Tissue uptake of human sex hormone-binding globulin and its influence on ligand kinetics in the adult female rat.

The distribution of human sex hormone-binding globulin (hSHBG) and its influence upon the kinetics of its ligands were assessed in the adult female rat, which lacks a comparable protein in serum. Purified hSHBG was administered i.v. to adult female rats as a single bolus. The plasma disappearance rate of immunoreactive hSHBG had one component with a half-life of 15 h. The estradiol (E2) binding activity of serum attributable to hSHBG was elevated 2-fold; during a continuous infusion of E2, hSHBG increased E2 serum levels above those of control infused animals. Treatment with hSHBG did not modify the plasma clearance of endogenous E2, but accelerated the disappearance rate of 5 alpha-dihydrotestosterone (DHT). In animals injected with a tracer dose of radioactive steroids, pretreatment with hSHBG increased uterine and oviductal accumulation of E2- but not DHT-associated radioactivity. This effect was specific to some E2 target tissues since hSHBG did not alter the concentration of E2- or DHT-associated radioactivity in the hypophysis, liver, diaphragm, or brain. Treatment with anti-E2 antibodies, which elevated E2 binding activity in serum, decreased the accumulation of E2-associated radioactivity in uterus and oviduct. Immunofluorescent localization of hSHBG revealed intense labeling of the uterine and oviductal epithelium. We conclude that this foreign hormone-binding globulin introduced in serum at concentrations that have minimal circulating reservoir effect on E2 can reach intracellular domains and affect the concentration of this ligand in target tissues.     

Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum

Many estrogenic chemicals found in the environment (xenoestrogens) show a lower affinity for plasma estrogen binding proteins relative to the natural estrogens such as estradiol. These binding proteins, which include alphafetoprotein in rats and mice, sex hormone binding globulin in humans, and albumin in all species, regulate estrogen uptake into tissues. Therefore, the in vivo estrogenic potency relative to estradiol of xenoestrogens that show lower binding to these serum proteins will thus be underestimated in assays that compare the potency of xenoestrogens to estradiol and do not take serum binding into account. We have examined the effects of the binding components in serum on the uptake of a number of xenoestrogens into intact MCF-7 human breast cancer cells. Since most estrogenic chemicals are not available in radiolabeled form, their uptake is determined by competition with [³H]estradiol for binding to estrogen receptors (ER) in an 18-h assay. Serum modified access (SMA) of cell uptake of xenoestrogens is calculated as the RBA in serum-free-medium ÷ the RBA in serum, and the bioactive free fraction of xenoestrogen in serum is then also calculated. We predicted the concentration of two xenoestrogens, bisphenol A and octylphenol, required to alter development of the prostate in male mouse fetuses. Whereas octylphenol was predicted to be a more potent estrogen than bisphenol A when tested in serum-free medium, our assay predicted that bisphenol A would be over 500-times more potent than octylphenol in fetal mice. The finding that administration of bisphenol A at a physiologically relevant dose predicted from our in vitro assay to pregnant mice from gestation day 11 to 17 increased adult prostate weight in male offspring relative to controls (similar to the effect of estradiol), while the same doses of octylphenol did not alter prostate development, provided support for our hypothesis.     

Genetic polymorphism of the human sex hormone-binding globulin: evidence of an isoelectric focusing variant with normal androgen-binding affinities

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein composed of two identical subunits. The protein, which has high affinity for testosterone and estradiol has been purified to homogeneity. In this study we have investigated, on neuraminidase-treated serum samples, the presence of genetic variations of hSHBG by polyacrylamide gel isoelectric focusing (IEF). Based on IEF analyses of 110 serum samples from adult Mexican individuals we have identified two distinct IEF-patterns. The most frequent phenotype (95.45%) was characterized by two IEF-bands with pIs of 6.50 and 6.63, respectively. In five serum samples, a different 4-band pattern with pIs of 6.50, 6.63, 6.70 and 6.76 was identified. Family studies showed that this pattern was genetically determined. The frequency of this variant was 4.55%, and the observed phenotypes were consistent with the expression of an autosomal genetic system. The estimated gene frequencies for both alleles were shown to be in genetic equilibrium. Affinity constants, binding kinetics and serum concentrations of hSHBG from individuals having a 4-band pattern were similar to those obtained in individuals with a 2-band pattern, thus suggesting that the mechanism responsible for the generation of polymorphic variants of hSHBG reported herein did not involve the steroid binding site of the molecule. These findings may be of broad interest, as other serum binding proteins express genetic variants, which may permit their further structural and functional subclassification.     

