scholarly journals Aldosterone antagonists destabilize the mineralocorticosteroid receptor

1992 ◽  
Vol 282 (3) ◽  
pp. 697-702 ◽  
Author(s):  
B Couette ◽  
M Lombes ◽  
E E Baulieu ◽  
M E Rafestin-Oblin
Keyword(s):  
Binding Sites ◽  
Gradient Centrifugation ◽  
Dissociation Rate ◽  
Receptor Interaction ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Aldosterone Antagonists ◽  
Low Salt ◽  
Idiotypic Antibody ◽  
Binding Subunit

To elucidate the mechanism of action of aldosterone antagonists, we studied the interaction of spironolactone with the chick mineralocorticosteroid receptor (MR). Intestinal cytosol contains specific spironolactone-binding sites (Kd approximately 3 nM; max. no. of binding sites approximately 100 fmol/mg of protein) that have been identified as MRs by competition experiments with steroid ligands and with the monoclonal anti-idiotypic antibody H10E that interacts with aldosterone-binding domain of the MR. Binding studies indicate that aldosterone and spironolactone bind to the MR through a common site that encompasses the epitope recognized by H10E. At 4 degrees C, spironolactone dissociates much more rapidly from the cytosol 8-9 S form of MR (t1/2 38 min) than does aldosterone (t1/2 3240 min). A high dissociation rate was also observed for progesterone, a natural aldosterone antagonist (t1/2 84 min). The covalent linkage of the 90 kDa heat shock protein (hsp90) to the ligand-binding subunit of MR with dimethyl pimelimidate did not notably modify the rate of dissociation of spironolactone from the receptor (t1/2 96 min), excluding the possibility that the rapid dissociation rate of the antagonist was related to hsp90 release. The effects of aldosterone and the two anti-mineralocorticosteroids on the 8-9 S heterooligomeric structure of the MR differed strikingly. Using low-salt density-gradient centrifugation analysis, aldosterone-labelled receptors were recovered as 8-9S complexes, whereas 4 S entities were detected after spironolactone and progesterone binding. This indicated that, under the experimental conditions used, aldosterone antagonists facilitate hsp90 release and thus do not stabilize the non-DNA-binding 8-9S form of MR. We propose that the combination of rapid dissociation of the ligand and a weakened hsp90-receptor interaction is involved in the anti-mineralococorticosteroid activity of aldosterone antagonists.

scholarly journals Chloroquine augments the binding of insulin to its receptor

1995 ◽  
Vol 311 (3) ◽  
pp. 787-795 ◽  
Author(s):  
A P Bevan ◽  
J R Christensen ◽  
J Tikerpae ◽  
G D Smith
Keyword(s):  
Binding Sites ◽  
Slow Component ◽  
Insulin Binding ◽  
Dissociation Rate ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Binding Model ◽  
Binding Data ◽  
Plasma Membrane Receptor ◽  
Dissociation Rate Constants

The effect of chloroquine on the interaction of insulin with its receptor has been investigated under both equilibrium and non-equilibrium conditions. Chloroquine was found to augment insulin binding in a pH-dependent manner between pH 6.0 and pH 8.5, with the maximum occurring at approximately pH 7.0. Analysis of the equilibrium binding data in terms of independent binding sites gave equivocal results but suggested an increase in the high-affinity component. Analysis using the negative co-operativity binding model of De Meyts, Bianco and Roth [J. Biol. Chem. (1976) 251, 1877-1888] suggested that the affinity at both high and low occupancy was increased equally. The kinetics of association of insulin with the plasma-membrane receptor indicated that, although the net rate of association increased in the presence of chloroquine, this was due to a reduction in the dissociation rate rather than an increase in the association rate. This was confirmed by direct measurement of the rates of dissociation. Dissociation was found to be distinctly biphasic, with fast and slow components. Curve fitting suggested that the decrease in dissociation rate in the presence of chloroquine was not due to a decrease in either of the two dissociation rate constants, but rather to an increase in the amount of insulin dissociating by the slow component. It was also found that the increase in dissociation rate in the presence of excess insulin, ascribed to negative co-operativity, could be accounted for by an increase in the amount of insulin dissociating by the faster pathway, rather than by an increase in the dissociation rate constant. Thus chloroquine appears to have the opposite effect to excess insulin, and evidence was found for the induction of positive co-operativity in the insulin-receptor interaction at high chloroquine concentrations. Evidence was also found for the presence of low-affinity chloroquine binding sites with binding parameters similar to the concentration dependence of the chloroquine-induced augmentation of insulin binding.

