Affinity labeling of steroid binding sites. Study of the active site of 20beta-hydroxysteroid dehydrogenase with 2alpha-bromoacetoxyprogesterone and 11alpha-bromacetoxyprogesterone.

To further characterize the active site of 20beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyced hydrogenans we synthesized 2alpha-bromoacetoxyprogesterone, a substrate for the enzyme in 0.05 M phosphate buffer at 25 degrees, pH 7.0, with Km and Vmax values of 1.90 X 10(-5) M and 6.09 nmol/min/mg of enzyme, respectively. This affinity labeling steroid inactivates 20beta-hydroxysteroid dehydrogenase in an irreversible and time-dependent manner which follows pseudo-first order kinetics with a t1/2 value of 4.6 hours. 2alpha-[2-3H]Bromoacetoxyprogesterone was synthesized and used to radiolabel the enzyme active site. Amino acid analysis of the acid hydrolysate of the radiolabeled enzyme supports a mechanism whereby the steroid moiety delivers the alkylating group to the steroid binding site of the enzyme where it reacts with a methionyl residue. Both 2alpha- and 11alpha-bromoacetoxyprogesterone alkylate a methionyl residue at the active site of 20beta-hydroxysteroid dehydrogenase. The enzyme was inactivated with a mixture containing both 2alpha-[2-3H]Bromoacetoxyprogesterone and 11alpha-2[2-14C]bromoacetoxyprogesterone. Following degradation of separate aliquots of the radiolabeled enzyme by cyanogen bromide or trypsin, the protein fragments were separated by gel filtration and ion exchange chromatography. Resolution of peptides carrying the 3H label from those possessing the 14C label demonstrates that 2alpha-bromoacetoxyprogesterone and 11alpha-bromoacetoxyprogesterone each label a different methionine at the steroid binding site of 20beta-hydroxysteroid dehydrogenase.     

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.     

A Leu----His substitution at residue 93 in human corticosteroid binding globulin results in reduced affinity for cortisol.

A steroid binding capacity assay and a radioimmunoassay were both used to measure corticosteroid binding globulin (CBG) in serum samples from 22 patients with sepsis. An approximately 50% discordancy between the two values in one patient suggested the presence of a CBG variant with reduced affinity for cortisol, and this was confirmed by Scatchard analysis. We therefore used the polymerase chain reaction to amplify exons that encode for human CBG from the genomic DNA of this patient. This revealed two mutations within the coding sequences: one of which results in a Leu----His substitution at residue 93 and another which encodes a Ser----Ala substitution at residue 224 of the human CBG polypeptide. To assess the impact of each substitution on the steroid binding affinity of CBG, each mutation was introduced separately into a normal human CBG cDNA, and the normal and mutated cDNAs were expressed in Chinese hamster ovary cells. Scatchard analysis of the CBG produced in culture indicated that the His93 mutation (Kd = 2.24 +/- 1.75 nM) reduced the cortisol binding affinity of CBG (mean +/- SD) significantly (P less than 0.024) when compared to normal CBG (Kd = 0.64 +/- 0.31 nM), while the Ala224 mutation (Kd = 0.63 +/- 0.33 nM) did not influence cortisol binding affinity. We therefore conclude that residue 93 may play an important role in determining the structure of the CBG steroid binding site.     

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.     

Nonexistence of Exo-cellobiohydrolase (CBH) in the Cellulase System of Trichoderma viride

Chemical modification of histidine residues in ricin E was studied with regard to saccharide binding. The analytical data indicate that 6 out of 7 histidine residues in ricin E are eventually modified with diethylpyrocarbonate (DEP) at pH 6.0 and 25°C in the absence of specific saccharides. Modification of histidine residues greatly decreased the cytoagglutinating activity of ricin E, and only 10% of the residual activity was found after modification of 6 histidine residues/mol. The data of affinity chromatography using lactamyl- and galactosamine-cellulofine columns suggest that modification of histidine residues does not have much effect on the binding ability at the low affinity saccharide-binding site of ricin E but abolishes the binding ability at the high affinity saccharide-binding site. In the presence of lactose, one histidine residue/mol was protected from the DEP modification with retention of a fairly high cytoagglutinating activity. Such a protective effect was also observed for specific saccharides such as galactose and A^-acetylgalactosamine, but not for glucose, a non-specific saccharide. On treatment with hydroxylamine, the modified ricin E restored 67 % of the cytoagglutinating activity. Based on these findings, it is suggested that in the high affinity saccharide- binding site of ricin E there exists one histidine residue responsible for saccharide binding.     