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

Effects of aggressive encounters on plasma corticosteroid-binding globulin and its ligands in white-crowned sparrows

In birds, corticosteroid-binding globulin (CBG) binds corticosterone, progesterone and testosterone. The concentration of each ligand can alter the binding of the other ligands through competitive interactions. Thus, an increase in corticosterone or progesterone may displace testosterone bound to CBG, leading to an increase in bioactive free testosterone levels without affecting total testosterone levels in the circulation. Aggressive interactions increase plasma total testosterone levels in some birds but not in others. Here, we tested the hypothesis that aggressive encounters in the late breeding season would not increase total testosterone levels in plasma, but would alter CBG, total corticosterone or total progesterone levels in such a way as to modify the number of available binding sites and therefore occupancy by testosterone. A marked decrease in CBG occupancy by testosterone would indirectly suggest an increase in free testosterone levels in plasma. Wild male white-crowned sparrows were exposed to a simulated territorial intrusion (STI) or control for 30 min. Subjects were then caught and bled. We measured CBG using a ligand-binding assay and corticosterone, progesterone and testosterone using highly sensitive radioimmunoassays. STI significantly increased aggressive behaviors but did not affect plasma total testosterone levels. STI significantly increased plasma CBG and total corticosterone levels and decreased plasma total progesterone levels. We predict that CBG occupancy by corticosterone will increase slightly following an aggressive encounter. However, this small change is unlikely to increase free testosterone levels, because of the large number of seemingly unoccupied CBG binding sites in these subjects.     

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

Intravenous injection of human sex steroid hormone-binding globulin in mouse decreases blood clearance rate and testicular accumulation of orally administered [2-125I]iodobisphenol A

Bisphenol A, an environmental compound with estrogenic activity, has been shown to bind human sex steroid hormone-binding globulin (hSHBG), the main plasma transport protein which regulates the metabolism of androgens and estrogens and limits their access to target organs. The present study was conducted to determine whether physiologically relevant concentrations of hSHBG can influence the blood clearance rate of bisphenol A and its accumulation in the testes. A radioactive [2-125I]iodobisphenol tracer was synthesized with an association constant (Ka) for binding to hSHBG of 0.14 +/- 0.01 x 10(6) M(-1) at 37 degrees C, a value much lower than for [2-125I]iodoestradiol, which was also synthesized. We used i.v. injection of immunopurified hSHBG in adult male mice to maintain hSHBG levels within the physiologically possible range for humans (27-267 nM) before gavage administration of [2-125I]iodobisphenol or [2-125I]iodoestradiol, for measuring the blood clearance rate of radioactive signal in blood samples taken during the following 120 min. Testicular accumulation of radioactivity was measured 24 h and 48 h after gavage of [2-125]iodobisphenol A. In mice receiving immunopurified hSHBG or vehicle, the time-dependent blood clearance of radioactivity exhibited a bi-exponential decrease which indicated alpha-diffusion and beta-elimination phases for both radioactive ligands. The presence of circulating hSHBG significantly and dose-dependently lowered the clearance rate of radioactivity. However, much higher circulating levels of hSHBG were required to retard the blood clearance of [2-125I]iodobisphenol A as compared to those required for [2-125I]iodoestradiol, in keeping with the important difference in their respective Ka value for binding to SHBG. In addition, mice treated with hSHBG exhibited significantly (P = 0.036) reduced testicular accumulation of radioactivity 24 h and 48 h after ingestion of [2-125I]iodobisphenol A. Provided that the binding properties of bisphenol A for hSHBG are not substantially different from those measured for [2-125I]iodobisphenol A, these findings suggest that, although hSHBG binds 2-mono-iodobisphenol A with a relatively low binding affinity, high enough concentrations of circulating hSHBG (range concentrations between 85 and 267 nM) are potentially able to exert a protective effect against exposure to bisphenol A.     

