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.     

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 testosterone, corticosterone and corticosterone binding globulin in the white-throated sparrow (Zonotrichia albicollis)

Plasma binding globulins bind steroid hormones and are thought to regulate hormone access to tissues. Mammals have both sex steroid binding globulin (SSBG) and corticosteroid binding globulin (CBG). Birds, however, have no detectable SSBG, leading to the early conclusion that birds have no plasma regulation of sex steroids. CBG, however, can bind androgens with relatively high affinity. In birds, therefore, the control of androgenic effects may be tightly regulated by glucocorticoid physiology because glucocorticoids compete with androgens for CBG binding sites. We report levels of total testosterone (T), total corticosterone, CBG, and estimated free T in the males, the more aggressive morph had higher levels of total T; female morphs did not differ. Approximately 96% of T was bound to CBG, but a lack of morph or sex-specific differences in corticosterone titers or CBG capacity caused patterns of free T to mirror those of total T. While CBG has the potential to greatly influence T availability to tissues, in this species interactions between T, CBG and corticosterone do not appear to alter general patterns of T availability to tissues.     

A new role for corticosteroid binding globulin (CBG), member of SERPIN superfamily]

Recent advances in molecular endocrinology have shed a new light on the role and mode of action of CBG. It is now not only demonstrated that this plasma glycoprotein, a steroid carrier, can be internalized by glucocorticoid target tissues, but it is also certain that CBG mRNA is synthesized by extra-hepatic tissues. Moreover, some authors have reported a modulation of CBG properties by free fatty acids. The existence of CBG receptors (or high affinity membrane-binding sites), and even a positive effect of CBG on adenylate cyclase activity, have also been reported. To progress in the understanding of these diverse results, one must first integrate them in a general scheme where it is considered that CBG is a member of the SERPIN (SERine Protease INhibitors) superfamily. In the case of CBG, that means a protein which functions as a substrate for elastase at the surface of neutrophils, for instance at sites of inflammation. CBG is specifically cleaved by this protease at a precise site close to its carboxy-terminus. This induces a conformation change and disrupts the binding between glucocorticoids and CBG, and promotes a significant and local release of glucocorticoids (over 90% of them are bound to CBG in human plasma). In this context, CBG directs glucocorticoids to sites of inflammation, and plays in consequence a crucial role in efficient glucocorticoid action in physiology. The elucidation of the CBG sequence, the knowledge of its gene structure, and the discovery of its chromosomal localization near two other SERPIN genes, are three sets of data in concordance to demonstrate that CBG is a SERPIN; and this has allowed the understanding of a new role for CBG, possibly with important consequences in pathology. Moreover, it could be more appropriate to say that CBG is a member of the SERine Protease INhibitors and Substrates superfamily (SERPINS).     

Interaction of human CBG with cell membranes

Specific binding sites for corticosteroid-binding globulin (CBG) and its pregnancy-associated variant (pCBG), having a modified carbohydrate moiety, were found in the plasma membranes of human liver, decidual endometrium and placental syncytiotrophoblast. The membrane binding was influenced by the conformation of the glycoprotein molecules and structure of their carbohydrate chains. CBG receptor was solubilized from the endometrium membrane and partially characterized. It was found to have a subunit structure, with a homooligomeric sialoglycoprotein consisting of four 20 kDa protomeric species being involved in the recognition of the CBG molecules complexed with progesterone or cortisol. A kinetic study using membrane microvesicles derived from the syncytiotrophoblast brush border revealed that neither CBG nor pCBG restricted cortisol accumulation in the intravesicular space, whereas only normal CBG could penetrate the syncytiotrophoblast membrane. Action of the CBG-cortisol complex on trophoblast cells resulted in the activation of membrane adenylate cyclase and growth of the cAMP accumulation within these cells. Collectively, these findings suggest that both normal CBG and pCBG are involved in the guided transport of steroid hormones to the target cells and transmembrane transfer of hormones and/or hormonal signals.     

