scholarly journals Acceptor sites for the oestrogen receptor in hen oviduct chromatin

1983 ◽  
Vol 210 (3) ◽  
pp. 905-912 ◽  
Author(s):  
T S Ruh ◽  
T C Spelsberg
Keyword(s):  
Receptor Binding ◽  
Oestrogen Receptor ◽  
Guanidine Hydrochloride ◽  
Scatchard Analysis ◽  
Oestrogen Receptors ◽  
Receptor Complexes ◽  
High Affinity ◽  
Acceptor Activity ◽  
Specificity Of Binding

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.

scholarly journals The binding of 3H-labelled oestradiol- and progesterone-receptor complexes to hypothalamic chromatin of male and female sheep

1978 ◽  
Vol 176 (3) ◽  
pp. 873-883 ◽  
Author(s):  
B N Perry ◽  
A Lopez
Keyword(s):  
Progesterone Receptor ◽  
Receptor Binding ◽  
Guanidine Hydrochloride ◽  
Male And Female ◽  
Receptor Complexes ◽  
Related Difference ◽  
Saturation Binding ◽  
Acidic Proteins ◽  
Female Sheep

Chromatin isolated from hypothalamic nuclei of sexually mature entire male and female sheep was linked to cellulose in u.v. light. The saturation binding of 3H-labelled oestrogen- and progesterone-receptor complexes, prepared by (NH4)2SO4 precipitation from the 105000g supernatant of hypothalamic cytosol, was then measured in vitro in 0.15m-KCl. Saturation binding was also measured after extraction of histones and masking acidic proteins. Salt + urea was observed to be more effective than guanidine hydrochloride in unmasking receptor acceptor sites, and the binding of labelled receptor complexes to dehistonized unmasked chromatin was shown to be largely resistant to 0.4m-KCl extraction. Whereas extents of receptor-complex binding were similar to published values for comparable preparations of hen oviduct chromatin, no sex-related difference was observed. However, binding of progesterone-receptor to chromatin was greater than that of oestradiol-receptor. Binding also increased more after removal of histones and masking acidic proteins, suggesting the presence of a greater number of progesterone-receptor acceptor sites in hypothalamic chromatin than of estradiol-receptor acceptor sites. The failure to demonstrate a sex-related difference in oestradiol-receptor binding to hypothalamic chromatin in vitro is discussed.

scholarly journals Nuclear acceptor sites for androgen-receptor complexes in seminal-vesicle epithelium

1980 ◽  
Vol 192 (1) ◽  
pp. 41-47 ◽  
Author(s):  
M J Weinberger ◽  
C M Veneziale
Keyword(s):  
Seminal Vesicle ◽  
Nuclear Dna ◽  
Steroid Receptor ◽  
Assay Method ◽  
Scatchard Analysis ◽  
Receptor Complex ◽  
Receptor Complexes ◽  
Constant Slope

An assay method in vitro was developed and applied to quantify acceptor binding of steroid-receptor complexes in nuclei from isolated epithelium of guinea-pig seminal vesicle. Steroid-receptor complex prepared from 1-day-castrated animals was incubated with purified nuclei from 1-28 day-castrated animals in a medium containing 0.15 M-KCl. Free and bound steroid-receptor complexes were measured and the data were submitted to Scatchard analysis. With nuclei from 1-day-castrated animals the Kd for binding of cytosolic [3H]dihydrotestosterone-receptor complexes was found to be 0.83 × 10(-10) M and the capacity for binding was 0.35 pmol/mg of nuclear DNA. Scatchard analysis consistently disclosed only a single line of constant slope and gave the same kinetic constants for nuclei obtained from animals castrated up to 28 days before assay. Administration of 2 mg of dihydrotestosterone, 3 alpha-androstanediol or androsterone or 100 microgram of oestradiol-17 beta 1 h before killing of the 1-day-castrated animals that provided the nuclei resulted in a significant decrease in nuclear acceptor binding of the steroid-receptor complex compared with untreated animals. Thus our assay method disclosed nuclear acceptor sites that may be involved in responses to androgens (and oestrogens) in vivo. We conclude that there is a class of nuclear accept or sites of high affinity and limited capacity that may be occupied by steroid-receptor complexes in vivo.

