Binding Sites For Testosterone Research Articles

The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells.

Simple SummaryPro-androgenic substances such as testosterone are often used to treat muscle- or bone-related disorders. Their interactions with the classical androgen receptor, however, can trigger a number of undesirable effects. It would therefore be of great benefit if the positive androgenic effects could be obtained by circumventing the classical androgen receptor. ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance. Using in silico methods, we identified and verified the extracellular localization of its androgen binding site and designed small peptides that fit in it that do not interact with the AR. All peptides were found to be pro-androgenic; they stimulate mineralization in osteoblastic cells and myogenesis in myoblasts. Thus, these peptides might serve as testosterone surrogates in the treatment of osteogenic or myogenic disorders.ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10(8) clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR-L3 and CDR-H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three-dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire.

Oestradiol and testosterone binding sites in mice tibiae and their relationship with bone growth.

High affinity oestradiol and testosterone binding sites were found in tibiae cytosol from entire male and female of different ages. Scatchard assay allowed to estimate a Kd of 2.7 X 10(-9) M for oestradiol binding sites indicating that the 3H-oestradiol binding was of high affinity. Oestradiol and testosterone binding sites abundance in mice tibiae are subject to change with age. It is not easy to establish a direct correlation between these changes and the values reported here on bone growth in weight and length, however seems possible to point a negative relationship between bone lengthening and oestradiol binding site levels in female, as well a positive relationship with testosterone in both sexes. The presence of oestradiol and testosterone binding sites in epiphyses and not in the diaphyses reinforces the hypothesis that both are playing some role in bone growth.

ISOLATION AND CHARACTERIZATION OF A cDNA FROM ATLANTIC CROAKER (Micropogonias undulatus) OVARIES ENCODING A NOVEL PROTEIN WITH THE CHARACTERISTICS OF A MEMBRANE ANDROGEN RECEPTOR

There is now extensive evidence that in addition to the wellcharacterized genomic actions mediated through nuclear receptors, steroids can also exert “nonclassical” rapid actions initiated at the cellsurface. However, the identities of the receptors that mediate many of these nonclassical steroid actions remain unresolved. Both nuclear receptors and novel receptors for estrogens and progestins have been identified in the plasma membranes of a variety of target cells. In contrast, there is a relative lack of information on the identities of membrane receptors mediating nonclassical androgen actions. In the present study, a protein displaying androgen binding characteristics typical of androgen membrane receptors was partially purified from the ovaries of Atlantic croaker. The purified protein was used to produce polyclonal antibodies that were subsequently used to screen an ovarian cDNA library. Positive clones were sequenced and a novel cDNA encoding a protein of approximately 40 kDa with several putative transmembrane domains was identified. Structural analyses revealed no sequence similarities on nucleotide or amino acid levels between this novel gene and nuclear steroid receptors or steroid membrane receptors. The novel cDNA was stably transfected into the breast cancer cell line SKBR-3 to examine androgen binding characteristics, expression patterns and cellsignaling. Western blot and immunocytochemical analyses of transfected cells showed that the recombinant protein had a molecular weight of approximately 40 kDa and was located in the plasma membranes. Testosterone (T) binding to plasma membranes prepared from transfected cells was increased 303 ± 87% compared to that in untransfected cells. Saturation and Scatchard analyses indicated the presence of a single, high affinity (Kd – 12.75 ± 3.22 nM), low-capacity (Bmax – 2.8 ± 0.5 pmol/mg protein) testosterone binding site on the membranes of transfected cells. The specific testosterone binding to the membrane fraction was readily displaceable with rapid association and dissociation kinetics (t½ – 1.5 ± 0.4 and 2.2 ± 0.7 minutes respectively). Steroid binding to the recombinant protein was specific for androgens. Progesterone displayed low affinity, and 17-beta estradiol (E2) and cortisol showed no affinity for the putative receptor in competitive binding assays. Highest binding affinity was observed with T, but other androgens such as 5alpha-dihydrotestosterone (DHT), androstenedione and 11-ketoandrostenedione also displayed significant binding affinities. Interestingly, the teleost androgen, 11- ketotestosterone, showed no binding affinity for the recombinant protein. Western blot and RT-PCR analyses of croaker tissues showed expression of the putative androgen membrane receptor protein and mRNA in reproductive tissues as well as in the brain and liver, whereas there was little or no expression in heart, gills, or intestine. Moreover, the expression of the novel protein in oocyte membranes varied during the ovarian cycle and was upregulated by gonadotropin and steroid treatments in vitro, suggesting it has a physiological role in croaker reproduction. Taken together, the results are consistent with the suggestion that the cDNA and protein discovered in Atlantic croaker ovaries is a novel androgen membrane receptor, unrelated to any known steroid receptors. (poster)