Distribution and percentages of non-protein bound contraceptive steroids in human serum

It has been shown that albumin bound steroids are taken up by the rat brain in addition to nonprotein bound steroids and it has also been suggested that cortisol binding globulin (CBG) may facilitate progesterone uptake by the rat uterus but not the brain. Recently serum sex-hormone binding globulin (SHBG) has been identified in the cytoplasm of sex steroid target cells. Thus the distribution of synthetic steroids between various protein bound and nonprotein bound components in serum may influence their bioavailability at different target tissues. The authors employed a newly developed technique, centrifugal ultrafiltration-dialysis. The results showed that there are no differences in percentages of nonprotein bound ethinyl estradiol (EE2), and cyproterone acetate (CA) with respect to sex or serum SHBG and CBG binding capacities. However serum percentages of nonprotein bound norethisterone (NET) (p0.05) are significantly lower in women than in men. Also the percentages of nonprotein bound NET and D-norgestrel are both very much lower (p0.001) in serum from pregnant women when compared to nonpregnant women. These differences appear to be inversely related to serum SHBG binding capacity. The percentages of nonprotein bound NET and D-norgestrel in heat treated serum from men and nonpregnant women are identical and largely represent the contribution of albumin binding alone. In addition heat labile binding proteins do not appear to influence the percentages of nonprotein bound EE2 and CA and it can be inferred that EE2 and CA are almost exclusively bound by albumin in native serum; 98.5% of EE2 and 93% of CA are bound to albumin. In contrast the percentages of nonprotein bound NET and D-norgestrel in native serum are inversely related to SHBG binding capacity. This data indicate that the nonprotein bound and albumin bound factors of NET and D-norgestrel may vary by as much as 2-3 fold between women who are known to have subnormal or supranormal levels of serum SHBG binding capacity and it is suggested that measurements of serum SHBG binding capacity may provide a method of assessing the lowest effective dose of these 2 progestins in individual subjects to help reduce side effects associated with their use. Future studies should address the effect of serum steroid concentrations on the actual nonprotein bound serum concentrations and distribution of these progestins.     

Effect of corticosteroid binding proteins on the steroidogenic activity of bovine adrenocortical cell suspensions

The possible role of steroid binding proteins in the hormonal secretion process of a steroidogenic tissue was examined using bovine adrenocortical cell suspensions, either under basal conditions or in the presence of half-maximally active concentration (1 × 10⁻⁹ M) of synthetic adrenocorticotropic hormone (ACTH). Three types of plasma cortisol binding proteins were used, namely bovine serum albumine (BSA), purified transcortin (CBG) and purified anticortisol immunoglobulins (IgG). When added to the incubation medium, CBG (at 1 × 10⁻¹⁰ to 2 × 1010⁻¹⁰ M cortisol binding sites) and anticortisol IgG (at 4.8 × 10⁻¹⁰ to 3 × 10⁻⁹M cortisol binding sites) did not influence either the basal nor the ACTH-stimulated net cortisol production of the cell preparations. Whereas crystallized and delipidated BSA showed also no effect, crude commercial BSA preparation (Cohn fraction V) exhibited an ACTH-like cofactor effect which resulted in a marked increase in the net cortisol production by stimulated cells. These observations might be explained by the presence in crude BSA of lipoprotein-cholesterol complexes, possibly acting as an extracellular source of cholesterol available for corticosteroidogenesis.It may be concluded that specific high affinity cortisol binding systems present outside adrenoeortical steroidogenic cells do not influence their secretory activity under short term in vitro condition. In addition, it can be stressed that use of ill defined protein preparations (e.g. crude BSA) may lead to artifactual observations in the study of the differentiated functions of isolated steroidogenic cells.     

Homology model of human corticosteroid binding globulin: a study of its steroid binding ability and a plausible mechanism of steroid hormone release at the site of inflammation

Corticosteroid binding globulin (CBG) and thyroxin binding globulin (TBG) both belong to the same SERPIN superfamily of serine-proteinase inhibitors but in the course of evolution CBG has adapted to its new role as a transport agent of insoluble hormones. CBG binds corticosteroids in plasma, delivering them to sites of inflammation to modify the inflammatory response. CBG is an effective drug carrier for genetic manipulation, and hence there is immense biological interest in the location of the hormone binding site. The crystal structure of human CBG (hCBG) has not been determined, but sequence alignment with other SERPINs suggests that it conforms as a whole to the tertiary structure shared by the superfamily. Human CBG shares 52.15% and 55.50% sequence similarity with alpha1-antitrypsin and alpha1-antichymotrypsin, respectively. Multiple sequence alignment among the three sequences shows 73 conserved regions. The molecular structures of alpha1-antitrypsin and alpha1-antichymotrypsin, the archetype of the SERPIN superfamily, obtained by X-ray diffraction methods are used to develop a homology model of hCBG. Energy minimization was applied to the model to refine the structure further. The homology model of hCBG contains 371 residues (His13 to Val383 ). The secondary structure comprises 11 helices, 15 turns and 11 sheets. The putative corticosteroid binding region is found to exist in a pocket between beta-sheets S4, S10, S11 and alpha helix H10. Both cortisol and aldosterone are docked to the elongated hydrophobic ligand binding pocket with the polar residues at the two extremities. A difference accessible surface area (DASA) study revealed that cortisol binds with the native hCBG more tightly than aldosterone. Cleavage at the Val379-Met380 peptide bond causes a deformation of hCBG (also revealed through a DASA study). This deformation could probably trigger the release of the bound hormone. Figure Stereoscopic view of the ribbon diagram of hCBG complexed with cortisol. The bound cortisol is shown in space filling model in blue. Helices and sheets are shown in red and magenta respectively. Turns are shown in yellow.     

