presence may significantly influence the concentration of certain steroids in saliva, and this may explain the occasional failure of salivary steroid concentrations to accurately reflect the plasma free hormone levels.