Receptors in Spermatozoa: Are They Real?

Receptors in Spermatozoa: Are They Real?

Testosterone-induced coronary vasodilatation occurs via a non-genomic mechanism: evidence of a direct calcium antagonism action.

Testosterone decreases myocardial ischaemia in men with coronary artery disease via a coronary vasodilatory action. However, long-term therapy may increase the risk of prostatic carcinoma via activation of the nuclear AR (androgen receptor). In the present study, we have investigated the mechanism of testosterone-induced vasodilatation using isolated rat coronary arteries and thoracic aortae from control and AR-deficient testicular-feminized mice. Vasodilatation induced by testosterone, T-3-OCMO [testosterone 3-(O-carboxymethyl)oxime] or T-3-OCMO conjugated to BSA was initially measured in preconstricted vessels that had undergone endothelial denudation or incubation with flutamide (10 microM). Cellular fluorescence was also measured in primary aortic SMCs (smooth muscle cells) following exposure to the above fluorescent-labelled agents. Subsequently, vessels were incubated with testosterone (100 microM) or vehicle prior to constriction with KCl (1-100 mM). Testosterone-induced vasodilatation was unaffected by endothelial denudation, flutamide treatment, AR deficiency or conjugation to BSA. Cells exposed to T-3-OCMO-BSA (10 microM) had a higher fluorescence than control cells (32.8+/-4.5 compared with 14.5+/-1.8 arbitrary units respectively; P<0.01). Incubation with testosterone (100 microM) reversibly attenuated coronary vasoconstriction to KCl (1-100 mM; 0.08+/-0.09 compared with 0.79+/-0.08 mN/mm respectively; P<0.0001). Testosterone-induced vasodilatation is independent of the vascular endothelium and nuclear AR, and is initiated at the SMC membrane, which contains testosterone binding sites. A direct calcium antagonistic action is implicated.

Membrane androgen binding sites are preferentially expressed in human prostate carcinoma cells.

BackgroundProstate cancer is one of the most frequent malignancies in males. Nevertheless, to this moment, there is no specific routine diagnostic marker to be used in clinical practice. Recently, the identification of a membrane testosterone binding site involved in the remodeling of actin cytoskeleton structures and PSA secretion, on LNCaP human prostate cancer cells has been reported. We have investigated whether this membrane testosterone binding component could be of value for the identification of prostate cancer.MethodsUsing a non-internalizable testosterone-BSA-FITC analog, proven to bind on membrane sites only in LNCaP cells, we have investigated the expression of membrane testosterone binding sites in a series of prostate carcinomas (n = 14), morphologically normal epithelia, taken from areas of the surgical specimens far from the location of the carcinomas (n = 8) and benign prostate hyperplasia epithelia (n = 10). Isolated epithelial cells were studied by flow cytometry, and touching preparations, after 10-min incubation. In addition, routine histological slides were assayed by confocal laser microscopy.ResultsWe show that membrane testosterone binding sites are preferentially expressed in prostate carcinoma cells, while BPH and non-malignant epithelial cells show a low or absent binding.ConclusionsOur results indicate that membrane testosterone receptors might be of use for the rapid routine identification of prostate cancer, representing a new diagnostic marker of the disease.