Lectin Binding Studies on RNA Tumor Virus-Infected Cells

1975 ◽  
Vol 33 ◽  
pp. 326-327
Author(s):  
D. C. Hixson
Keyword(s):  
Binding Sites ◽  
Lectin Binding ◽  
Culture Conditions ◽  
3T3 Cells ◽  
Binding Studies ◽  
Con A ◽  
Microscopic Studies ◽  
Tumor Virus ◽  
Infected Cells ◽  
Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

scholarly journals Ultrafiltration Method for Plasma Protein Binding Studies and Its Limitations

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 382
Author(s):  
Camelia-Maria Toma ◽  
Silvia Imre ◽  
Camil-Eugen Vari ◽  
Daniela-Lucia Muntean ◽  
Amelia Tero-Vescan
Keyword(s):  
Protein Binding ◽  
Plasma Protein Binding ◽  
Plasma Protein ◽  
Specific Binding ◽  
Critical Role ◽  
Volume Ratio ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Key Aspects

Plasma protein binding plays a critical role in drug therapy, being a key part in the characterization of any compound. Among other methods, this process is largely studied by ultrafiltration based on its advantages. However, the method also has some limitations that could negatively influence the experimental results. The aim of this study was to underline key aspects regarding the limitations of the ultrafiltration method, and the potential ways to overcome them. The main limitations are given by the non-specific binding of the substances, the effect of the volume ratio obtained, and the need of a rigorous control of the experimental conditions, especially pH and temperature. This review presents a variety of methods that can hypothetically reduce the limitations, and concludes that ultrafiltration remains a reliable method for the study of protein binding. However, the methodology of the study should be carefully chosen.

The physicochemical and DNA binding studies of some medicinal compounds in solutions

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Abbas Khan ◽  
Naila ◽  
Muhammad Humayun ◽  
Muhammad Sufaid Khan ◽  
Luqman Ali Shah ◽  
...  
Keyword(s):  
Flow Behavior ◽  
Aqueous Media ◽  
Research Work ◽  
Binding Constants ◽  
Physicochemical Study ◽  
Solution Temperature ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Medicinal Compounds ◽  
Association Behavior

Abstract To understand the expected mode of action, the physicochemical study on the solution properties of medicinal compounds and their interaction with deoxyribonucleic acid (DNA), under varying experimental conditions, is of prime importance. The present research work illustrates the physicochemical study and interaction of certain medicinal compounds such as; Levofloxacin, Ciprofloxacin, and Ibuprofen with DNA. Density, viscosity and surface tension measurements have been performed in order to determine, in a systematic manner, the physicochemical, volumetric and thermodynamic properties of these compounds; and most of these parameters have shown different behavior with varying concentration of solution, temperature of the medium and chemical nature/structure of the compound. In addition, these drugs showed a spontaneous surface-active and association behavior in aqueous solutions. The flow behavior, surface properties, volumetric behavior and solute–solvent interaction of these drugs were prominently influenced by experimental variables and addition of DNA to their solutions. UV–Visible spectroscopy was also used to examine the interaction of these drugs with DNA in aqueous media in detail. Calculated values of binding constants (Kb) for all complexes of drug-DNA are positive, indicating a fruitful binding process. It is seen that a smaller Kb value reflects weaker binding of the drug with DNA and vise versa. Due to the difference in the chemical structure of drugs the values of binding constant are different for various drug-DNA complexes and follow the order Kb(Levofloxacin-DNA) > Kb(Ciprofloxacin-DNA) > Kb(Ibuprofen-DNA). On the basis of spectral changes and Kb it can be said that the binding of all these drugs with DNA may be of physicochemical nature and the dominating binding force be of hydrogen bonding between oxygen of drugs and hydrogen of DNA units and the drug having more oxygen atoms showed stronger binding ability. The data further suggest a limited possibility of chemical type attachment of these drugs with DNA.

scholarly journals Cytosol oestrogen receptor of lactating mammary gland. Effect of heparin on the aggregation of the receptor and interaction of the receptor with heparin-Sepharose

