scholarly journals A high-affinity oestrogen-binding protein in cockerel liver cytosol

1979 ◽  
Vol 180 (2) ◽  
pp. 347-353 ◽  
Author(s):  
C B Lazier ◽  
A J Haggarty
Keyword(s):  
Binding Sites ◽  
Proteinase Inhibitors ◽  
Specific Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Single Class ◽  
High Affinity ◽  
Significant Concentration

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1–4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

Prostaglandin E2 binding sites in porcine oxyntic mucosa: effects of salicylates

1986 ◽  
Vol 64 (5) ◽  
pp. 515-520 ◽  
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Prostaglandin E ◽  
Affinity Binding ◽  
Variable Degree ◽  
Single Class ◽  
Oxyntic Mucosa ◽  
High Affinity Binding ◽  
Mucosal Ulceration ◽  
High Affinity

These studies were designed to examine the changes in the characteristics of prostaglandin E2 (PGE2) binding to porcine oxyntic mucosa in the response to oral ingestion of salicylates. Either acetylsalicylic acid (ASA) or salicylic acid (SA) was administered to conscious pigs (100 mg/kg in 30 mL of an equimolar concentration of NaHCO3) once a day for 1, 3, 10, or 20 days. In control experiments a similar volume of 0.3 M NaHCO3 was administered for similar durations. Mucosal ulceration and the characteristics of the binding of [3H]PGE2 to a 30 000 × g membrane preparation of oxyntic mucosa were examined. Generation of mucosal PGE2 was measured by radioimmunoassay. ASA treatment resulted in an increase in the number and severity of mucosal ulcers and a decrease in PGE2 levels within the first treatment day. By day 20 the degree of ulceration had decreased in spite of a persistent reduction of mucosal PGE2 generation. A variable degree of ulceration was observed in SA-treated animals. In control animals only a single class of binding sites for [3H]PGE2 was evident. After 3 days of ASA treatment a second class of binding sites with a high affinity dissociation constant appeared. There was a decrease in the high affinity binding of [3H]PGE2 after 20 days of ASA ingestion. Low affinity binding was not altered. ASA treatment resulted in a significant increase in specific binding capacities for both families of binding sites. SA treatment did not consistently alter PGE2 binding characteristics from control at any time period studied. These data suggest that SA treatment results in a small degree of mucosal damage in the absence of a significant reduction in tissue generation of PGE2 or changes in PGE2 binding. Damage in response to ASA ingestion was associated with a reduction in both endogenous synthesis of PGE2 and an increase in the concentration of both low and high affinity binding sites for PGE2. The reduction in mucosal ulceration on day 20 in spite of depressed endogenous PGE2 coincides with an increase in PGE2 binding.

scholarly journals Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum

1991 ◽  
Vol 274 (3) ◽  
pp. 861-867 ◽  
Author(s):  
R A J Challiss ◽  
A L Willcocks ◽  
B Mulloy ◽  
B V L Potter ◽  
S R Nahorski
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Binding Activity ◽  
Inositol Polyphosphate ◽  
Homogeneous Population ◽  
Site Model ◽  
High Affinity ◽  
Pentosan Polysulphate

1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude ‘P2’ cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.

scholarly journals Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum

1990 ◽  
Vol 265 (2) ◽  
pp. 393-398 ◽  
Author(s):  
D L Nunn ◽  
B V L Potter ◽  
C W Taylor
Keyword(s):  
Binding Sites ◽  
Radioligand Binding ◽  
Inositol Phosphate ◽  
Rank Order ◽  
Binding Protein ◽  
Molecular Target ◽  
Binding Studies ◽  
Single Class ◽  
Surface Receptors ◽  
High Affinity

Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein.

scholarly journals Loss of Striatal [76Br]Bromospiperone Binding Sites Demonstrated by Positron Tomography in Progressive Supranuclear Palsy

1986 ◽  
Vol 6 (2) ◽  
pp. 131-136 ◽  
Author(s):  
J. C. Baron ◽  
B. Mazière ◽  
C. Loc'h ◽  
H. Cambon ◽  
P. Sgouropoulos ◽  
...  
Keyword(s):  
Progressive Supranuclear Palsy ◽  
Binding Sites ◽  
Matched Control ◽  
Specific Binding ◽  
Neuroleptic Drug ◽  
Uptake Ratio ◽  
Positron Tomography ◽  
High Affinity ◽  
Da Receptors