A HIGH-AFFINITY BINDING SITE FOR THE ANTIOESTROGENS, TAMOXIFEN AND CI 628, IN IMMATURE RAT UTERINE CYTOSOL WHICH IS DISTINCT FROM THE OESTROGEN RECEPTOR

1981 ◽  
Vol 91 (1) ◽  
pp. 155-161 ◽  
Author(s):  
L. C. MURPHY ◽  
R. L. SUTHERLAND
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Oestrogen Receptor ◽  
Dissociation Constants ◽  
Significant Proportion ◽  
Immature Rat ◽  
High Affinity ◽  
Saturable Binding ◽  
Receptor Sites

A high-affinity, saturable antioestrogen binding site, which does not bind oestradiol, has been reported to exist in a number of oestrogen target tissues but not in the immature rat uterus. This study reports the results of a more thorough search for this site in immature rat uterine cytosol. When concentrations of uterine cytoplasmic oestrogen receptor were selectively depleted by translocation of 90–95% of the cytoplasmic oestrogen receptor to the nucleus, saturation analysis studies revealed that the antioestrogens, tamoxifen and CI 628, were bound to high-affinity, saturable binding sites which were present at about 2·5 times the concentration of the residual oestrogen receptor sites. Oestradiol could only partially inhibit the binding of tritiated antioestrogens to their saturable binding sites in this material indicating that a significant proportion of these sites were distinct from the oestrogen receptor sites. This was confirmed in experiments where oestrogen receptor sites were saturated in vitro with oestradiol and high-affinity, saturable sites for CI 628 and tamoxifen were still present. The CI 628 and tamoxifen had high affinity for these sites with dissociation constants of 1·0–1·6 nmol/l. These specific antioestrogen binding sites were present at about 5% of the concentration of oestrogen receptors in normal immature rat uterine cytosol which probably explains their previous lack of detection in this material.

Effect of theophylline on nuclear retention of oestrogen-receptor complexes: correlation with oestrogen responses

1985 ◽  
Vol 105 (3) ◽  
pp. 397-403
Author(s):  
J. Steinsapir ◽  
A. M. Rojas ◽  
M. E. Bruzzone ◽  
A. White ◽  
O. Alarcón ◽  
...  
Keyword(s):  
Body Weight ◽  
Oestrogen Receptor ◽  
Nuclear Retention ◽  
Adult Rat ◽  
Receptor Complexes ◽  
Wet Weight

ABSTRACT In the ovariectomized adult rat uterine oedema induced by 0·01 and 0·1 μg oestradiol-17β/100 g body weight increased further in the presence of theophylline. Nuclear retention of oestrogen-receptor complexes also increased in response to theophylline both in vivo and in vitro. Theophylline decreased the number of eosinophils in the blood and concurrently decreased oestrogen-induced uterine eosinophilia at doses of 0·001, 0·01, 0·1, 1, 10 or 30 μg oestradiol/100 g body weight, through a mechanism independent of glucocorticoids. There was, therefore, no correlation between changes in the number of uterine eosinophils and changes in uterine wet weight induced by theophylline and oestrogen. It is suggested that the presence of oestrogen-receptor complexes in the nucleus for at least 4 h is a prerequisite for the induction of uterine oedema and growth in the presence of theophylline and oestradiol-17β. J. Endocr. (1985) 105, 397–403

Abnormal polarization of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD

1995 ◽  
Vol 269 (2) ◽  
pp. C487-C495 ◽  
Author(s):  
J. Du ◽  
P. D. Wilson
Keyword(s):  
Epithelial Cell ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Scatchard Analysis ◽  
Egf Receptors ◽  
Renal Epithelial Cell ◽  
Western Blots ◽  
High Affinity ◽  
Cyst Fluids

The underlying mechanism of the hyperproliferative response of human autosomal dominant polycystic kidney disease (ADPKD) epithelia was studied. Epidermal growth factor (EGF) protein is highly expressed in ADPKD cyst epithelia in vivo, and primary cultures are hyperesponsive to mitogenic stimulation by EGF in vitro. Doses of > 1 ng/ml EGF were highly mitogenic to ADPKD epithelia. 3H-labeled thymidine proliferation assays showed that cyst fluids and ADPKD epithelial cell-conditioned media also stimulated renal epithelial cell proliferation and contained EGF immunoreactivity (6, 30, and 37 kDa) as detected by Western blots. Radioimmunoassays detected mean levels of 2.87 and 1.4 ng/ml EGF in cyst fluids from early (proliferative) and end-stage ADPKD cysts, respectively. Scatchard analysis of 125I-labeled EGF binding to apical and basolateral membrane showed high-affinity binding to basolateral membranes of normal and ADPKD kidneys but additional unique high-affinity receptor binding to apical membranes of ADPKD but not normal kidneys. Cross-linking analysis and antiphosphotyrosine Western analysis demonstrated functionally active apical EGF receptors at 150-170 kDa. These results suggest mediation of cyst expansion via an autocrine loop involving EGF synthesis and processing by cyst epithelial cells, apical secretion into cyst lumens, and subsequent binding to and phosphorylation of apical membrane EGF receptors. These findings are consistent with a membrane protein polarization defect in ADPKD cyst epithelia.