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.     

Steroid-protein interactions. XXXIV. Chemical modification of alpha1-acid glycoprotein for characterization of the progesterone binding site.

The nature of the steroid binding site in alpha1-acid glycoprotein (orosomucoid) was investigated by chemical modification of individual amino acids and subsequent examination of the binding affinity for progesterone. Equilibrium dialyses were performed under conditions that excluded contact with human skin. Reaction of the lysyl residues with trinitrobenzenesulfonic acid or arylisocyanates resulted in a reduction of active sites. In an alternate approach, one lysyl residue of alpha1-acid glycoprotein was protected from modification by trinitrobenzenesulfonic acid when progesterone was present to form the complex with alpha1-acid glycoprotein. We conclude that a lysyl residue is located in the binding site. Reaction of tetranitromethane with the tyrosine groups in alpha1-acid glycoprotein also reduced the number of active binding sites for progesterone. Again, a partial protection of this modification was seen in the presence of progesterone and other delta4-3-ketosteroids. The progesterone binding activity observed in the tyrosine-modified alpha1-acid glycoprotein by equilibrium dialysis and by fluorescence quenching titration can be interpreted best by the presence of one tyrosyl residue in the binding site, and involvement of a second tyrosine nearby. Modification of tryptophan in alpha1-acid glycoprotein by mild acid hydrolysis, N-bromosuccinimide, hydroxynitrobenzylbromide, and formic acid resulted in a decreased steroid binding; the formylation reaction was fully reversible. The approximate distance between progesterone and the tryptophan involved in the binding was calculated to be between 9.1 A and 14.1 A. When alpah1-acid glycoprotein was cleaved by the cyanogen bromide procedure according to Ikenaka et al. (1972, Biochemistry 11, 3817-3829), both the amino and the carboxyl fragment had a weak progesterone binding affinity which could be measured in 4 M NaCl. This result thus failed to specify the location of the steroid binding site in alpha1-acid glycoprotein. However, the closeness of tryptophan, lysine and tyrosine in the primary and presumably the tertiary structure of alpha1-acid glycoprotein is in agreement with the properties of the binding site suggested by our studies.     

Titratable effects of p-chloromercuriphenyl sulfonate, a thiol-attacking reagent, on glucocorticoid receptor binding

Cytosol prepared from cultured AtT-20 mouse pituitary cells or mouse liver was treated with concentrations of p-chloromercuriphenyl sulfonate (PCMPS) which reduced but did not abolish receptor-binding activity. Scatchard analysis of triamcinolone acetonide binding to the treated cytosol showed that the PCMPS effect was caused by a reduction of binding affinity with little effect on the apparent binding site concentration. The effect on affinity was dose-dependent. Binding specificity appeared unaffected since the relative abilities of triamcinolone acetonide, dexamethasone, cortisol, progesterone, and corticosterone to compete with labeled triamcinolone were similar at various PCMPS concentrations which caused a progressive reduction of detectable cytosol binding. The PCMPS effect was reversible since cytosol treated with up to 200 microM PCMPS followed by dithiothreitol 15 min later showed nearly complete recovery of binding sites (62-100%). The possibility that several sulfhydryl groups were involved in this phenomenon was further explored in experiments using AtT-20 cytosol labeled with [3H]dexamethasone-mesylate, a glucocorticoid affinity label which binds covalently to sulfhydryl groups. Chromatography of dexamethasone-mesylate labeled receptor on a sulfhydryl affinity column resulted in binding, indicating that the receptor had at least two sulfhydryl groups, one bound to the mesylate moiety of the steroid and the other capable of binding to the affinity column.     

