Ligand properties of diethylstilbestrol: studies with purified native and fatty acid-free rat alpha 1-fetoprotein and albumin.

We report the equilibrium binding parameters for the interactions of the estrogen analogue diethylstilbestrol (DES) with highly purified rat alpha 1-fetoprotein (AFP) and serum albumin preparations. At 25 degrees C and pH 7.4, an association constant (Ka) of about 1.5 X 10(6)M-1 and 2 sites/mole are measured with the DES-AFP system, whereas for the DES-albumin interaction, we find a Ka of approximately 2 X 10(5)M-1 and about 11 sites/mole of protein. The removal of fatty acids from pure AFP causes a reversible 3 fold increase of the number of DES binding sites; the same delipidation procedure applied to albumin slightly diminishes its DES binding parameters. We also demonstrate the capability of DES to displace competitively estradiol-17 beta (E2) from its high affinity sites on the estrophilic rat AFP. Finally, the binding behaviour of the two serum proteins towards the synthetic estrogen is compared to their interaction with the natural hormones. The physiological and pharmacological relevance of these data is discussed.     

A surprising increase in the affinity of estrogen for rat alpha-fetoprotein after it is treated with N-ethylmaleimide

Incubation of rat alpha-fetoprotein (AFP) with mM concentrations of N-ethylmaleimide (MalNEt) at 22 degrees, pH7.8 for several hours yields a form of AFP with an equilibrium dissociation constant of 0.1 nM for estrone. This affinity for estrone is about 10 fold higher than that for untreated rat AFP. MalNEt-treated AFP still binds inhibitors and substrates of serine proteases suggesting that the binding of these compounds to AFP does not involve an interaction with a thiol group. MalNEt-treated AFP may be useful for studying the mechanism of estrogen action in vitro and in vivo, estrogen-protein interactions, and the functioning of AFP.     

A stoichiometric analysis of bovine serum albumin-gossypol interactions: a fluorescence quenching study.

The interaction of gossypol with bovine serum albumin, human serum albumin and n-bromosuccinimide-modified bovine serum albumin has been followed by fluorescence quenching measurements. The presence of a high affinity site (association constant K = 2.2 x 10(6) M-1) for gossypol on bovine serum albumin and human serum albumin is indicated. The stoichiometry of binding for the high affinity site was evaluated using Job's method of continuous variation, thereby suggesting the formation of 1:1 complex. Modification of the tryptophan residues on bovine serum albumin does not affect the binding of gossypol to either high or low affinity site of albumin.     

A diphenylmethane derivative specific for the antiestrogen binding site found in rat liver microsomes

A new para-diphenylmethyl derivative, N,N-diethyl-2-[(4-phenylmethyl)-phenoxy]-ethanamine·HCl (N,N-DPPE) has been synthesized which binds with high affinity to the anti-estrogen binding site found in male rat liver microsomes. However, no evidence of significant interaction with the estrogen receptor can be observed at or below 10 μM in rat uterine cytosols; 10 nM N,N-DPPE fails to significantly induce progesterone receptor in MCF-7 cells. Tamoxifen also binds to anti-estrogen binding site but, unlike N,N-DPPE, binds significantly to estrogen receptor at much loeer concentrations and induces MCF-7 progesterone receptor. This property of high affinity for anti-estrogen binding site but not for estrogen receptor may make N,N-DPPE an important probe for the study of anti-estrogen binding site and its biological relevance.     

Evidence for a common high affinity binding site on glutathione S-transferase B for lithocholic acid and bilirubin

Binding of lithocholic acid, bilirubin, and gossypol to glutathione S-transferase B (ligandin or transferase YaYc) was compared using four methods. Tryptophan quenching revealed a single high affinity site for bilirubin and gossypol but could not be used for lithocholic acid. Both displacement of the fluorescent probe, 1-anilino-8-naphthalenesulfonate, and spectral changes induced by bilirubin binding demonstrated a common high affinity site for which all three ligands compete. Similar results were obtained by equilibrium dialysis. The dissociation constants for the binding of both bilirubin and lithocholic acid were comparable with the various methods (range 0.2-0.7 microM). Thus, lithocholic acid and bilirubin share a high affinity binding site on gluthathione S-transferase B that appears to be separate from the binding site for substrates.     