No Direct Mitogenic Effect of Sex Hormones on Antlerogenic Cells Detected in Vitro

Deer pedicles, antecedents of antlers, develop from a specialized periosteum (antlerogenic periosteum) which overlies the lateral crest of the deer frontal bone. The initiation of pedicle growth is triggered by androgen hormones. Thus far, it is not known whether pedicle initiation is caused by direct stimulation of androgen hormones on the antlerogenic periosteum or whether some intermediate mechanisms are necessary. The present study took an in vitro approach to investigate whether sex hormones have direct mitogenic effects on primary cultured antlerogenic periosteal cells (antlerogenic cells). Antlerogenic cells were obtained from two 5-month-old red deer calves. The cells were passaged twice and then treated with testosterone, dihydrotestosterone, and estradiol. The proliferation assays showed that no direct mitogenic effects on the second passage antlerogenic cells could be detected with any of the sex hormone treatments (P > 0.05). Testosterone-binding studies showed that at the second passage, specific testosterone-binding sites were present in the antlerogenic cells. Therefore, we conclude that androgens do not have mitogenic effects on antlerogenic cells in vitro. Our results suggest that pedicle formation may not be the result of direct stimulation of androgen hormones on antlerogenic tissue. Instead, androgen hormones may only allow the process to proceed by increasing the sensitivity of antlerogenic cells to mitogens, e.g., some growth factors.

Effects of testosterone either alone or with IGF-I on growth of cells derived from the proliferation zone of regenerating antlers in vitro.

Deer antlers are male secondary sexual characters and are the fastest growing mammalian tissue. As such, both androgens and growth factors play a major role in antler development. The timing of the antler cycle is controlled by the seasonal fluctuations of testosterone, and the actual growth of antlers is mainly stimulated by growth factors including insulin-like growth factor-1 (IGF-I). However, whether or not testosterone at low levels plays a growth-promoting role during antler formation is controversial. In the present study, we took an in vitro approach to investigate whether testosterone either alone or with IGF-I had mitogenic effects on mesenchymal or cartilaginous cells derived from the proliferation zone of regenerating antlers. In addition, a binding assay was carried out to determine whether the specific binding sites for testosterone were preserved after cell disaggregation. The results showed that testosterone either in physiological concentrations or at low levels did not exert direct mitogenic effects on antler cells derived from the proliferation zone in serum-free medium in vitro (P>0.05), even if the specific binding sites for testosterone in these cells were well preserved. Likewise, testosterone in a very wide range of concentrations not only failed to enhance (P>0.05), but at certain levels (0.1-5 nM) impaired the mitogenic effects of IGF-I on these antler cells in vitro (P<0.001). Therefore, these results support neither a conclusion that low level testosterone has growth-promoting effects on antler formation nor the hypothesis that testosterone effects may be achieved through sensitizing these antler cells to the mitogenic effects of IGF-I.

Effects of the mammalian antiandrogen vinclozolin on development and reproduction of the fathead minnow ( Pimephales promelas)

Previous work with the chlorinated fungicide vinclozolin and its metabolites, 2-{[(3,5-dichlorophenyl)-carbamoyl]oxy}-2-methyl-3-butenoic acid (M1) and 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), indicated antiandrogenic properties expressed in vivo as abnormalities in sexual differentiation of male rats after maternal exposures. In this study, we attempted to determine whether vinclozolin might also exhibit antiandrogenic properties in a model fish species, the fathead minnow, Pimephales promelas. In one study, embryonic (<6 h old) fathead minnows were exposed for approximately 34 days to five toxicant concentrations, ranging from 90 to 1200 μg l −1, delivered via a flow-through diluter. The embryos were periodically sampled to determine survival, growth and gross pathology, and then placed in clean water for 4–6 months to assess long-term effects on sexual differentiation and subsequent reproductive success. Except for slightly reduced growth after 34 days in the highest vinclozolin concentration, no adverse effects were noted with respect to any of these endpoints. In a second experiment, adult fathead minnows were exposed to vinclozolin concentrations of approximately 200 or 700 μg l −1 for 21 days, following which, gonadal morphology was assessed and serum sex steroid concentrations determined. Tissue samples from the exposed adults were assayed for vinclozolin and its metabolites. There was a slight increase in the serum β-estradiol concentration of the male fathead minnows exposed to 700 μg vinclozolin l −1, and a marked reduction in gonadal condition of female fish from this treatment. The possibility that vinclozolin and its metabolites would bind to androgen receptors in the fathead minnow was investigated through competitive radioligand binding studies. Vinclozolin, M1 and M2 failed to compete for high-affinity, low-capacity testosterone binding sites in fathead minnow brain and ovary cytosolic fractions, suggesting that these chemicals might not act as antiandrogens in the fathead minnow. More experimentation is necessary to determine whether responses observed in vivo might be due to the effects of vinclozolin (or its metabolites) on some other aspect of endocrine function.

Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages.

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca(2+)], which is due to release of Ca(2+) from intracellular Ca(2+) stores. This Ca(2+) mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

Functional testosterone receptors in plasma membranes of T cells.

T cells are considered to be unresponsive to testosterone due to the absence of androgen receptors (AR). Here, we demonstrate the testosterone responsiveness of murine splenic T cells in vitro as well as the presence of unconventional cell surface receptors for testosterone and classical intracellular AR. Binding sites for testosterone on the surface of both CD4(+) and CD8(+) subsets of T cells are directly revealed with the impeded ligand testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Binding of the plasma membrane impermeable testosterone-BSA conjugate induces a rapid rise (<5 s) in [Ca2+]i of Fura-2-loaded T cells. This rise reflects influx of extracellular Ca2+ through non-voltage-gated and Ni2+-blockable Ca2+ channels of the plasma membrane. The testosterone-BSA-induced Ca2+ import is not affected by cyproterone, a blocker of the AR. In addition, AR are not detectable on the surface of intact T cells when using anti-AR antibodies directed against the amino and carboxy terminus of the AR, although T cells contain AR, as revealed by reverse transcription-polymerase chain reactions and Western blotting. AR can be visualized with the anti-AR antibodies in the cytoplasm of permeabilized T cells by using CLSM, though AR are not detectable in cytosol fractions when using the charcoal binding assay with 3H-R1881 as ligand. Cytoplasmic AR do not translocate to the nucleus of T cells in the presence of testosterone, in contrast to cytoplasmic AR in human cancer LNCaP cells. These findings suggest that the classical AR present in splenic T cells are not active in the genomic pathway. By contrast, the cell surface receptors for testosterone are in a functionally active state, enabling T cells a nongenomic response to testosterone.

Characterization of putative steroid receptors in the membrane, cytosol and nuclear fractions from the olfactory tissue of brown and rainbow trout

Specific binding sites for testosterone have been detected in three compartments of olfactory tissue from brown and rainbow trout. Binding of3H-testosterone to the membrane fraction of olfactory tissue is of high affinity (Kd = 0.5–1.9 nM) and limited capacity (Nmax = 30–60 fmol mg+1 protein). Binding is reversible, and is eliminated by protease treatment. The membrane binding site exhibits a high degree of ligand specificity; 11β-hydroxytestosterone, 11-ketotestosterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, cortisol, and estradiol-17β all fail to displace testosterone at 20-fold excess while testosterone itself competes successfully. These attributes are consistent with the presence of specific steroid receptor proteins. Binding of testosterone within the cytosol is of moderate affinity (Kd = 9.0–23.0 nM) and high capacity (Nmax = 0.5–2.9 pmol mg+1 protein) and is more readily displaced by a number of steroid competitors than is the case for the membrane site. The rate of association and dissociation of testosterone from the cytosolic binding site is markedly more rapid than the equivalent processes in the membrane fraction. Binding of testosterone to the nuclear extract is of high affinity (Kd ∼3.0 nM) and limited capacity (Nmax ∼50 fmol mg+1 protein).There are no substantial differences between species or between sexes in the affinity or capacity of testosterone-binding sites in nuclear extract or membrane fraction. However, cytosolic testosterone-binding sites are three- to four-fold more abundant in rainbow trout than in brown trout, and female rainbow trout have more cytosolic binding sites than male rainbow trout, but a lower affinity for testosterone than male sites.Preliminary evidence supports the involvement of the membrane-associated testosterone-binding site in olfactory processes. Rainbow trout display an EOG response to testosterone at a concentration (≥ 10+9 M) which is consistent with the equilibrium dissociation constant (Kd) of the membrane-associated testosterone-binding site. Binding of3H-testosterone to the membrane-associated site shows a pH dependency which is comparable to the effects of pH on the EOG response to testosterone in intact fish. The attributes of the intracellular testosterone-binding sites are common to testosterone receptors in other fish tissues which are known androgen target tissues. This suggests that the development and/or function of salmonid olfactory tissue may be susceptible to influence by endogenous testosterone.