1978 ◽  
Vol 171 (1) ◽  
pp. 137-141 ◽  
Author(s):  
F Auricchio ◽  
A Rotondi ◽  
P Sampaolo ◽  
E Schiavone
Keyword(s):  
Mammary Gland ◽  
Oestrogen Receptor ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Large Aggregate ◽  
Low Salt ◽  
Lactating Mammary Gland ◽  
Stokes Radius

1. An oestrogen receptor is present in low-salt cytosol of the mammary gland of lactating mice as a large aggregate; it is excluded from gel matrix when filtered on a Sephadex G-200 column and sediments at 7S in sucrose gradients. After incubation of cytosol with heparin, the receptor is dissociated. On a Sephadex G-200 column, it is included in the gel matrix and eluted as a protein with mol.wt. 260000 and a Stokes radius of 6.8nm; it sediments at 6S in sucrose gradients. 2. Dissociation of the mammary-gland cytosol oestrogen receptor seems to be the result of interaction of the oestrogen-receptor complex with heparin. This receptor interacts with heparin covalently bound to Sepharose, thereafter sedimenting at 6S. By using this interaction, the cytosol receptor was purified 200-fold compared with the homogenate, with a yield of 70%. 3. The cytosol receptor that was not incubated or was incubated with heparin was much smaller during sucrose-gradient centrifugation than during gel filtration. This discrepancy can be explained by pressure-induced dissociation during high-speed centrifugation. This possibility is supported by the decrease in the sedimentation coefficient of the receptor with increased duration of centrifugation.

scholarly journals Hybrid Dynamic Pharmacophore Models as Effective Tools to Identify Novel Chemotypes for Anti-TB Inhibitor Design: A Case Study With Mtb-DapB

2020 ◽  
Vol 8 ◽  
Author(s):  
Chinmayee Choudhury ◽  
Anshu Bhardwaj
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Chemical Space ◽  
Pharmacophore Model ◽  
Model Systems ◽  
Receptor Interaction ◽  
Hybrid Molecules ◽  
Wide Range ◽  
Binding Pockets ◽  
Pharmacophore Models

Antimicrobial resistance (AMR) is one of the most serious global public health threats as it compromises the successful treatment of deadly infectious diseases like tuberculosis. New therapeutics are constantly needed but it takes a long time and is expensive to explore new biochemical space. One way to address this issue is to repurpose the validated targets and identify novel chemotypes that can simultaneously bind to multiple binding pockets of these targets as a new lead generation strategy. This study reports such a strategy, dynamic hybrid pharmacophore model (DHPM), which represents the combined interaction features of different binding pockets contrary to the conventional approaches, where pharmacophore models are generated from single binding sites. We have considered Mtb-DapB, a validated mycobacterial drug target, as our model system to explore the effectiveness of DHPMs to screen novel unexplored compounds. Mtb-DapB has a cofactor binding site (CBS) and an adjacent substrate binding site (SBS). Four different model systems of Mtb-DapB were designed where, either NADPH/NADH occupies CBS in presence/absence of an inhibitor 2, 6-PDC in the adjacent SBS. Two more model systems were designed, where 2, 6-PDC was linked to NADPH and NADH to form hybrid molecules. The six model systems were subjected to 200 ns molecular dynamics simulations and trajectories were analyzed to identify stable ligand-receptor interaction features. Based on these interactions, conventional pharmacophore models (CPM) were generated from the individual binding sites while DHPMs were created from hybrid-molecules occupying both binding sites. A huge library of 1,563,764 publicly available molecules were screened by CPMs and DHPMs. The screened hits obtained from both types of models were compared based on their Hashed binary molecular fingerprints and 4-point pharmacophore fingerprints using Tanimoto, Cosine, Dice and Tversky similarity matrices. Molecules screened by DHPM exhibited significant structural diversity, better binding strength and drug like properties as compared to the compounds screened by CPMs indicating the efficiency of DHPM to explore new chemical space for anti-TB drug discovery. The idea of DHPM can be applied for a wide range of mycobacterial or other pathogen targets to venture into unexplored chemical space.