Characteristics of glucocorticoid-binding by inflammatory tissue of rats

Glucocorticoid-binding activities of the granuloma cytosol were compared with those of the liver cytosol and of the serum in vitro. The granuloma cytosol bound cortisol (HC) about 4-fold higher than dexamethasone (DX) and triamcinolone acetonide (TA); the liver cytosol bound these two synthetic agonists more than HC. The kinetic parameters of the glucocorticoid-binding components of the granuloma and the liver cytosols were studied by the Scatchard method. The binding components of the granuloma cytosol had a single class of binding sites with high affinity for these three steroids, whereas the binding site of the liver cytosol had negative cooperativity or consisted of two distinct classes, because its Scatchard plot showed a hyperbolic curve. The granuloma glucocorticoid-binding components will be protein since their binding was prevented by a trypsin treatment and completely lost by heating at 60 C for 5 min. Heating at 25 or 37 degrees C for 30 min did not affect the HC-binding activity of the granuloma cytosol, regardless of prelabeling with the steroid. The binding activity for DX and TA were heat-labile and completely lost by heating the cytosol at 37 degrees C for 30 min without the respective steroid. The results of thermal inactivation and ammonium sulfate fractionation show the granuloma HC-binding protein closely resembles corticosteroid-binding globulin (CBG). From enzymatic determination of hemoglobin in tissue cytosols, attribution of the contaminating blood to the HC-binding activity of the cytosol is considered to be negligible.     

Towards Engineering Hormone-Binding Globulins as Drug Delivery Agents

The treatment of many diseases such as cancer requires the use of drugs that can cause severe side effects. Off-target toxicity can often be reduced simply by directing the drugs specifically to sites of diseases. Amidst increasingly sophisticated methods of targeted drug delivery, we observed that Nature has already evolved elegant means of sending biological molecules to where they are needed. One such example is corticosteroid binding globulin (CBG), the major carrier of the anti-inflammatory hormone, cortisol. Targeted release of cortisol is triggered by cleavage of CBG''s reactive centre loop by elastase, a protease released by neutrophils in inflamed tissues. This work aimed to establish the feasibility of exploiting this mechanism to carry therapeutic agents to defined locations. The reactive centre loop of CBG was altered with site-directed mutagenesis to favour cleavage by other proteases, to alter the sites at which it would release its cargo. Mutagenesis succeeded in making CBG a substrate for either prostate specific antigen (PSA), a prostate-specific serine protease, or thrombin, a key protease in the blood coagulation cascade. PSA is conspicuously overproduced in prostatic hyperplasia and is, therefore, a good way of targeting hyperplastic prostate tissues. Thrombin is released during clotting and consequently is ideal for conferring specificity to thrombotic sites. Using fluorescence-based titration assays, we also showed that CBG can be engineered to bind a new compound, thyroxine-6-carboxyfluorescein, instead of its physiological ligand, cortisol, thereby demonstrating that it is possible to tailor the hormone binding site to deliver a therapeutic drug. In addition, we proved that the efficiency with which CBG releases bound ligand can be increased by introducing some well-placed mutations. This proof-of-concept study has raised the prospect of a novel means of targeted drug delivery, using the serpin conformational change to combat the problem of off-target effects in the treatment of diseases.     