The effect of short term confinement stress on binding characteristics of sex steroid binding protein (SBP) in female black bream (Acanthopagrus butcheri) and rainbow trout (Oncorhynchus mykiss)

The effect of short term confinement stress on sex steroid binding protein (SBP) binding characteristics was examined in female black bream (Acanthopagrus butcheri), and rainbow trout (Oncorhynchus mykiss). Black bream were sampled immediately after capture from the wild and again after 1, 6 or 24 h confinement. Rainbow trout were sampled before and after 5 h confinement. Confinement of black bream for 6 h after capture significantly reduced the binding capacity of SBP. Binding affinity also tended to be lower after confinement. There were no differences in binding affinity or capacity of black bream SBP after 1 or 24 h confinement, or rainbow trout SBP after 5 h confinement. Plasma from rainbow trout at 3 and 6 h after treatment with cortisol was compared to plasma from saline-injected controls. No significant differences in binding characteristics were detected, but there was a trend of decreased binding capacity in cortisol-injected fish compared to controls at 6 h post-injection. Relative binding studies indicated that plasma cortisol at concentrations 100x or more greater than plasma estradiol (E(2)) may displace E(2) from SBP in black bream, and act to reduce circulating levels of E(2) through increased clearance of free steroid. Physiological levels of cortisol did not displace E(2) from SBP in trout. The observed changes in SBP and the competition of physiological concentrations of cortisol for SBP binding sites may generate a component of the stress-induced falls in plasma levels of E(2) reported across a range of species.     

Reversible dissociation of cortisol-transcortin complex by sodium para-chloromercuribenzoate

Mercurials are considered as sulphydryl group specific reagents and one of them, sodium para-chloromercuribenzoate (PCMB), is currently used for SH titration. It has been shown that cellular steroid receptors are reversibly inactivated by mercurials even when the binding site is occupied by the steroid (Coty, W.A. (1980) J. Biol. Chem. 255, 8035-8037). This is a striking difference with alkylating SH reagents such as iodoacetic acid or N-ethylmaleimide, since these reagents inactivate only steroid-free receptors. In order to explain this discrepancy, we tested, in the present study, the specificity of PCMB on a blood plasma steroid binding protein: human transcortin. This protein presents the advantage, over cellular receptors, of being well characterized and to be available in a pure state. The transcortin-cortisol complex was also reversibly inactivated by PCMB when the reaction was carried out at a high excess of reagent over protein; such conditions are those previously used with steroid receptors. The reversibility was obtained not only with a reducing agent (dithiothreitol) but also with EDTA, which suggests a poor stability of the protein mercurial bond and therefore a nonspecific action. The decrease of activity was the result of a loss of binding sites and Scatchard plot analysis did not reveal any detectable decrease of the affinity constant for cortisol. Transcortin possesses two SH groups per molecule, one of these being buried in native conformation. After blockage of the accessible SH group by aminoethylation, transcortin kept the same activity, but when this aminoethylated transcortin was incubated with PCMB a loss of activity was obtained, although the residual buried SH group was again titrable with Ellman's reagent. Therefore, we can conclude that the action of PCMB on proteins must be interpreted with precaution, since it can induce an inactivation that is SH-independent.     

An amino acid substitution in biobreeding rat corticosteroid binding globulin results in reduced steroid binding affinity

BioBreeding (BB) rats are derived from an outbred colony of Wistar rats and are used as a model of autoimmune diabetes mellitus. A corticosteroid binding globulin (CBG) variant with reduced affinity for glucocorticoids has now been found in the blood of these animals. The dissociation rate constants of BB CBG for cortisol (4.42 nM) and corticosterone (1.43 nM) are both about 50% higher than those associated with Wistar CBG, but no obvious difference in the steroid binding specificity of BB and Wistar CBGs was detected. Purified BB and Wistar CBGs exhibit the same size heterogeneity when examined by polyacrylamide gel electrophoresis under denaturing conditions, and the sizes of their respective hepatic mRNAs are identical. The genetic basis for this abnormality was therefore determined by comparing the cDNA sequences for BB and Wistar CBG, and this revealed a point mutation that results in a single amino acid substitution at residue 276 (Ile in BB CBG and Met in Wistar CBG). To confirm that this mutation is responsible for the reduced steroid binding affinity associated with BB CBG, the cDNAs for rat CBG-Ile276 and CBG-Met276 were expressed in Chinese hamster ovary cells. The steroid binding affinities of the CBGs secreted by these cells were essentially identical with those observed in the corresponding serum samples from these two rat strains. The amino acid substitution identified in BB rat CBG therefore clearly accounts for the reduction in its steroid binding affinity, and further analysis of this and other natural CBG variants may reveal important information about the CBG steroid binding site. It is also possible that this mutation may contribute to the etiology of pathological abnormalities that are characteristic of the BB rat.     

