scholarly journals High affinity binding of anti-oestrogen to the chick liver nuclear oestrogen receptor

1982 ◽  
Vol 206 (2) ◽  
pp. 387-394 ◽  
Author(s):  
Catherine B. Lazier ◽  
V. Craig Jordan
Keyword(s):  
Nuclear Receptor ◽  
Oestrogen Receptor ◽  
Potent Inhibitor ◽  
Inhibitory Effect ◽  
Affinity Binding ◽  
Soluble Receptor ◽  
High Affinity ◽  
Receptor Concentration ◽  
Liver Nuclei

Tamoxifen is a potent inhibitor of specific oestrogen-induced yolk protein synthesis by chicken liver. The oestradiol receptor in salt extracts of liver nuclei from oestrogen-treated chicks has a Kd for oestradiol of 0.7±0.2nm. Tamoxifen and its metabolite, monohydroxytamoxifen, compete for binding to the salt-soluble nuclear receptor with Ki values of 2.6 and 0.1nm respectively. The anti-oestrogens show much less inhibition of [3H]oestradiol binding when assays are carried out using intact nuclei. The competition by unlabelled oestradiol for [3H]oestradiol binding to receptor is identical in both salt extracts and intact nuclei. This suggests that intact nuclei contain components which bind anti-oestrogens, but not oestradiol. While tamoxifen and desmethyltamoxifen will readily dissociate from the salt-soluble nuclear oestrogen receptor, monohydroxytamoxifen does not dissociate under the conditions generally used for exchange assays. A modified assay was developed in which 60–70% of monohydroxytamoxifen-bound sites were shown to be exchangeable for [3H]oestradiol. Soluble receptor preparations were first incubated in a 1.7% charcoal suspension at 37°C for 15min before assay of specific oestradiol binding. This technique was used in examining the effects of tamoxifen and monohydroxytamoxifen given in vivo on the nuclear oestrogen receptor concentration. Despite their 30-fold difference in binding affinity for the receptor, both anti-oestrogens increase nuclear receptor levels to about the same degree. When given with oestradiol, both compounds have the same apparent partial inhibitory effect on the oestrogen-induced increase in nuclear receptor. These data are consistent with the metabolic hydroxylation of tamoxifen before binding to the hepatic oestrogen receptor.

scholarly journals The Identification of Naturally Occurring Neoruscogenin as a Bioavailable, Potent, and High-Affinity Agonist of the Nuclear Receptor RORα (NR1F1)

2013 ◽  
Vol 19 (3) ◽  
pp. 399-406 ◽  
Author(s):  
Stéphane Helleboid ◽  
Christian Haug ◽  
Kai Lamottke ◽  
Yijun Zhou ◽  
Jianbing Wei ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Screening Method ◽  
Structural Diversity ◽  
Orphan Receptor ◽  
Compound Identification ◽  
High Affinity ◽  
Specific Selectivity ◽  
Reporter Assays

Plants represent a tremendous structural diversity of natural compounds that bind to many different human disease targets and are potentially useful as starting points for medicinal chemistry programs. This resource is, however, still underexploited due to technical difficulties with the identification of minute quantities of active ingredients in complex mixtures of structurally diverse compounds upon raw phytomass extraction. In this work, we describe the successful identification of a novel class of potent RAR-related orphan receptor alpha (RORα or nuclear receptor NR1F1) agonists from a library of 12,000 plant extract fractions by using an optimized, robust high-throughput cell-free screening method, as well as an innovative hit compound identification procedure through further extract deconvolution and subsequent structural elucidation of the active natural compound(s). In particular, we demonstrate that neoruscogenin, a member of the steroidal sapogenin family, is a potent and high-affinity RORα agonist, as shown by its activity in RORα reporter assays and from its effect on RORα target gene expression in vitro and in vivo. Neoruscogenin represents a universal pharmacological tool for RORα research due to its specific selectivity profile versus other nuclear receptors, its excellent microsomal stability, good bioavailability, and significant peripheral exposure in mouse.

scholarly journals The Arg-Gly-Asp–Containing, Solvent-Exposed Loop of Ptr ToxA Is Required for Internalization