alpha-fetoprotein binding specificity for arachidonate, bilirubin, docosahexaenoate, and palmitate. A spin label study

A dianionic spin label, 1-L-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-i,4-dinitrobenzene, has been used to probe the relative binding specificity of a single anionic ligand site on bovine alpha-fetoprotein (AFP) to arachidonate, bilirubin, docosahexaenoate, and plamitate. The binding isotherm of the spin label with AFP, as shown by a Scatchard plot, indicates the presence of a single high affinity binding site. The site-site relationship of the four endogenous ligands, arachidonate, bilirubin, docosahexaenoate, and palmitate, was determined by studying their effectiveness in competing for this anionic ligand binding site on AFP. Scatchard plots of the spin label in the presence of 1 to 3 molar equivalents of arachidonate, bilirubin, and docosahexaenoate and up to 6 molar equivalents of palmitate have been determined. The effectiveness of the four endogenous ligands in displacing the spin label from its primary binding site is bilirubin greater than or equal arachidonate approximately equal to docosahexaenoate greater than palmitate. These results indicate that polyunsaturated essential fatty acids and bilirubin share a high affinity binding site on AFP. We propose that the function of this anionic ligand binding site on AFP is for the transport of bilirubin and polyunsaturated fatty acids in fetal serum, as well as for the cross-placental transfer of this metabolite and of essential fatty acids.     

Diethyl pyrocarbonate, a histidine selective reagent, inhibits estrogen binding to receptor protein in rat uterus cytosol

We find that at pH 6.1 diethyl pyrocarbonate inhibits estrogen binding to its receptor protein in rat uterus. Hydroxylamine partially reverses this inhibition and estrogen partially protects its receptor protein from this inhibition. We suggest that the estrogen receptor protein in rat uterus contains a nucleophilic site that either overlaps or is near the estrogen binding site. Based on the pH of inhibition reaction, the receptor concentration in the experiment, and the partial reversal of the inhibition by hydroxylamine, we suggest that this site contains a histidine residue or possibly an unusually reactive tyrosine residue that is important for estrogen binding.     

Immunological demonstration of alpha-fetoprotein in uterine cytosol from immature rats

An immunological demonstration of alpha-fetoprotein (AFP) in uterine cytosol from immature rats is reported. The identity between serum AFP and the estrogen binding 4.5 S macromolecular complex of uterine cytosol was demonstrated by the use of a specific immunoadsorbent to AFP. Analysis of the sedimentation profile in glycerol gradients of uterine cytosol incubated with tritiated estrone or estradiol suggests that the total estrogen binding capacity of the 4.5 S complex is provided by AFP. Changes of AFP content in rat uterus with the age of the animals suggest that this protein is probably present in the cytosol as a serum contaminant.     

Differential binding of antiestrogens by rat uterine and chick oviduct cytosol

Analysis of the interactions of two synthetic estrogen antagonists, tamoxifen and CI 628, with rat uterine and chick oviduct cytosol revealed significant differences in the antiestrogen binding properties of these tissues. In the rat uterus CI 628, tamoxifen and estradiol were bound to a similar number of saturable binding sites and estradiol could completely inhibit the binding of tritiated antiestrogens to these sites. In contrast, high affinity, saturable antiestrogen binding sites in chick oviduct were present at three times the concentration of estradiol binding sites and estradiol could only partially inhibit the binding of tritiated antiestrogens to these sites. It is concluded that antiestrogens bind to the estrogen receptor in both tissues and that chick oviduct has an additional saturable antiestrogen binding site distinct from the classical estrogen receptor site.     