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.     

Role of Histidine Residues in the Adenosine A2a Receptor Ligand Binding Site

Abstract: The pH dependency of the binding of ligands to adenosine A_2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two- to fourfold increase in affinity was observed for CGS-21680, 5'- N -ethylcarboxamidoadenosine, adenosine, 3'-deoxyadenosine, 5'-deoxyadenosine, inosine, and N ⁶-methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2'-deoxyadenosine, although 2'-methoxyadenosine binding was pH dependent. This effect on the affinity of CGS-21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2'-hydroxyl group of adenosine agonists.     

Novel nuclear corticosteroid binding in rat small intestinal epithelia

When small intestinal epithelial cells are incubated with [(3)H]corticosterone, nuclear binding is displaced neither by aldosterone nor RU-28362, suggesting that [(3)H]corticosterone is binding to a site distinct from mineralocorticoid receptor and glucocorticoid receptor. Saturation and Scatchard analysis of nuclear [(3)H]corticosterone binding demonstrate a single saturable binding site with a relatively low affinity (49 nM) and high capacity (5 fmol/microg DNA). Competitive binding assays indicate that this site has a unique steroid binding specificity, which distinguishes it from other steroid receptors. Steroid specificity of nuclear binding mirrors inhibition of the low 11beta-dehydrogenase activity, suggesting that binding may be to an 11beta-hydroxysteroid dehydrogenase (11betaHSD) isoform, although 11betaHSD1 is not present in small intestinal epithelia and 11betaHSD2 does not colocalize intracellularly with the binding site. In summary, a nuclear [(3)H]corticosterone binding site is present in small intestinal epithelia that is distinct from other steroid receptors and shares steroid specificity characteristics with 11betaHSD2 but is distinguishable from the latter by its distinct intracellular localization.     

Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.     

Affinity labelling of the NADP+-binding site of glucose 6-phosphate dehydrogenase from Candida utilis.

1. Periodate-oxidized NADP+ inhibits the catalytic activity of glucose 6-phosphate dehydrogenase from Candida utilis, competing with NADP+. 2. Incubation of the enzyme with the coenzyme analogue causes partial reversible inactivation of the enzyme as a result of affinity labelling of the coenzyme-binding site. 3. Some kinetic values of the reaction were calculated. 4. The inactivation can be made irreversible by treatment with NaBH4, which reduces a Schiff base formed between an aldehyde group on the coenzyme analogue and a lysine residue on the enzyme. 5. Complete inactivation can be correlated with the binding of only one inhibitor to each enzyme subunit. 6. The lysine residue involved in the binding of the inhibitor is present at the coenzyme-binding site.     

The catalytic site of Escherichia coli aspartate transcarbamylase: interaction between histidine 134 and the carbonyl group of the substrate carbamyl phosphate

Previous pKa determinations indicated that histidine 134, present in the catalytic site of aspartate transcarbamylase, might be the group involved in the binding of the substrate carbamyl phosphate and, possibly, in the catalytic efficiency of this enzyme. In the present work, this residue was replaced by an asparagine through site-directed mutagenesis. The results obtained show that histidine 134 is indeed the group of the enzyme whose deprotonation increases the affinity of the catalytic site for carbamyl phosphate. In the wild-type enzyme this group can be titrated only by those carbamyl phosphate analogues that bear the carbonyl group. In the modified enzyme the group whose deprotonation increases the catalytic efficiency is still present, indicating that this group is not the imidazole ring of histidine 134 (pKa = 6.3). In addition, the pKa of the still unknown group involved in aspartate binding is shifted by one unit in the mutant as compared to the wild type.     