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

Epinephrine induces Ca2+ uptake in human blood platelets

1980 ◽  
Vol 239 (4) ◽  
pp. H483-H483
Author(s):  
N. E. Owen ◽  
H. Feinberg ◽  
G. C. Le Breton
Keyword(s):  
Human Blood ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Receptor Interaction ◽  
Induced Aggregation ◽  
Selective Increase ◽  
Dose Dependent ◽  
Primary Platelet ◽  
Human Blood Platelets

Human blood platelets isolated by albumin density gradient centrifugation take up Ca2+ during 10-6M epinephrine-induced primary aggregation but not during 10-6 M ADP-induced primary aggregation. Platelet uptake of Ca2+ is dose-dependent over a range of 10-7) to 10-5 M epinephrine. Antagonism of the platelet α-receptor by phentolamine (10-6 M) results in inhibition of both epinephrine-stimulated Ca2+ uptake and aggregation. The Ca2+ antagonist verapamil (50 μM) blocks Ca2+ uptake and epinephrine-induced aggregation, but not ADP-induced aggregation. The verapamil inhibition of aggregation is reduced on Ca2+ addition. These results suggest that epinephrine acts to stimulate primary platelet aggregation through a specific receptor interaction that results in a selective increase in platelet membrane permeability to Ca2+.

Stroma of human benign prostatic hyperplasia: preferential tissue for androgen metabolism and oestrogen binding

1981 ◽  
Vol 96 (3) ◽  
pp. 422-432 ◽  
Author(s):  
M. Krieg ◽  
G. Klötzl ◽  
J. Kaufmann ◽  
K. D. Voigt
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Binding Sites ◽  
Reductase Activity ◽  
Enzyme Reaction ◽  
Prostatic Hyperplasia ◽  
Binding Studies ◽  
Normal Prostate ◽  
Layer Chromatography ◽  
Androgen Binding ◽  
Tissue Fraction

Abstract. Because of the well known stromal-epithelial interaction of various urogenital organs, it was of interest to compare quantitatively steroid metabolism and binding in epithelium (E) and stroma (S) of the human benign prostatic hyperplasia (BPH). Testosterone 5α-reductase activity was determined by thin-layer chromatography and androgen as well as oestrogen binding sites by a charcoal adsorption technique after a steroid incubation period of 18 h at 0°C, using methyltrienolone (R1881) and oestradiol-17β as tritiated ligands and unlabelled R1881 and diethylstilboestrol as the respective competitors. The main results were as follows: (1) using biochemical markers (acid phosphatase, hydroxyproline), an on average 17% contamination of E by S and 6% of S by E was found, (2) the molar optimum of NADPH for the enzyme reaction was nearly identical in E and S, ranging between 1 and 0.1 mm, (3) the apparent Michaelis constant (Km) of 5α-reductase was in both fractions identical, the mean being 0.15 (μm, (4) the maximal rate of 5α-reductase activity (pmol 5α-reduced metabolites · mg protein−1 · 1 h−1) was 161 ± 28 (sem; n = 20), 66 ± 4.6 and 148 ± 6.6 in S, E and whole tissue fraction of BPH, respectively. In two normal prostates the means were: 88, 53 and 73, respectively, (5) the androgen binding sites were evenly distributed between the cytosol of E and S, while measurable oestrogen binding sites were found in 42% of the analyzed S but only in 5% of analyzed E. In conclusion: the 2.4 times higher 5·-reductase activity in S compared to E of the BPH is responsible for the about 2 to 2.5 times higher activity in the whole tissue fraction of BPH if compared with the normal prostate. Furthermore, due to our preliminary binding studies, oestrogens might play an important role in the S fraction of BPH.

scholarly journals Internalization and down-regulation of the prostacyclin receptor in human platelets

1997 ◽  
Vol 325 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Serenella GIOVANAZZI ◽  
Maria R. ACCOMAZZO ◽  
Ornella LETARI ◽  
Daniela OLIVA ◽  
Simonetta NICOSIA
Keyword(s):  
Binding Sites ◽  
Organic Anion ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Organic Anion Transporters ◽  
Camp Production ◽  
Binding Studies ◽  
Anion Transporters ◽  
Prostacyclin Receptor ◽  
High Affinity Binding Site

The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 °C, addition of unlabelled ligand at either 37 °C or 4 °C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 °C or 4 °C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 °C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 °C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 °C, two classes of binding sites were present, whereas after preincubation at 37 °C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.

Sign in / Sign up

Export Citation Format

Share Document