2008 ◽  
Vol 21 (3) ◽  
pp. 315-325 ◽  
Author(s):  
Viola A. Manning ◽  
Sara M. Hamilton ◽  
P. Andrew Karplus ◽  
Lynda M. Ciuffetti
Keyword(s):  
Phosphorylation Site ◽  
Affinity Binding ◽  
Mesophyll Cells ◽  
High Affinity ◽  
Treatment Zone ◽  
Ptr Toxa ◽  
High Affinity Binding Site ◽  
Toxin Uptake

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 × 106 per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.

scholarly journals The Xenobiotic Compound 1,4-Bis[2-(3,5-Dichloropyridyloxy)]Benzene Is an Agonist Ligand for the Nuclear Receptor CAR

2000 ◽  
Vol 20 (9) ◽  
pp. 2951-2958 ◽  
Author(s):  
Iphigenia Tzameli ◽  
Pavlos Pissios ◽  
Erin G. Schuetz ◽  
David D. Moore
Keyword(s):  
Ligand Binding ◽  
Nuclear Receptor ◽  
Metabolic Response ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Inverse Agonists ◽  
Wide Range ◽  
Xenobiotic Compound

ABSTRACT A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

scholarly journals The effects of altered sterol composition on the mitochondrial adenine nucleotide transporter of Saccharomyces cerevisiae

1977 ◽  
Vol 166 (3) ◽  
pp. 559-563 ◽  
Author(s):  
J M Haslam ◽  
A M Astin ◽  
W W Nichols
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Adenine Nucleotide ◽  
Tween 80 ◽  
Sterol Composition ◽  
Affinity Binding ◽  
Sterol Content ◽  
Macromolecular Synthesis ◽  
High Affinity

1. The membrane sterol composition of mitochondria of the ole-3 mutant of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of Tween 80 (1%, W/V) plus defined supplements o- delta-aminolaevulinate. 2. Changes in mitochondrial sterol content induced considerable changes in the adenine nucleotide transporter. 3. As the sterol content was decreased, the affinity of the transporter for ATP did not alter significantly, but the rate of ATP uptake was greatly decreased, the total number of atractylate-sensitive binding sites diminished, and the proportion of high-affinity binding sites was decreased. 4. Since sterol depletion also uncouples oxidative phosphorylation [Astin & Haslam (1977) Biochem. J., 166, 287-298] and prevents the intramitochondrial generation of ATP, the decrease in the rate of ATP uptake by sterol-depleted mitochondria will cause a decrease in intramitochondrial ATP concentrations in vivo. This probably explains the inhibition of mitochondrial macromolecular synthesis that has previously been reported in lipid-depleted yeast mitochondria.

Possible Mode of Action of Ticlopidine: A Novel Inhibitor of Platelet Aggregation

1979 ◽  
Author(s):  
H. Johnson ◽  
J. B. Heywood
Keyword(s):  
Platelet Aggregation ◽  
Mode Of Action ◽  
Potent Inhibitor ◽  
Platelet Reactivity ◽  
Inhibitory Effect ◽  
Thromboxane Synthetase ◽  
Sustained Effect ◽  
Dependent Inhibition

Ticlopidine (T) is weakly active in vitro, but is a potent inhibitor of platelet aggregation induced by ADP, collagen, thrombin, adrenaline, arachidonic acid, prostaglandin (PG) endoperoxide and thromboxane A2 with a sustained effect, when administered to a variety of animal species, including man. Platelets from treated animals were normal in ultrastructure and 14C-ADP binding was not modified by T. Basal PG synthesis was unaffected, whereas aspirin (A) had a marked inhibitory effect. Platelet cyclo-oxygenase and thromboxane synthetase activities were 90.6±12.9 and 96.1±5.3% of control following T treatment. In contrast to A, T had no effect on vascularprostacyclin (PGI2) synthesis, this being 1.4±0.1, 0.5±0.1 and 1.3±0. 3ng/mg wet weight aorta in T and A-treated and control animals respectively. Platelets from T-treated rats were significantly more responsive to inhibition by exogenous PGI2 (0.2-4 ng/ml) and PGE1 (4- 20 ng/ml). when compared with controls. T administration (30-300 mg/kg) resulted in a dose-dependent inhibition of ADP-induced platelet aggregation (26.0- 87. 5%) and enhancement of platelet reactivity to PGI2 (37.0-159.8%). There was a good correlation between these parameters (r=+0.994). T is a potent inhibitor of platelet aggregati on with a novel mode of action. It is not aspirin-like, but may act to potentiate endogenous PGI2 in vivo, possibly through an effect on platelet adenylate cyclase.