Serum corticosteroid binding globulin expression is modulated by fasting in polar bears (Ursus maritimus)

Polar bears (Ursus maritimus) from several subpopulations undergo extended fasting during the ice-free season. However, the animals appear to conserve protein despite the prolonged fasting, though the mechanisms involved are poorly understood. We hypothesized that elevated concentrations of corticosteroid binding globulin (CBG), the primary cortisol binding protein in circulation, lead to cortisol resistance and provide a mechanism for protein conservation during extended fasting. The metabolic state (feeding vs. fasting) of 16 field sampled male polar bears was determined based on their serum urea to creatinine ratio (>25 for feeding vs. <5 for fasting). There were no significant differences in serum cortisol levels between all male and female polar bears sampled. Serum CBG expression was greater in lactating females relative to non-lactating females and males. CBG expression was significantly higher in fasting males when compared to non-fasting males. This leads us to suggest that CBG expression may serve as a mechanism to conserve protein during extended fasting in polar bears by reducing systemic free cortisol concentrations. This was further supported by a lower serum glucose concentration in the fasting bears. As well, a lack of an enhanced adrenocortical response to acute capture stress supports our hypothesis that chronic hunger is not a stressor in this species. Overall, our results suggest that elevated serum CBG expression may be an important adaptation to spare proteins by limiting cortisol bioavailability during extended fasting in polar bears.     

Genome Wide Association Identifies Common Variants at the SERPINA6/SERPINA1 Locus Influencing Plasma Cortisol and Corticosteroid Binding Globulin

Variation in plasma levels of cortisol, an essential hormone in the stress response, is associated in population-based studies with cardio-metabolic, inflammatory and neuro-cognitive traits and diseases. Heritability of plasma cortisol is estimated at 30–60% but no common genetic contribution has been identified. The CORtisol NETwork (CORNET) consortium undertook genome wide association meta-analysis for plasma cortisol in 12,597 Caucasian participants, replicated in 2,795 participants. The results indicate that <1% of variance in plasma cortisol is accounted for by genetic variation in a single region of chromosome 14. This locus spans SERPINA6, encoding corticosteroid binding globulin (CBG, the major cortisol-binding protein in plasma), and SERPINA1, encoding α1-antitrypsin (which inhibits cleavage of the reactive centre loop that releases cortisol from CBG). Three partially independent signals were identified within the region, represented by common SNPs; detailed biochemical investigation in a nested sub-cohort showed all these SNPs were associated with variation in total cortisol binding activity in plasma, but some variants influenced total CBG concentrations while the top hit (rs12589136) influenced the immunoreactivity of the reactive centre loop of CBG. Exome chip and 1000 Genomes imputation analysis of this locus in the CROATIA-Korcula cohort identified missense mutations in SERPINA6 and SERPINA1 that did not account for the effects of common variants. These findings reveal a novel common genetic source of variation in binding of cortisol by CBG, and reinforce the key role of CBG in determining plasma cortisol levels. In turn this genetic variation may contribute to cortisol-associated degenerative diseases.     

N-glycans modulate the function of human corticosteroid-binding globulin

Human corticosteroid-binding globulin (CBG), a heavily glycosylated protein containing six N-linked glycosylation sites, transports cortisol and other corticosteroids in blood circulation. Here, we investigate the biological importance of the N-glycans of CBG derived from human serum by performing a structural and functional characterization of CBG N-glycosylation. Liquid chromatography-tandem MS-based glycoproteomics and glycomics combined with exoglycosidase treatment revealed 26 complex type N-glycoforms, all of which were terminated with α2,3-linked neuraminic acid (NeuAc) residues. The CBG N-glycans showed predominantly bi- and tri-antennary branching, but higher branching was also observed. N-glycans from all six N-glycosylation sites were identified with high site occupancies (70.5-99.5%) and glycoforms from all sites contained a relatively low degree of core-fucosylation (0-34.9%). CBG showed site-specific glycosylation and the site-to-site differences in core-fucosylation and branching could be in silico correlated with the accessibility to the individual glycosylation sites on the maturely folded protein. Deglycosylated and desialylated CBG analogs were generated to investigate the biological importance of CBG N-glycans. As a functional assay, MCF-7 cells were challenged with native and glycan-modified CBG and the amount of cAMP, which is produced as a quantitative response upon CBG binding to its cell surface receptor, was used to evaluate the CBG:receptor interaction. The removal of both CBG N-glycans and NeuAc residues increased the production of cAMP significantly. This confirms that N-glycans are involved in the CBG:receptor interaction and indicates that the modulation is performed by steric and/or electrostatic means through the terminal NeuAc residues.     

