pH-dependence of hormone-binding and enzymatic activity of estrophilic hydroxysteroid dehydrogenase from the rabbit liver

The effects of pH on the ability of NADP-dependent estrophilic hydroxysteroid dehydrogenase (HSD) from the soluble fraction of rabbit liver to bind steroids and catalyze their 3 alpha, 3 beta, 17 beta- and 20 alpha-oxidoreduction were studied. The pH optima for enzymatic transformations of various steroids were found to differ significantly by more than two units. These differences do not seem to be related to the localization of the modified group in the steroid molecule. Kinetic data suggest that pH influences the catalytic efficiency, steroid affinity for the protein and, perhaps, the degree of interdependence of steroid and cofactor binding to the protein. These assumptions were confirmed by the results of direct 3H-steroid-HSD binding studies. Furthermore, the maximal levels of binding of various steroids to the protein were found to occur at pH values differing by more than 4 units. Scatchard analysis revealed the effects of hydrogen ion concentrations both on the steroids affinity for the protein and on the concentration of steroid-binding sites of HSD. The data obtained are suggestive of some "superfluity" of the protein steroid-binding site which, in turn, ensures the multifunctionality of estrophilic HSD including a possibility of an alternative orientation of steroids in their binding site.     

Isolation of estrophilic fractions of hydroxysteroid dehydrogenase from rabbit liver

Estrophilic forms of rabbit liver cytosolic hydroxysteroid-dehydrogenase (HSD) were obtained as a highly purified preparations by means of fractionation with ammonium sulfate, gel-filtration, ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on estradiol-Sepharose and ion-exchange chromatography on DEAE-Toyopearl 650M. The protein express 4 different kinds of NADP-dependent activities: 3 alpha, 3 beta- and 17 beta-HSD activities with androgens and 20 alpha-HSD with progesterone as substrates. Revealed multiplicity of HSD enzymatic activity is demonstrated here for the first time. 17 beta-HSD activity of the protein preparations with estradiol is extremely low. Absence of a real metabolic activity of the protein with a ligand interacting with it rather intensively suggests that the isolated HSD forms can act not only as an enzyme, but also as a buffer-reserving mechanism for some steroids.     

The effect of cofactors on the binding of steroid substrates with extrophilic hydroxysteroid dehydrogenase in the rabbit liver

The isolated NADP (H)-dependent extrophilic hydroxysteroid dehydrogenase (EHSD) catalyzes oxidative-reductive reactions of 3 alpha-, 3 beta-, 17 beta-, and 20 alpha-hydroxy groups of androgens and progestagens, and binds these steroids and estrogens with relatively high affinities. In three series of experiments of the enzymatic kinetics, binding of 3H steroids to the protein, and inhibitory analysis an influence of cofactors on the affinity of steroid substrates and their analogs to the protein was demonstrated. A degree and direction of this influence are dependent on the steroid structure and cofactor form. It is proposed that cofactors may be important physiological regulators of enzymatic and steromoduline functions of the isolated EHSD.     

Spectrophotometric study of cytochrome P-450 (11 beta) interaction with physiological effectors

Using the optical absorbance spectroscopy method, the interaction of a number of biospecific ligands (steroids, adrenodoxin) with homogeneous cytochrome P-450 (11 beta) from bovine adrenal mitochondria was investigated. The parameters of the steroid-protein interaction in a number of substrates and products of the 11 beta- and 18 (19)-hydroxylation with the active site of cytochrome P-450 (11 beta) were determined. A sharp decrease in the cytochrome affinity for steroids upon the insertion of the first hydroxy group was observed, which provides for a predominant formation of monohydroxylated products from the substrate and minimum amounts of dihydroxylated ones, despite the presence of more than one position for the substrate hydroxylation by cytochrome P-450 (11 beta). Some structural elements of the steroid molecule were determined as any alterations in these strongly affect the enzyme affinity for the steroid. These structures are: 1) delta 4-3-oxo structure; 2) either 21-hydroxy group of pregnen steroids or the one fulfilling its functions, 17 beta-hydroxy or 17-oxo group of androsten steroids, and 3) the 11th position of all the substrates under study. It was shown that the binding of various substrates into stoichiometric (1:1) steroid-protein complexes provides a transition to high spin state from 30-40% (cortisol, corticosterone) to 90-95% (11-deoxycorticosterone) of hemoprotein iron. Using the experimental system containing individual cytochrome P-450 (11 beta) and adrenodoxin, as well as the steroid and nonionic detergent Tween 20, it was shown that the parameters of substrate binding and hemoprotein spin equilibrium did not differ from the corresponding parameters of the cytochrome-adrenodoxin dienzyme complex. The peculiarities of the multiligand interactions in the 11 beta-hydroxylase system, involving cytochrome, substrates and ferredoxin demonstrate some analogy with a bacterial camphor hydroxylase system and some differences from the mitochondrial system for the side chain cleavage of cholesterol.     