RAT MYOMETRIAL ACTIVITY IN VIVO: EFFECTS OF OESTRADIOL-17β AND PROGESTERONE IN RELATION TO THE CONCENTRATIONS OF CYTOPLASMIC PROGESTERONE RECEPTORS

1978 ◽  
Vol 78 (1) ◽  
pp. 103-117 ◽  
Author(s):  
SANDRA J. DOWNING ◽  
S. J. LYE ◽  
JANE M. C. BRADSHAW ◽  
D. G. PORTER
Keyword(s):  
Binding Protein ◽  
Progesterone Receptors ◽  
Affinity Binding ◽  
Pregnant Rats ◽  
Pregnant Rat ◽  
High Affinity Binding ◽  
High Affinity ◽  
Repeated Doses ◽  
In Vivo Effects

The amplitude, frequency and rate of rise of intra-uterine pressure cycles in rats (postpartum, ovariectomized) were unaffected by treatment with progesterone. Amplitude was also unaffected by a combination of treatments with progesterone and oestradiol-17β, which was adequate to ensure the survival of 84% of foetuses in ovariectomized pregnant rats. The failure of progesterone to influence myometrial activity could not be attributed to a lack of 'true' progesterone receptors since these were present in the myometria of the test animals in concentrations exceeding those of oestrous animals. Evidence was obtained which suggested that a high-affinity binding protein, different from the 'true' receptor may predominate in the myometrium of the pregnant rat. Oestradiol-17β in single or repeated doses of from 0·25 to 5 μg, however, was found to reduce the frequency of pressure cycles but to increase significantly their rate of rise of pressure. There was a latency of 6–8 h in these effects of oestradiol. The possibility that inhibition of the myometrium by oestrogen may play a part in the preparation for parturition is discussed.

scholarly journals Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein.

1981 ◽  
Vol 91 (2) ◽  
pp. 545-550 ◽  
Author(s):  
P Walter ◽  
I Ibrahimi ◽  
G Blobel
Keyword(s):  
Wheat Germ ◽  
Inhibitory Effect ◽  
Secretory Protein ◽  
Affinity Binding ◽  
Signal Recognition ◽  
Cytoplasmic Protein ◽  
High Affinity Binding ◽  
High Affinity ◽  
Selective Translation ◽  
Recognition Protein

An 11S protein composed of six polypeptide chains was previously purified from a salt extract of dog pancreas microsomal membranes and shown to be required for translocation of nascent secretory protein across the microsomal membrane (Wistar and Blobel 1980 Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). This 11S protein, termed signal recognition protein (SRP), has been shown here (a) to inhibit translation in the wheat germ cell-free system selectively of mRNA for secretory protein (bovine preprolactin) but not of mRNA for cytoplasmic protein (alpha and beta chain of rabbit globin); (b) to bind with relatively low affinity (apparent KD less than 5 x 10(-5)) to monomeric wheat germ ribosomes; and (c) to bind selectively and with 6,000-fold higher affinity (apparent KD less than 8 x 10(-9)) to wheat germ ribosomes engaged in the synthesis of secretory protein but not to those engaged in the synthesis of cytoplasmic protein. Low- and high-affinity binding as well as the selective translation-inhibitory effect were abolished after modification of SRP by N-ethyl maleimide. High-affinity binding and the selective translation-inhibitory effect of SRP were largely abolished when the leucine (Leu) analogue beta-hydroxy leucine was incorporated into the nascent secretory polypeptide.

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