Effects of neurotrophic stress on maternal metabolism and binding of plasma cortisol in late pregnant guinea-pigs and their fetuses

The maternal metabolic clearance rate (MCR) and the binding of cortisol in the plasma of fetus and mother were estimated 8 days before term in conscious pregnant guinea-pigs, control or subjected to a neurotrophic stress (they were immobilized for 3 h in a dark room in front of an intermittent luminous flash). The maternal MCR of cortisol dropped during pregnancy related to an increase in corticosteroid binding globulin (CBG) and the stress mimicked these changes with a decrease in MCR and a rise in CBG. The increase in cortisol concentrations occurring in the fetal plasma after maternal stress could be related to the increase in free cortisol in the stressed mother. Nevertheless, although the fetal CBG did not rise after maternal stress, the increase in free cortisol in the fetal plasma remained moderate, due to the numerous free sites of CBG and albumin for cortisol.     

Mineralcorticoid receptor from rat kidney. Interaction with heparin and purification to a CBG-free stage

Purification of the mineralcorticoid receptor is a particularly challenging problem. This receptor is present in target tissues at concentrations lower and is less stable than any other steroid receptor. Addition of molybdate ions (20 mM) to rat kidney cytosol enhances stability of mineralcorticoid-specific binding sites: the inactivation rate at 0 degrees C decreases from 7.2 to 1.7% per hour in the absence of aldosterone, and from 1.8 to 0.3% per hour in the presence of hormone. Rates of inactivation in the presence of molybdate are thus compatible with purification procedures. Also, the corticosteroid-binding globulin (CBG) is an important contaminating component of kidney cytosol because it cannot be specifically blocked preliminarily to affinity chromatography. We show that when kidney cytosol is incubated with heparin covalently linked to Sepharose (Sepharose-heparin), after 30 min at 0 degrees C more than 80% of the mineralcorticoid-specific binding sites interact strongly with Sepharose-heparin while CBG is not bound at all. The mineralcorticoid-specific binding sites can be recovered from Sepharose-heparin by washing with heparin (2 mg/ml; recovery up to 90%), KCl (0.3 M; recovery up to 90%); and, less efficiently, with total liver RNA (2 mg/ml; recovery up to 55%) and dextran sulfate (2 mg/ml; recovery up to 40%); little or no recovery is achieved with chondroitin sulfate, sonicated DNA, pyridoxal-5-phosphate, dextran, d-glucosamine and d-glucuronic acid. With demonstration that also the mineral-corticoid receptor binds to heparin, this property has become a general hallmark of steroid receptors. If the "heparin" binding site of steroid receptors is of physiological significance it remains to be established. By application of the newly found property of the mineralcorticoid receptor, an overall 10-fold purified, CBG-free preparation of this receptor can be obtained from kidney cytosol with a single chromatography on Sepharose-heparin.     

Corticosteroid-binding globulin status at the fetomaternal interface during human term pregnancy

The status of the corticosteroid-binding globulin (CBG) at the fetomaternal interface, especially in the maternal intervillous blood space (I), was investigated and compared to that of CBG in the maternal (M) and fetal (umbilical arteries [A] and vein [V]) peripheral circulations at term. Immunoquantitation of plasma CBG showed that the CBG concentration in I was 30% less than that in M (P < 0.001) and threefold higher than that in umbilical cord blood (P < 0.001). The microheterogeneity of CBG studied by immunoaffinoelectrophoresis in the presence of concanavalin A and Western blotting indicated that the CBG in I was mainly of maternal origin and different from fetal CBG. A CBG mRNA, but no classic 50- to 59-kDa CBG, was found in isolated term trophoblastic cells. The steroid environment of the CBG in I differed greatly from that in the peripheral maternal and fetal circulations, because the progesterone:cortisol molar ratio in I was 75-fold higher than that in M and 7- to 10-fold higher than that in the fetal circulation. Binding studies revealed that the affinity constants of CBG for cortisol in I, A, and V were significantly lower than that in M plasma (P < 0.02) in their respective hormonal contexts. The binding parameters for I-CBG stripped of endogenous steroids and lipids were close to those for M-CBG but different from those of fetal CBG (P < 0.001). These data reflect the physiological relevance of the CBG-steroid interaction, especially with very CBG-loaded progesterone at the fetomaternal interface during late pregnancy.     