scholarly journals Saturation Analysis in PET—Analysis of Errors Due to Imperfect Reference Regions

1994 ◽  
Vol 14 (2) ◽  
pp. 358-361 ◽  
Author(s):  
J.-E. Litton ◽  
H. Hall ◽  
S. Pauli
Keyword(s):  
Receptor Binding ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Position Emission Tomography ◽  
Reference Region ◽  
Nonspecific Binding ◽  
Binding Studies

In the determination of specific binding in receptor binding techniques in vitro as well as in vivo, determination of the nonspecific binding as well as the free component is of crucial importance. If a low proportion of specific binding is included when determining the nonspecific binding, relatively large errors may be obtained. In the present study, benzodiazepine (BZ) receptor binding in the human brain was determined in vivo using position emission tomography (PET) by applying a saturation procedure using [11C]flumazenil as an example of this problem. Analysis of the errors in Bmax and KD obtained using Scatchard analysis in PET was performed using a priori information from in vitro [3H]flumazenil binding in the pons, used normally as a reference region in BZ receptor binding studies. Even if the density of BZ receptors in the reference region pons is only 2% compared to that in the frontal cortex, this small proportion of specific binding sites will result in a 10% error in the Bmax and KD values. Simulation of a number of Scatchard plots was performed at varying ratios between the nonspecific and the specific binding.

scholarly journals The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

1992 ◽  
Vol 12 (4) ◽  
pp. 1451-1459 ◽  
Author(s):  
A Klippel ◽  
J A Escobedo ◽  
W J Fantl ◽  
L T Williams
Keyword(s):  
Growth Factor ◽  
Sh2 Domain ◽  
Full Length ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Sh2 Domains ◽  
High Affinity ◽  
Src Homology ◽  
Specificity Of Binding

Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor.

Oestrogen receptor-like binding in the surface germinal epithelium of the rat ovary

1982 ◽  
Vol 95 (3) ◽  
pp. 377-385 ◽  
Author(s):  
T. C. Hamilton ◽  
P. Davies ◽  
K. Griffiths
Keyword(s):  
Oestrogen Receptor ◽  
Cultured Cells ◽  
Sedimentation Coefficient ◽  
Germinal Epithelium ◽  
Sedimentation Analysis ◽  
High Affinity ◽  
Rat Ovary

The surface germinal epithelium of the rat ovary was isolated and grown in vitro. Cytosol from cultured cells contained a saturable component, of sedimentation coefficient 7·5–8·5S, which bound oestrogenic and antioestrogenic substances with high affinity (Kd 400 pmol/l). Nuclei isolated from cells exposed to [3H]oestradiol contained radioactivity partially susceptible to KC1 extraction. Sedimentation analysis of the KC1 extract showed that [3H]oestradiol was associated with a moiety of sedimentation coefficient 4·5–5S.

scholarly journals Uterine oestrogen-receptor binding to oligo(dT)-cellulose. An inhibitor from hypothalamic cytosol

1979 ◽  
Vol 182 (1) ◽  
pp. 241-243 ◽  
Author(s):  
G Shen ◽  
S Thrower ◽  
L Lim
Keyword(s):  
Receptor Binding ◽  
Oestrogen Receptor ◽  
Potent Inhibitor

The binding of rat uterine cytosol oestrogen receptor in vitro to oligo(dT)-cellulose is mediated by an activating factor in the cytosol [Thrower, Hall, Lim & Davison (1976) Biochem. J. 160, 271-280]. A potent inhibitor of this binding is present in hypothalamic cytosol. This inhibitor may have a role in vivo in regulating receptor translocation in the hypothalamus.

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