Molecular dynamic investigation of the interaction of supported affinity ligands with monoclonal antibodies

Diagnostics and therapeutic treatments based on monoclonal antibodies have been attaining an increasing importance in the past decades, but their large scale employment requires the optimization of purification processes. To obtain this goal, research is focusing on affinity chromatography techniques and the development of new synthetic ligands. In this work we present a computational investigation aimed at obtaining some guidelines for the rational design of affinity ligands, through the study of their interactions with both monoclonal antibodies (modeled as the FC domain of human IgG) and a model support material (agarose). The study was carried out performing molecular dynamics simulations of the support-spacer-ligand-IgG complex in explicit water. Binding energies between IgG and two supported ligands, a disubstituted derivative of trichlorotriazine and a tetrameric peptide, were determined with the linear interaction energy and MM-GBSA approaches. A detailed study of the possible binding sites of the considered ligands was performed exploiting docking protocols and MD simulations. It was found that both ligands bind IgG in the same site as protein A, which is the hinge region between the CH2 and CH3 domains of IgG. However this site is not easily accessible and requires a high mobility of the ligands. The energetic analysis revealed that van der Waals and electrostatic energies of interaction of the triazine ligand with the support are significant and comparable to those with the protein, so that they limit its capability to reach the protein binding site. A similar result was found also for the tetrameric peptide, which is however able to circumvent the problem; for steric reasons only two of its arms can interact at the same time with the agarose support, thus leaving the remaining two available to bind the protein. These results indicate that the interaction between ligand and support material is an important parameter, which should be considered in the computational and experimental design of ligands for affinity chromatography.     

A method for characterization of endogenous ligands to orphan receptors belonging to the steroid hormone receptor superfamily--isolation of progesterone from pregnancy plasma using progesterone receptor ligand-binding domain.

An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.     

Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa

The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.     

Structural requirements for the binding of dexamethasone to nuclear envelopes and plasma membranes

The specificity of dexamethasone binding sites on nuclear envelopes (NE) and plasma membranes (PM) was determined in competition studies with natural and synthetic steroids. The binding affinities for nuclear envelopes and plasma membranes were then correlated with the three-dimensional structures of the ligands. Three major factors are implicated in the ability of the steroid to bind to the membrane sites: (1) the separation between the terminal oxygen atoms substituted at atoms C3 and C17, or attached to the substituent at C17, is found to be longer than 10 A for the medium and high affinity steroids; (2) the beta-orientation of the oxygen atom in the C17-substituent to the D-ring is favored over alpha-orientation; and (3) bulky substituents and nontypical configurations are not accepted by the binding sites. A nearly linear correlation between the O3...O (substituted at C17) distance and the binding affinity of the tested steroids is observed; explanations for the lack of linear correlation of some steroids are given. A preliminary model for the interaction of steroids with these membrane sites is proposed which requires two hydrogen bonding regions that interact with the 2 oxygen atoms and some steric restriction sites that prevent the binding of steroids with large substituents. The hydrophobicities of the steroids do not correlate with binding affinities to the dexamethasone binding sites; hydrophobicity seems to play a minor role in these steroid-membrane interactions. Comparisons of the specificity of the dexamethasone binding sites on membranes to the specificity of various steroid receptors are also presented.     

Auxin binding site in tobacco cells

A specific, high affinity, IAA binding site was demonstrated in both a cytosolic fraction, and in isolated nuclei, fromNicotiana tabacum cv. Wisconsin No. 38 cells grown in suspension culture. The amount of the binding site detected in both these fractions changed during the culture cycle according to a strict pattern. The molecular mass of the binding site was estimated by gel filtration to be approximately 175 000 and it appears to be a protein. When partially purified by affinity chromatography and allowed to pre-incubate with IAA, the site had a significant stimulatory effect on total RNA synthesis, as measured by a cell-free assay system. Unpurified extracts had no such effects. The system behaves rather like the steroid hormone-receptor system in animals.     

Sex hormone-binding globulin: Anatomy and physiology of a new regulatory system

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds a number of circulating steroid hormones (testosterone, dihydrotestosterone and estradiol) with high affinity, thus regulating their free concentration in plasma. In addition to binding steroids, SHBG itself binds to receptor sites on plasma membranes with somewhat unusual kinetics. Both the off and on rates are quite slow. The steroid-binding and membrane-binding functions are interwined in what is clearly an allosteric relationship. Occupation of SHBG's steroid-binding site by a steroid inhibits its ability to bind to its membrane receptor-binding site. This inhibition is not related to a steroid's biological activity. Metabolites of steroids without biological activity, e.g. 2-methoxyestradiol, actively inhibit SHBG's interaction with its membrane receptor. However, if unliganded SHBG is allowed to bind to its receptor on intact cells, and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases. This function is specific for steroids with biological activity, 2-methoxyestradiol has no activity in this arena. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.     