Interactions between human plasma sex hormone-binding globulin and xenobiotic ligands

Human sex hormone-binding globulin (SHBG) binds sex steroids with high affinity. In plasma, the number of SHBG steroid-binding sites far exceeds the molar concentrations of sex steroids, and will accommodate other ligands such as phytoestrogens and fatty acids. We have therefore developed a screening assay to identify ligands for SHBG, which exist in our diet or environment. This assay allows the binding of potential ligands to SHBG to be assessed under physiological conditions, and is sensitive to the effects of plasma constituents. Several classes of endocrine active compounds were tested, including hydroxy-polychlorinated biphenyls (HO-PCBs), phthalate esters, monoesters, chlorinated pesticides, as well as synthetic estrogens and phytoestrogens. The relative binding affinities (RBAs) of various compounds to SHBG were determined in competitive displacement assays, by comparison with 17 beta-estradiol (RBA=100). Synthetic estrogens bound SHBG with RBAs of 0.4 (ethinylestradiol)-0.2 (diethylstilbestrol), while some phytoestrogens bound with RBAs of 0.12 (coumestrol)-0.04 (naringenin). Many compounds did not bind to SHBG with sufficient affinity to allow RBA measurements, and these include: several phytoestrogens, such as genistein and kaempferol, polychlorinated biphenyls, phthalate esters and monoesters. Of nine HO-PCB congeners tested only 4-OH-2', 3', 4', 5'-tetraCB and 4-OH-2, 2', 3', 4', 5'-pentaCB bound SHBG in undiluted serum with RBAs of 0.05 and 0.11. Although all test compounds bound to SHBG with much lower affinity than endogenous sex steroids, these interactions may be physiologically relevant in situations where plasma SHBG levels are high and endogenous sex steroid levels are low, such as in pre-pubertal children and women taking oral contraceptives.     

Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein that binds sex steroids with high affinity. Variations in hSHBG glycosylation contribute to its electrophoretic microheterogeneity, but the functional significance of different SHBG glycoforms is unknown. Carbohydrates may influence the biological activities and half-lives of glycoproteins and we have examined how oligosaccharides at specific sites influence the plasma clearance of hSHBG in vivo. To accomplish this, fully-glycosylated hSHBG, or hSHBG mutants lacking specific oligosaccharides chains, were expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography. The purified recombinant proteins were then biotinylated to study their plasma half-lives after intravenous injection into rabbits. When compared to hSHBG isolated from serum, recombinant hSHBG migrates with a slightly larger average molecular size during denaturing polyacrylamide gel electrophoresis. This is due to a greater proportion (33–39% vs. 3%) of more highly branched N-linked oligosaccharides on the recombinant proteins. When injected into rabbits, the disappearance of recombinant hSHBG showed two exponential components, as previously shown for natural hSHBG in the same animal model. The mean±S.E.M. plasma half-lives of recombinant hSHBG (t_1/2 0.11±0.03 h and t_1/2β 18.94±1.65 h) are shorter than previously measured for natural hSHBG (t_1/2 3.43±0.72 h and t_1/2β 38.18±7.22 h) and this is likely due to differences in the composition of their N-linked oligosaccharides. An O-linked chain at Thr⁷ does not influence the plasma clearance of hSHBG in the presence or absence of N-linked carbohydrates at Asn³⁵¹ and Asn³⁶⁷. However, a 1.5–1.6 fold (p<0.03) increase in plasma half-life of variants lacking both N-glycosylation sites was observed and this is probably due to the fact these variants are not recognized by the asialoglycoprotein receptor-mediated clearance system. Removal of either N-glycosylation consensus site also increased (p<0.0001) the plasma half-life of hSHBG by 2.3–2.4 fold. Thus, the metabolic clearance of hSHBG appears to be determined by the number of N-linked oligosaccharides rather than their location.     

Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.)