Equivalent affinity of aldosterone and corticosterone for type I receptors in kidney and hippocampus: direct binding studies

In studies from several laboratories evidence has been adduced that renal Type I (mineralocorticoid) receptors and hippocampal "corticosterone-preferring" high affinity glucocorticoid receptors have similar high affinity for both aldosterone and corticosterone. In all these studies the evidence for renal mineralocorticoid receptors is indirect, inasmuch as the high concentrations of transcortin (CBG) in renal cytosol make studies with [3H]corticosterone as a probe difficult to interpret, given its high affinity for CBG. We here report direct binding studies, with [3H]aldosterone and [3H]corticosterone as probes, on hippocampal and renal cytosols from adrenalectomized rats, in which tracer was excluded from Type II dexamethasone binding glucocorticoid receptors with excess RU26988, and from CBG by excess cortisol 17 beta acid. In addition, we have compared the binding of [3H]aldosterone and [3H]corticosterone in renal cytosols from 10-day old rats, in which CBG levels in plasma and kidney are extremely low. Under conditions where neither tracer binds to type II sites or CBG, they label an equal number of sites (kidney 30-50 fmol/mg protein, hippocampus approximately 200 fmol/mg protein) with equal, high affinity (Kd 4 degrees C 0.3-0.5 nM). Thus direct tracer binding studies support the identity of renal Type I mineralocorticoid receptors and hippocampal Type I (high affinity, corticosterone preferring) glucocorticoid receptors.     

Characteristics of aldosterone binding in rat and human serum

Binding of cortisol and corticosterone by serum proteins is well established, but discrepancies exist regarding aldosterone. We have observed that approximately 1% of 3H-aldosterone incubated with rat serum was bound in a time-dependent process, although it was not competed by a large excess of non-radioactive aldosterone, assessed by Florisil separation or gel filtration on Sephadex G-50 columns. After electrophoresis on cellulose acetate of rat serum incubated with 3H-aldosterone, specific or non-specific binding to protein fractions was not obtained. Further, a 10 000-fold molar excess of aldosterone (10 microM) displaced only 34% of the bound 3H-aldosterone to rat serum, preventing the calculation of the IC50 value. Increasing concentrations of aldosterone (3-83 nM) did not displace 3H-corticosterone bound in rat serum to presumably corticosterone binding globulin (CBG). In contrast, inhibition of this binding by 3-83 nM corticosterone was concentration dependent, showing an IC50 value of 10(-8) M. In normal human serum, binding of 3H-aldosterone demonstrated competition by a 100 and 1 000-fold excess of aldosterone. Displacement curves of 3H corticosterone bound to human serum by 1.7-75 nM corticosterone or 0.05-8.8 microM aldosterone yielded IC50 values in the range of 10(-8) M for corticosterone and 10(-6) M for aldosterone. With horse serum, aldosterone's binding affinity was three orders of magnitude lower than that of corticosterone. These studies suggest that in the rat aldosterone was loosely and weakly bound to a high capacity binder, possibly albumin. In agreement with the work of others, in humans aldosterone may be bound to both CBG and albumin. The current data do not substantiate for the presence of specific aldosterone binding proteins in serum.     

Polymorphism of blood serum transcortin during column chromatography

Serum protein bound progestins, androgens, estrogens, and cortexolone, were fractionated on a number of chromatographic systems. Contrary to earlier suggestions of a homogeneous unit by competition binding and Scatchard analysis, polymorphism and heterogeneity in the molecular nature of the corticosteroid binding globulin (CBG) were evident with progesterone on Sephadex A-25 columns. Components in the 55 000 and 67 000 molecular weight regions were obtained with cortexolone, estradiol, progesterone and testosterone on Ultrogel columns. Separation on DEAE-cellulose-52 resin revealed a fraction in the low ionic prewash followed by another, highly charged entity eluted only with 0.06 M phosphate buffers. Thus, under these conditions, serum protein bound sex steroids eluted in the same position as transcortin glucocorticoid complexes. These results are presented as a caution against the universal use of association dissociation assays to study steroid ligand binding and biological response. The techniques here detailed may fruitfully be employed to explore the hydrodynamic characteristics of protein ligand interactions. In addition, they support the model, earlier proposed with tissue specific steroid receptors, that calls for the various subunits of the ligand as a prerequisite for the expression of all grades of agonist and antagonist activity of a given hormone.