Using positron tomography and 76Br-labeled bromospiperone, a neuroleptic drug with high affinity for the dopamine (DA) receptors, we have estimated the specific binding of the radiotracer to striatal DA receptors in seven patients suffering from progressive supranuclear palsy. Compared with age- and sex-matched control subjects, we found a significant (p < 0.02) decrease of the striatum–cerebellum uptake ratio in progressive supranuclear palsy patients, suggesting loss of striatal DA receptors. This in vivo study confirms recent postmortem data on progressive supranuclear palsy patients and provides an explanation for the lack of benefit from l-DOPA and DA agonists in this condition, despite reduced nigrostriatal dopaminergic function.

scholarly journals Contribution of gangliosides to thyroxin binding with plasma membranes

1995 ◽  
Vol 41 (2) ◽  
pp. 28-30
Author(s):  
T. S. Saatov ◽  
F. Ya. Gulyamova ◽  
G. U. Usmanova
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Specific Binding ◽  
Brain Cell ◽  
Cell Recognition ◽  
High Affinity ◽  
Specific Binding Sites

Besides intracellular receptors of thyroid hormones, specific binding sites for T3 and T4 were detected on plasma membranes (PM) of some cells and a relationship between membrane reception .and lipid composition of membranes shown. The parameters of 125I-T4 binding to highly purified PM of hepatic and cerebral cells of rats were studied. The hepatic and cerebral cellular membranes were found to contain two sites of hormone binding each, one of these sites being characterized by a high affinity and low capacity, and the other by low affinity and a higher binding capacity. The association constant of highly affine site of hepatocyte membranes was found to be higher than that of brain cell membranes. T4 membranous receptors may be significant in the process of cell “recognition" by the hormone. In vivo and in vitro experiments with 125I-T4 and 14C-labeled thyroxin in ganglioside fractions showed appreciable binding of the hormone to Gm3 fraction, this evidently pointing to participation of this, ganglioside in T4 interaction with membrane receptor. It is possible that gangliosides situated on membranous surface are components of or function as receptors.

scholarly journals Properties of soluble somatostatin-binding protein

1977 ◽  
Vol 165 (2) ◽  
pp. 269-277 ◽  
Author(s):  
Norio Ogawa ◽  
Tom Thompson ◽  
Henry G. Friesen ◽  
Joseph B. Martin ◽  
Paul Brazeau
Keyword(s):  
Blood Cells ◽  
Anterior Pituitary ◽  
Specific Binding ◽  
Binding Protein ◽  
Gel Filtration ◽  
Binding Activity ◽  
Rat Tissues ◽  
Peripheral Blood Cells ◽  
Tissue Extracts ◽  
Human Anterior Pituitary

A soluble somatostatin-binding protein was detected in the cytosol fractions of various rat, human and bovine tissues. Maximum binding occurred at pH8.0–8.5 and was Ca2+-dependent. The specific binding of somatostatin per 10μg of cytosol protein from 12 rat tissues ranged between 36 and 15%, and 3% for peripheral blood cells. There was also substantial binding in cytosol from human anterior pituitary and liver, and bovine anterior pituitary. The specific binding in rat and human plasma in the presence of EDTA was only 1%. Gel filtration suggested a molecular weight of approx. 80000 for the somatostatin-binding protein from several sources. Exposure of the binding protein to trypsin eliminates somatostatin-binding activity but ribonuclease and deoxyribonuclease have no effect. The binding protein is thermolabile, ethanol-precipitable, and not completely specific for somatostatin. Bound125I-labelled [Tyr1]somatostatin is not easily displaced by excess of unlabelled somatostatin. The effects of dithiothreitol and mercaptoethanol on the binding of125I-labelled [Tyr1]somatostatin to the binding protein suggests that binding involves two sequential steps, first loose binding, then disulphide linkage. Since semipurified somatostatin-binding protein causes a dose-related inhibition of the binding of125I-labelled [Tyr1]somatostatin in radioimmunoassays for somatostatin, estimates of somatostatin content of tissue extracts by radioimmunoassay in some cases may be spuriously high. It is not yet clear whether the binding protein is a true cytosol protein or an easily solubilized membrane protein.

A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

scholarly journals Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle

1991 ◽  
Vol 276 (1) ◽  
pp. 41-46 ◽  
Author(s):  
V Shoshan-Barmatz ◽  
T A Pressley ◽  
S Higham ◽  
N Kraus-Friedmann
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Specific Binding ◽  
Dna Amplification ◽  
Single Class ◽  
High Affinity ◽  
Sodium Dantrolene

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.

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