A specific sex steroid-binding protein (SBP) is believed to be involved in regulation of circulating sex steroids, steroid delivery to target cells and intracellular signalling in sex steroid-sensitive tissues. In the present work, interactions between xenoestrogens and the plasma SBP in Arctic charr (Salvelinus alpinus L.) were determined using ligand-protein binding studies. The test compounds were all able to displace tritiated 17 beta-estradiol (E2) from the Arctic charr SBP (acSBP) in a competitive and dose-dependent manner. The acSBP affinities for the xenoestrogens ranged over several orders of magnitude (17 beta-estradiol>ethynylestradiol (EE2)>zearalenone (ZEA)>diethylstilbestrol (DES)>genistein (GEN)>bisphenol A (BPA), 4-t-octylphenol (OP)>o,p'-DDT, and dieldrin (DIN)), but were consistently lower than that of 17 beta-estradiol (about 4 x 10(2) -10(6)-fold less potent). The relative binding affinity (RBA) for selected chemicals were independent of both gender, age and maturation status, as well as variations of acSBP binding affinity. The affinity of endogenous steroids and estrogen mimics for the acSBP shows a high correlation to the affinity for the rainbow trout SBP, thus suggesting a phylogenetically conserved ligand-binding site between closely related species. Furthermore, it is argued that interaction with the acSBP- and SBP-mediated processes may introduce novel pathways for endocrine disruption, which may work in concert with the classical receptor-mediated effects.     

Endogenous estradiol and testosterone levels are associated with cognitive performance in older women and men

Relatively few studies have investigated the relationship between endogenous sex steroid levels and cognition in older people and the reported results have been inconsistent. A number of experimental hormone replacement studies have suggested that estrogen replacement in older women enhances cognition, especially verbal memory. In contrast, little research has been done focusing on men. In the current study the association between endogenous sex steroids (estradiol and testosterone) and cognition was investigated in 38 healthy older women (mean age 68 years) and 30 healthy older men (mean age 69 years). Five cognitive tests measuring verbal memory, spatial memory, verbal fluency, mental rotation, and susceptibility to interference were administered. Results revealed that in women higher estradiol levels as well as testosterone levels were associated with better verbal memory (paired associates and estradiol; r =.38, P < 0.05; paired associates and testosterone; r =.33, P < 0.05;). Moreover estradiol, but not testosterone was associated with less susceptibility to interference (Stroop color word test; r = -0.34, P < 0.05). In men the only significant association was a negative correlation between testosterone and verbal fluency (r = -0.38, P < 0.05). The associations observed in this small study support the notion that estradiol is protecting verbal memory and possibly also frontal lobe mediated functions in older women. In contrast to the positive findings in women endogenous sex steroids do not appear to be closely linked to better cognition in older men.     

Characterization and partial purification of an estrogen type II binding site in chick oviduct cytosol

An estrogen binding site of moderate affinity (Kd approximately 10 nM) and high capacity (approximately 25-70 pmol/g of tissue) was measured in DES-stimulated chick oviduct cytosol. Saturation analysis by [3H]estradiol exchange demonstrated that this binding site displayed sigmoidal binding characteristics suggesting a cooperative binding mechanism. Competition analysis with a number of compounds demonstrated that the bioflavonoid luteolin was a better competitor for binding to type II sites in chick than either estradiol or DES. Steroid specificity was demonstrated by the inability of 17 alpha-estradiol, progesterone, testosterone, corticosterone, and the triphenylethylene antiestrogen nafoxidine (U-1100A) to compete for [3H]-17 beta-estradiol binding to chick oviduct cytosol preparations. In addition, the binding site appeared to be sensitive to sulfhydryl reducing reagents as evidenced by a 75% reduction in binding activity in the presence of dithiothreitol. Both prelabeling and postlabeling procedures used in conjunction with Sephacryl S-300 chromatography resulted in a single major peak of type II binding activity representing a molecular weight in the 40,000 range. Type II binding activity was recoverable after precipitation with ammonium sulfate, and this material was subjected to a variety of column chromatography procedures in order to achieve further purification of the type II site. Significant purification of the site was achieved with a bioflavonoid-Sepharose (quercetin-Sepharose) affinity matrix. The purified type II sites eluted from quercetin-Sepharose displayed the same sigmoidal binding curves characteristic of native cytosol.     