Anhydrotrypsin: new features in ligand interactions revealed by affinity chromatography and thionine replacement.

Anhydrotrypsin was isolated in high purity from the product of base elimination from phenylmethanesulfonyl-trypsin, by a single operation of affinity chromatography. The adsorbent used for the chromatography was an agarose derivative coupled with peptides containing C-terminal arginine residues. As the affinity of the adsorbent for anhydrotrypsin was high compared with that for trypsin, purification of the enzyme derivative was easily achieved without the prior inactivation of trypsin which had been regenerated during the elimination reaction. Comparative studies of the ligand interaction specificities with anhydrotrypsin and trypsin confirmed the stronger interaction of the former protein with product-type ligands such as Bz-Arg-OH. No marked differences were observed between them in affinities toward substrate-type ligands such as Bz-Arg-NH2. The higher affinity of anhydrotrypsin was found to be limited to product-type ligands of L-configuration, i.e., the protein displayed an ability to discriminate the L-ligand from its optical isomer. THE PKa value for the ionization form of anhydrotrypsin responsible for the interaction with Bz-Arg-OH was estimated to be 7.60+/-0907     

Metabolism of the 16-androstene steroids in primary cultured porcine hepatocytes

The hepatic metabolism of the 16-androstene steroids was investigated using isolated porcine hepatocytes. This study demonstrated that the liver is capable of producing both phase I and phase II steroid metabolites from 16-androstene steroid precursors. 16-Androstene metabolites were recovered by solid-phase extraction and identified by gas chromatography-mass spectrometry (GC-MS). When 5alpha-androstenone was provided as a substrate, both 3beta- and 3alpha-androstenol were produced as well as a metabolite that showed evidence of hydroxylation. Incubations with the various 16-androstene steroids produced metabolic profiles which suggested that the major role of the liver is phase II conjugation. Sulfoconjugated 16-androstene steroids included androstadienol, 5alpha-androstenone, 3beta-, 3alpha-androstenol, and possibly the hydroxylated metabolite of 5alpha-androstenone. It was determined that hydroxysteroid sulfotransferase (HST) is the likely candidate for the sulfoconjugation of the 16-androstene steroids within the liver. Despite the capacity of the hepatocytes to sulfoconjugate the 16-androstene steroids, the principle metabolites produced from incubations with 5alpha-androstenone, 3beta-, and 3alpha-androstenol were glucuronide conjugates, accounting for approximately 68% of all phase II metabolism. These findings underline the importance of steroid conjugation and suggest that hepatic metabolism of the 16-androstene steroids may influence the levels of 5alpha-androstenone present in the circulation, and thus, capable of accumulating in fat.     

Interaction of steroids with the nuclear envelope

Three approaches have been taken to determine the molecular mechanism by which steroid hormones traverse the nuclear envelope on their way to the genome. The first approach involved characterization of steroid binding to nuclear envelope preparations. We have characterized androgen binding to nuclear envelopes isolated from the rat ventral prostate, the rat liver, and androgen-responsive and androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma and glucocorticoid binding to rat liver. Relatively high affinity binding sites for steroids have been identified on nuclear envelopes. Importantly, the number and specificity of the sites correlates with the responsiveness of the tissue to the steroid. In the second approach, we have undertaken to identify the steroid binding site directly. As the characteristics of the rat ventral prostate site resembled those of the nuclear androgen receptor, we have begun purifying that receptor and have found fast protein liquid chromatography to be very effective. By affinity labelling studies, the dexamethasone binding site on the rat liver nuclear envelope has been identified as a peptide of molecular weight of approximately 90,000. The third approach we have used is to identify androgen-dependent peptides in nuclear envelope preparations. In both the rat ventral prostate and an androgen-responsive cell line of the Shionogi mouse mammary carcinoma, we have identified abundant androgen-dependent peptides. The relationship of these peptides to the binding sites identified by the first two approaches and their role in steroid transport is being investigated.     

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.

RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX. Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome. The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein. These include a tetraloop of primary sequence AUCA and a variable-length stem with a bulged adenosine residue at a specific stem position. The predicted consensus agrees well with the highest-affinity RNA binding site of the R17 coat protein, identified through classical but laborious techniques. These results demonstrate the value of SELEX as a tool for isolating high affinity RNA ligands to a specific target protein, and the further value of those ligands to point the researcher toward natural sequences for that target protein.     