Affinity chromatography of mouse and rat alpha-fetoproteins on immobilized diethylstilbestrol]

Alpha-fetoproteins (AFP) from amniotic fluid of mouse and rat demonstrate high affinity and specificity during their binding with immobilized diethylstilbestrol, which allows to isolate these two proteins by one step using the method of affinity chromatography on Sepharose with immobilized diethylstilbestrol. Meanwhile the yield of mouse AFP was 42%, and rat AFP--75%. The preliminary incubation of the amniotic fluid of rat and mouse with free estradiol results in abrupt fall of AFP outcome, which may testify to the binding of estradiol and diethylstilbestrol by the same receptor sites on AFP molecule.     

Photoaffinity labeling of rat alpha-fetoprotein

Two photosensitive estrogen derivatives, 16-diazoestrone and 4-azidoestradiol, have been studied as photoaffinity-labeling agents for the estrogen-binding site of rat alpha-fetoprotein (AFP). 16-Diazoestrone has a high affinity for AFP (121%, relative to 17 beta-estradiol), and photolysis of the 16-diazo[3H]estrone . AFP complex for 30 min at 300 nm results in the covalent attachment of 19% of the ligand bound reversibly to the estradiol site at the time of irradiation. The photocovalent attachment appears to result from both a "chromophore-dependent" process (photoaffinity labeling), whose time course follows the photolytic consumption of the diazoketone chromophore and is not susceptible to scavenging by nucleophiles, and a "chromophore-independent" process (pseudophotoaffinity labeling) that results from covalent attachment of an electrophilic photoproduct and can be intercepted by 20 mM mercaptoethanol. AFP covalently labeled with 16-diazo[3H]estrone has the same electrophoretic mobility as unlabeled AFP on normal and sodium dodecyl sulfate-polyacrylamide gels; labeled AFP has an apparent molecular weight of 69,400 and is distinguishable from albumin (which is also labeled by 16-diazo[3H]estrone, but not in a site-specific manner). While 4-azido[3H]estradiol undergoes extensive photoinduced covalent attachment to AFP, little of this is site-specific.     

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.     

Estrogen-binding properties of rat serum alpha 1-fetoprotein and its isoforms. Investigation of the apparent non-integrality of sites on the unfractionated protein.

Rat fetal serum alpha 1-fetoprotein (AFP), a heterogeneous glycoprotein, binds estrogens with high affinity but at a fractional number of sites even after treatment with charcoal (n = 0.6), which may mean 60% of the protein has 1 site and the remainder none. To investigate the origin of this fractional number of sites the "native" protein (purified by negative affinity chromatography) was further purified (step 1) and fractionated (step 2) into its two main charge variants (electrophoretically "slow" and "fast") by a two-step fast-protein liquid chromatography method. The binding parameters for estrone and estradiol-17 beta of the "native" and "repurified" proteins and of each charge variant were determined by equilibrium microdialysis. The molar extinction coefficient at 278 nm of each sample was also determined. (1) The "repurified" AFP and each charge variant had a number of binding sites for estrogens close to unity. This increase in the number of sites could neither be explained by the loss of a non-binding isoform (corresponding to 40% of the protein) during chromatography, nor by the existence of complex negative modulatory interactions between isoforms. (2) The affinities for estrogens of the "repurified" protein and the two charge variants were slightly decreased compared to that of "native" AFP, except that the "fast" form had the "native" protein's high affinity for estrone--but not for estradiol-17 beta. (3) The molar extinction coefficients at 278 nm of the "repurified" AFP and the isoforms were much lower than that of the "native" protein. These results suggest that the presence of (an) inhibitor(s) of estrogen binding on the "native" protein which is/are removed by the ion-exchange fast protein liquid chromatography (FPLC) column. A ligand absorbing at 278 nm, which may or may not be the inhibitor, is also removed. The isoform heterogeneity with respect to estrone binding is discussed.     

Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.     