Quantification of cytosolic steroid receptors in secretory and non-secretory epithelial cells of the canine prostate

Radiolabelled methyltrienolone, dihydrotestosterone and estradiol were used as ligands to identify and quantify androgen and estrogen receptors in freshly dispersed cells from the canine prostate. Soluble extracts (cytosols) were obtained from secretory and non-secretory epithelial cells separated on the basis of their density in Percoll gradients. For both cell types, as well as for the whole prostate, Scatchard plot analyses were linear and showed a single class of high affinity binding sites: Kd values of 3.6 +/- 2.2 X 10(-9) M and 3.0 +/- 1.2 X 10(-10) M were measured for the androgen and estrogen receptors, respectively. The number of binding sites for the cytosolic androgen receptor, expressed per mg of protein or per mg of DNA, was 2.4- to 6.7-fold higher in the non-secretory cells compared to the secretory cells. However, these two cell types contained a similar number of specific sites for the estrogens. The specificities of the androgen and estrogen receptors were shown to be identical for the two cell types: the binding of [3H]R1881 was strongly inhibited by unlabelled R1881, 5 alpha-androstane-3 alpha, 17 beta-diol and dihydrotestosterone, while 5 alpha-androstane-3 beta, 17 beta-diol, estradiol and estrone did not displace bound R1881. The addition of triamcinolone acetonide did not alter the binding of R1881 in extracts of either cell type or in the whole prostate. The binding of [3H]estradiol to the estrogen receptor was highly specific since a strong displacement was only observed with estradiol (83%).     

Radiochemical determination of estrogen receptors in cryostat sections of target tissues

A radioreceptor assay on unfixed cryostat sections has been developed. Mounted and dried sections were incubated with radiolabeled estradiol in the absence and presence of an excess of diethylstilbestrol and washed with buffer. Binding of radiolabel to sections was measured by direct liquid scintillation counting. Also protein-bound radioactivity which eluted from the sections was determined with a dextran-coated charcoal assay. Parallel sections were used for histological staining and protein determination. Scatchard analysis showed the presence of specific saturable binding sites for estradiol with dissociation constants in the 0.1-1.5 nM range. It is concluded that these high affinity and limited capacity (type I) binding sites represent estrogen receptors. The ligand-binding activity of section-bound estrogen receptors did not decrease upon dry storage up to 20 h at 4 or 23 degrees C prior to assay. During aqueous incubation a significant amount of receptor, representing about 40-60% of the total tissue content, elutes from the sections. Steroid specificity was proven by incubation with excess androgen, progestogen or corticoid instead of diethylstilbestrol or estradiol. With these ligands no significant competition was found. Tissue specificity was demonstrated by a very low level of specific estradiol binding to cryostat sections of rat skeletal muscle, spleen and intestine and by a moderate level in rat liver.     

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.     

Concentration of estradiol,testosterone and progesterone in cerebrospinal fluid compared to plasma unbound and total concentrations

Estradiol, progesterone and testosterone are measured in plasma and CSF in 17 women and 11 men. The results show a transfer from plasma to CSF of about 4% for estradiol, 10% for progesterone and 2.5% for testosterone. There was found to be a clear correlation between the plasma and CSF levels of these steroids. A comparison is also made between the calculated levels of unbound estradiol and testosterone in plasma and the levels in the CSF. The results show approximately the same concentrations of these steroids in the CSF as the calculated levels of unbound steroids in the plasma.     

Relative binding affinity of norgestimate and other progestins for human sex hormone-binding globulin

The relative binding affinity of norgestimate for human sex hormone-binding globulin was compared with that of its metabolites and other progestins by measuring their abilities to displace [3H]testosterone from this carrier protein in vitro. Norgestimate and its 17-deacetylated and 3-keto metabolites did not significantly displace [3H]testosterone from sex hormone-binding globulin at concentrations up to 10,000 nM, whereas gestodene, levonorgestrel, and 3-keto desogestrel displaced [3H]testosterone from sex hormone-binding globulin with IC50 concentrations of 23.1, 53.4, and 91.0 nM, respectively. Since it is believed that a progestin may exert androgenic effects by displacing testosterone from sex hormone-binding globulin, thereby increasing circulating levels of free, active testosterone, these data are consistent with the results of preclinical and clinical studies demonstrating the selective progestational activity of norgestimate.     