Effects of Estradiol and Testosterone on the Synthesis, Expression and Degradation of Androgen Receptor in Human Uterine Endometrial Fibroblasts.

The mechanism of known receptor-mediated androgen effects on the endometrial stroma was studied in endometrial fibroblasts derived from human uterus. 17beta-Estradiol (E) induced the expressions of androgen receptor (AR) mRNA, and predominantly increased the level of testosterone-binding sites (TBS) in uterine endometrial fibroblasts. The effect on the level of dihydrotestosterone-binding sites (DHTBS) was similar but smaller. This result suggests that the AR mRNA expressed might encode TBS, but probably not DHTBS. The TBS level increased by estrogen was down-regulated by testosterone (T) + E, but the AR mRNA expression increased by E was not down-regulated by E + T in the fibroblasts. Although the synthesis rate of AR was slightly increased (p < 0.05) by E alone or E + T, the degradation rate of AR was significantly accelerated (p < 0.05) by E + T in the fibroblasts. This result suggests that T might stimulate the metabolic rate of TBS, but does not inhibit the synthesis rate of AR mRNA to TBS in endometrial fibroblasts. Copyright 1995 S. Karger AG, Basel

Biological implications of estrogen and androgen effects on androgen receptor and its mRNA levels in human uterine endometrium.

It has been shown that some effects of testosterone are different from those of its 5 alpha-reduced metabolite, dihydrotestosterone. Briefly, activities of testosterone might be related to cellular differentiation, whereas dihydrotestosterone acts on cellular proliferation. The number of testosterone binding sites in the uterine endometrium was increased by estradiol dipropionate, and this increase was down-regulated by testosterone cypionate. Dihydrotestosterone-specific binding sites in the endometrium were not modulated by estradiol dipropionate and testosterone cypionate. The dissociation constants of the binding sites for testosterone and dihydrotestosterone were not altered by these steroids. Estradiol dipropionate with or without testosterone cypionate induced androgen receptor mRNA expression in the endometrium. In conclusion, testosterone might predominantly affect cellular differentiation in the endometrium.

Analysis of hypothalamo-hypophyseo-gonadal relationships in female rats with experimental diabetes

Hypothalamo-hypophyseo-gonadal system functional activity was studied in rats with streptozotocin diabetes. In intact rats concentrations of sex hormones nuclear receptors were measured in the hypothalamic preopticoanterior, mediobasal segments and in the adenohypophysis, as were blood serum gonadotropins and sex hormones. Estradiol and progesterone were injected to ovariectomized females and LH-RH levels measured in preopticoanterior segment of the hypothalamus, arcuate nucleus, and median eminence, as well as LH and FSH concentrations in the blood in order to detect disorders in basal and cyclic gonadotropin secretion. Streptozotocin injection to cycling females disordered the estral cycle and was associated with reduction of LH, FSH, and sex hormones basal and cyclic secretion. Estradiol nuclear receptors concentrations reduced in the preopticoanterior hypothalamus and hypophysis, the count of nuclear testosterone-binding sites reduced only in the hypophysis. Gonadotropin wave stimulated with sex steroids in ovariectomized females was reduced in diabetes because of changed activity of LH-RH-producing system. We believe that changes in basal and cyclic secretion of gonadotropins in rat females with experimental diabetes is explained by reduced activity of LH-RH-producing system and receptor binding at the level of the hypothalamo-hypophyseal complex.