The metabolism of cholesterol and certain hormonal steroids by Treponema denticola

The aim was to investigate whether reference cultures and fresh isolates of Treponema denticola are able to 5alpha-reduce and further metabolise testosterone, 4-androstenedione, progesterone, corticosterone, cortisol or cholesterol. Two reference and five freshly isolated cultures of T. denticola were incubated with either radiolabeled or unlabeled steroid substrates; in the first case products were identified by thin layer chromatography and in the latter by gas chromatography-mass spectroscopy. All the substrates were 5alpha-reduced. Both reference cultures and fresh isolates of T. denticola presented 3beta- and 17beta-hydroxy steroid dehydrogenase activity. It was concluded that T. denticola was capable of steroid metabolism and hypotheses are discussed regarding the in vivo function of this metabolism including, T. denticola utilising host supplied steroids as growth factors and T. denticola steroid metabolism acting as a virulence factor.     

Investigation of Testosterone,Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

Neuroactive steroids.

Neuroactive steroids are natural or synthetic steroids that rapidly alter the excitability of neurons by binding to membrane-bound receptors such as those for inhibitory and (or) excitatory neurotransmitters. The best-studied neuroactive steroids are a series of sedative-hypnotic 3 alpha-hydroxy ring A-reduced pregnane steroids that include the major metabolites of progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC), respectively. These 3 alpha-hydroxysteroids do not interact with classical intracellular steroid receptors but bind stereoselectively and with high affinity to receptors for the major inhibitory neurotransmitter in brain, gamma-amino-butyric acid (GABA). Biochemical and electrophysiological studies have shown that these steroids markedly augment GABA-activated chloride ion currents in a manner similar (but not identical) to that of anesthetic barbiturates. Several steroids have also been observed to have convulsant or proconvulsant properties, including the synthetic amidine 3 alpha-hydroxy-16-imino-5 beta-17-azaandrostan-11-one (RU5135) and the natural sulfate esters of pregnenolone and dehydroepiandrosterone. Several of these have been shown to be bicuculline or picrotoxin-like GABAA receptor antagonists. Examples of steroids that alter neuronal excitability rapidly by augmenting or inhibiting excitatory amino acid receptor-mediated responses have also been reported. Recently, allopregnanolone and allotetrahydroDOC have also been measured in brain and plasma where their levels have been shown to fluctuate in response to stress and during the estrous and menstrual cycles of rats and humans, respectively. Although the major fraction of allopregnanolone in tissue, including brain, is of adrenal and/or ovarian origin, appreciable levels of allopregnanolone can still be measured in the brains of adrenalectomized and/or oophorectomized animals. Receptor-active neurosteroids may represent an important class of neuromodulators that can rapidly alter central nervous system excitability via novel nongenomic mechanisms.     

Purification and identification of the heat-stable factor required for pregnenolone-binding protein activity. Evidence that the factor is adenosine 3',5'-diphosphate.

This paper presents data identifying adenosine 3',5'-diphosphate (3',5'-ADP) as the small heat-stable factor essential for the active steroid binding complex of the adrenocortical pregnenolone-binding protein (PBP). Factor activity obtained from the boiled supernatant of partially purified PBP was isolated by high performance liquid chromatography using weak anion-exchange and hydrophobic (C18) chromatography sequentially. The purified material retained characteristic factor activity and presented a UV spectrum identical to that for authentic 3',5'-ADP. Mass spectroscopic analysis of the isolated factor revealed an M-H ion of appropriate mass (m/z = 426) and a decomposition pattern for the M-H ion that was consistent with the structure of 3',5'-ADP. The studies presented here demonstrate that authentic 3',5'-ADP can categorically substitute for factor prepared from the soluble fraction of the guinea pig adrenal. Specifically, 3',5'-ADP potentiated ligand binding of partially purified native PBP and restored binding capacity to alkaline phosphatase-inactivated PBP in a dose-dependent manner. As is the case for adrenocortical factor activity, these effects were negated by pretreating the 3',5'-ADP with calf intestinal alkaline phosphatase. Other nucleotides similarly tested, including ADP isomers, were ineffective as factor substitutes. The sulfated form of 3',5'-ADP (i.e. 3'-phosphoadenosine 5'-phosphosulfate) demonstrated some potential for restoring binding capacity to phosphatase-inactivated PBP; however, this compound was clearly inhibitory rather than stimulatory for native PBP activity. Taken collectively, the data overwhelmingly demonstrate that 3',5'-ADP is in fact the molecule required by the PBP for high affinity steroid binding complex formation. It is not yet known whether 3',5'-ADP acts allosterically or contributes directly to the structure of the steroid binding site.     

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-beta-(4-dimethylaminophenyl)-17a-hydroxy-17 beta-(3- hydroxypropyl)-13 alpha-methyl-4,9-gonadien-3-one]). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [3H]ZK98299 with affinity approximately equal to that of [3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0 degrees C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [3H]ZK98299 and [3H]triamcinolone acetonide binding. While [3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide (NEM), [3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 degrees C) and receptor transforming (23 degrees C) conditions, and exhibited a faster rate of dissociation at 23 degrees C when compared with [3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.