Methyl p-hydroxyphenyllactate. An inhibitor of cell growth and proliferation and an endogenous ligand for nuclear type-II binding sites

We previously described and partially characterized endogenous ligands for nuclear type II sites in normal and malignant tissues. Chromatography of these ligands on Sephadex LH-20 revealed that two peaks with binding activity (alpha and beta) could be resolved. The beta-peak component was present in all normal tissues that we examined, but not in malignant tissues, and it inhibited the growth of MCF-7 human breast cancer cells in vitro. Conversely, the alpha-peak component was found to be present in both normal and malignant tissues, and did not inhibit MCF-7 cell growth. The present studies describe the purification and identification of the alpha-peak and beta-peak components in bovine serum and an assessment of the effects of these compounds on normal and malignant cell growth. Gas chromatography-mass spectroscopy analysis of the purified beta-peak component demonstrated that the compound was methyl p-hydroxyphenyllactate (MeHPLA). Competition analysis revealed that MeHPLA binds to nuclear type II sites with a high binding affinity, while physiological levels of this compound blocked estradiol stimulation of uterine growth in vivo and inhibited the growth of MCF-7 human breast cancer cells in vitro. The alpha-peak component was found to be the corresponding acid, p-hydroxyphenyllactic acid (HPLA). This compound interacted with nuclear type II sites with a relatively low affinity and did not block uterotropic response to estradiol or inhibit MCF-7 cell growth. These studies demonstrate that HPLA and MeHPLA are ligands for nuclear type II sites and that MeHPLA may be a very important regulator of normal and malignant cell growth.     

The temperature dependence of the binding of 5 alpha-dihydrotestosterone, testosterone and estradiol to the sex hormone globulin (SHBG) of human plasma

The binding of 5 alpha-dihydrotestosterone (DHT), testosterone and estradiol to the sex hormone binding globulin (SHBG) and albumin in human plasma has been studied at 4, 20 and 37 degrees C using the method of equilibrium partition in an aqueous two-phase system based on dextran, poly(ethylene glycol) and water. The intrinsic association constants for the binding to SHBG and the apparent association constant for the binding to albumin have been determined from Scatchard-type binding plots. The affinity of SHBG for DHT is 1.2-1.3 times higher than that for testosterone and 4 times higher than that for estradiol. The affinity of SHBG for the steroids decreases with increasing temperature. The mean values of the free energy of binding, delta G degree, in the temperature range used are -52.3, -51.7 and -48.9 kJ X mol-1 for the binding of DHT, testosterone and estradiol, respectively, to SHBG. The corresponding values of the enthalpy change, delta H degree, are 73.7, 70.0 and 99.0 J X mol-1 X K-1. These values are discussed in terms of the difference in the structure of the steroids. The affinity of albumin for testosterone and estradiol is almost equal and is lower than that for DHT. The delta G degree for the binding to albumin is about 55% lower than that for the binding to SHBG.     

19F NMR studies of tryptophan/serum albumin binding

19F NMR provides direct measures of the Trp binding avidity of 'fatty acid free' bovine serum albumin when D- and L-6-fluorotryptophan are used as the probes. Both a high and low affinity binding site are present. The addition of octanoate either displaces the ligand from both sites or greatly decreases the affinity such that little binding occurs at 2 mM levels. In the case of L-6-fluorotryptophan separate signals are observed for the high and low affinity binding sites and titrations with competing ligands can be used to establish the relative affinities of ligands at the high affinity site. Binding at this site appears to be hydrophobic and shape specific with L-Phe being a very poor ligand (K(D)[L-Phe]/K(D)[L-Trp]=800) while both GHKalphaNal and GHKW displace L-6-fluorotryptophan from this site. In tripeptides of the general formula GHK[ epsilon NH(CH(2))(n)(CO)W], affinity increases with tether length and binding at the low affinity site is restored. This NMR assay appears well-suited for the discovery of selective binding agents in this and other biorecognition phenomena.