Hydroxylated 2,3-diarylindenes: synthesis, estrogen receptor binding affinity, and binding orientation considerations

In order to develop high affinity, fluorescent ligands for the estrogen receptor based on 2-arylindenes, it is important to understand how this non-steroidal estrogen is oriented within the binding site and to know how hydroxyl substituents affect binding. To investigate these issues a series of dihydroxyl-substituted 2,3-diphenylindenes were prepared by the cyclization of appropriately substituted alpha-benzyldesoxybenzoins, and their binding affinities for the estrogen receptor measured by a competitive radiometric binding assay. Introduction of a p-hydroxyl group in the 2-phenyl ring of two 2,3-diphenyl-6-hydroxyindene systems causes a 3-fold increase in binding affinity, whereas, p-hydroxylation in the 3-phenyl ring of these systems causes a 2-fold reduction in binding affinity. The parallel change in binding affinity in these two systems suggests a consistent binding orientation of the 2,3-diarylindene systems, which, on the basis of earlier studies, has the indene system corresponding to the A/B-ring system of estradiol. This orientation model and the enhanced affinity of the p-hydroxy 2-ring derivatives are suggestive of a new hydrogen bonding site below the D-ring binding site. Changes in receptor binding affinity upon hydroxylation in triphenylacrylonitrile ligands for the estrogen receptor, reported by others, do not show such parallelism, suggesting that different derivatives may not be bound in congruent orientations. A m-hydroxyl substituent in ring-3 of the 2,3-diarylindene has very little effect on receptor binding. In designing fluorescent 2,3-diarylindene ligands for the estrogen receptor, 3-ring hydroxylation may be useful in reducing non-specific binding and in modifying electron donation to the fluorophore with only modest or no reduction in binding affinity. p-Hydroxylation of the 2-ring, although increasing receptor binding, is not consistent with the electron accepting nature required of this ring.     

Alpha-fetoprotein favours accumulation of estrone but not arachidonic acid into the fetal and new-born rat brain

Tritriated estrone or arachidonic acid, two high affinity ligands for rat alpha-fetoprotein (AFP), were injected into adult, pregnant or newborn Sprague Dawley rats in order to evaluate their possible transfer into the brain. This study shows that the developing brain accumulates the estrogen but not the fatty acid, suggesting that the uptake of AFP by the developing brain is a mechanism for transporting estrogens, but not fatty acids.     

Estrogen binding sites in the sea scallop: characterization and possible involvement in reproductive regulation

Previous reports have suggested that estrogen is involved in bivalve reproduction and have also hypothesized that its effects are mediated through binding sites on specific receptors. In this study, we provide initial characterization of the estrogen binding sites in the gonads of both female and male sea scallops (Placopecten magellanicus). Saturation analyses indicated two binding sites in fractions which have classically been used to represent the cytosol and the nucleus. One binding site is characterized by high affinity and limited binding capacity while the other site is characterized by low affinity and high capacity. Competitive binding analyses demonstrated that these sites can bind natural and synthetic estrogens with high affinity but only bind testosterone and progesterone at high concentrations. Comparison of binding capacity in scallops at different sexual maturation stages suggested that these sites may be involved in reproductive regulation in sea scallops.     

Binding of radiolabelled luteinizing hormone to intact and ovariectomised rat uterus.

Binding of ovine LH to uterine tissue preparation from intact and ovariectomised rat clearly indicates that uterus possesses specific binding sites for LH. Binding characteristics of LH to uterine tissue preparation from intact rat showed saturability with high affinity and low capacity. Scatchard plot analysis showed dissociation constant of the specific binding site to be 0.12 x 10(-9) mol/l and the number of binding sites was 2.31 +/- 0.05 f mol/mg protein. Ovariectomy did not change the binding affinity but effected a decrease in the number of binding sites (1.7 +/- 0.08 f mol/mg protein). LH treatment of ovariectomized (ovx) rat had no effect on binding affinity but significantly increased the number of binding sites (3.23 +/- 0.1 f mol/mg protein). Reduction of uterine weight due to ovariectomy and marked increase of ovx rat uterine weight by LH administration indicate a source of estrogen in ovx rat. An in vitro uterine tissue slice (from intact and ovx rat) incubation showed depletion of 17 beta-estradiol (E2) content in ovx rat which significantly elevated on LH addition. Data suggest that LH binding to rat uterine tissue has biological relevance.