Antibodies against cortisol block suppressive effects of corticosteroids on lymphocytes in vitro.

Lymphocyte transformation assays were used to test the ability of antibodies against cortisol to reduce bioactivity of corticosteroids in vitro. Mononuclear cells were separated from whole bovine blood and cultured in the presence of PHA alone, PHA + steroid, PHA + steroid + anticortisol, or PHA + steroid + anti-bovine serum albumin. Tritiated thymidine uptake was determined for all groups during the last 24 hr of a 72-hr culture period by scintillation counting. Polyclonal anticortisol against cortisol-bovine serum albumin conjugated in the 21 position was more effective in blocking cortisol activity than monoclonal anticortisol built against conjugates in the 3 position. The steroids that suppressed PHA-induced lymphocyte proliferation in a concentration-dependent manner were: cortisol, corticosterone, dexamethasone, prednisolone, 11-deoxycortisol, and 11-deoxycorticosterone. Aldosterone, cortisone, cholesterol, estradiol, and progesterone did not exhibit concentration-dependent effects and, thus, were not considered suppressive. These concentration-independent steroids were also the least suppressive (with the exception of aldosterone). Anticortisol was able to reduce bioactivity of suppressive corticosteroids that had an 11-hydroxy group, suggesting the antibody was primarily made against this site. Anti-BSA was not effective in blocking corticosteroid activity, but it did enhance proliferation of lymphocytes if added in combination with weakly suppressive steroids. Anticortisol also had an enhancing effect when added with some weakly suppressive steroids. We conclude that antibodies against cortisol are capable of reducing bioactivity of steroids that strongly suppress lymphocyte proliferation. Additionally, the 11-hydroxy group may be an important antigenic determinant of steroid molecules.     

Steroid hormone modulation of 3H-prostaglanding E1 binding to bovine corpus leteum cell membranes.

The specific binding of 3H-prostaglandin E1 (3H-PGE1) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0x10-minus-6M. At concentrations above 2.5 x 10-minus-6M; estrone, 17beta-estradiol (but not 17alpha-estradiol or 17beta-estradiol glucuronide), estroil, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of 3H-PGE1. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced 3H-PGE1 binding. These findings permitted the following correlations between steroid structure and modulation of 3H-PGE1 binding: steroids with a free phenolic ring and a 17beta-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of 3H-PGE1. PGE receptors are heterogeneous with respect to affinity for 3H-PGE1. The steroids that decreased 3H-PGE1 binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased 3H-PGE1 binding caused appearance of new low affinity PGE receptors. Association rate constants for 3H-PGE1 binding were decreased by 17beta-estradiol (61%) and increased by DHT (59%).     

Affinity of tixocortol pivalate (JO 1016), tixocortol, cortisol acetate and cortisol for dexamethasone receptors of mouse thymus cells and rat renomedullary interstitial cells in culture. Correlation with their biological activities

Affinity for the dexamethasone binding sites of tixocortol pivalate, (the ester of the 21 thiol derivatives of cortisol), a steroid with local anti-inflammatory activity similar to cortisol acetate and with no systemic activity, was investigated in comparison with other steroids: tixocortol (the thiol derivative yielded by esterase hydrolysis), cortisol acetate and cortisol. The rank order of relative affinity for dexamethasone receptor of mouse thymocytes (37 degrees C) was: dexamethasone (1), cortisol (0.20), tixocortol pivalate (0.16), tixocortol (0.065), cortisol acetate (0.05). The corresponding 21 oxygenated ester (cortisol pivalate) was found less potent (0.080). Using rat renomedullary interstitial cells in culture, tixocortol pivalate showed also a higher receptor affinity than tixocortol. Cortisol acetate was as potent as tixocortol pivalate. The biological activity of tixocortol pivalate measured by the inhibition of PGE2 secretion on the same model, was similar to cortisol acetate and cortisol (10(-6) M, 24 h incubation, 38-55% inhibition). Tixocortol was less active (26%). These results with tixocortol pivalate are in good agreement with previously reported in vivo studies and show a good correlation between its binding ability and biological effect.