Testosterone binding sites in the brain, plasma sex hormones and reproductive behaviour in males of the toad Bufo bufo

The central action of steroid hormones of the regulation of reproductive behaviour has been recognized in Amphibia, as in, other Vertebrates. Central and peripheral endocrine aspects of reproductive behaviour were studied in the toad Bufo bufo during the breeding aspects of reproductive behaviour were studied in the toad Bufo bufo during the breeding (DHT) and estradiol (E) levels. In the first experiment the effect of early and late entrance into the breeding pond and the difference between animals entering and leaving was established: DHT and E plasma levels, as well as T binding capacity in the brain were lower in animals leaving the pond at the end of the breeding season; animals entering early in the season showed higher levels of E than those entering late. In the second experiment the hormonal effect of amplexus and spawning was established: single males showed lower plasma DHT, higher plasma E and higher T binding values in the brain than males paired with a female. Males in amplexus with a non-spawning female showed higher plasma E levels than those with a spawning female. These results show that it is possible to relate the different reproductive success to different T binding in the brain. The effect of amplexus on potential responsiveness to steroid hormones at the central level and on peripheral hormone concentrations suggests the presence of a regulatory mechanism which is more active when both amplexus and spawning occur. On the other hand, the data concerning animals entering and leaving the pond indicate that the hormonal variations are not due solely to the end of sexual behaviour since the difference is already significant between groups entering early and late in the breeding season.

Sexual dimorphism of binding sites of testosterone and dihydrotestosterone in rabbit model

1. 1. Binding sites for testosterone (TR) and dihydrotestosterone (DHTR) were separately detected in various organs of the rabbit. 2. 2. Possibilities obtained were suggested as follows. 3. 3. Sexual dimorphism of TR or DHTR level was present among organs. 4. 4. The responsiveness of TR or DHTR levels to estradiol-17β with testosterone, but not estradiol-17β alone is different among organs, with sexual minor dimorphism.

Steroid binding to human serum albumin and fragments thereof: Role of protein conformation and fatty acid content

The binding properties of the steroids testosterone and pregnenolone to human serum albumin (HSA) and derived fragments of albumin have been investigated by means of equilibrium dialysis and circular dichroism. The 46 kDa peptic fragment (P46) of HSA comprises domains one and two of the HSA structure, whereas the other fragment, the 45 kDa tryptic fragment (T45) is composed of domains two and three. A comparison of the binding behaviour of the steroid ligands to HSA and its fragments showed that the single primary testosterone binding site in all probability is located in the second domain of the HSA molecule. For pregnenolone it was found that at least two primary binding sites are present, also located in domain two. Both steroids show pH dependent binding profiles in the case of HSA and the P46 fragment. The binding of the steroids to the T45 fragment seems to be pH independent. The same phenomenon was observed with the circular dichroism experiments, indicating a link between the steroid binding properties and the structural behaviour of the proteins. In fact, the binding properties of the steroids can be assigned to the neutral-to-base (N-B) transition. The possible role of fatty acids as modulators in the transport processes of steroids in the body is discussed.

Isolation and characterization of a 50 kDa testosterone-binding protein from Pseudomonas testosteroni

A testosterone-binding protein (M r = 50,500) has been isolated from the Gram-negative bacterium Pseudomonas testosteroni. The protein was partially purified by a combination of ion exchange chromatography and chromatofocusing. Final purification was achieved by electroelution of the 50 kDa protein from SDS-polyacrylamide gels. Following renaturation from a diluted solution of guanidine-HCl, specific binding of [ 3H]testosterone to the purified protein was observed. The native protein has a pI of 6.8. It appears to contain 428 amino acids, 39% of which are hydrophobic. There is only one cysteine residue. Both chymotrypsin and V8 protease were used to produce peptide maps of the protein for use in future identification. The first 10 amino acids situated at the N-terminal of the protein were Ser-Pro-Phe-Asp-Leu-Arg-Pro-Leu-Ser-Gly. Testosterone binding to the protein was saturable at ~3.8 nmol/mg protein; the binding constant was ~25 nM. Unlabelled testosterone, androstenedione, 5α-dihydrotestosterone and 5β-dihydrotestosterone were able to compete for [ 3H]testosterone bound to the protein; 17β-estradiol also competed for [ 3H]testosterone but to a lesser degree. Neither progesterone nor desoxycorticosterone competed for the testosterone-binding site. Binding of testosterone to the protein was stable at pH's ranging from 5.5 to 9.0 and at various temperatures ranging from 4 to 30°C. The protein was unable to metabolize testosterone in either the presence or absence of the cofactor NAD.