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At least 649 records · Page 36Linked to original sources
PubMed1979

Synthesis and mode of action on axonal membranes of photoactivable derivatives of tetrodotoxin.

Two photoactivable derivatives of tetrodotoxin have been synthesized. Electrophysiological experiments on crab giant axons and competitive binding with [3H]-tetrodotoxin for the tetrodotoxin receptor indicate that they are only 4.5 to 7.5 times less active than tetrodotoxin itself. These compounds give a reversible block of the sodium channel in the dark but after ultraviolet irradiation they provoke an irreversible blockade of the channel.

Animals↗
PubMed2004

Analysis of paralytic shellfish poisoning toxin congeners by a sodium channel receptor binding assay.

This study was carried out to characterize the detection and quantitation of several paralytic shellfish poisoning (PSP) toxin congeners using a receptor binding assay (RBA). This involved competitive binding of the toxin congeners against tritium-labeled STX for receptor sites on rat brain sodium channels. Competitive binding curves were described by a four-parameter logistic equation. Half-saturation values (EC(50)) ranged from 4.38 nM for STX to 142 nM for GTX5. Receptor binding affinity was in the order STX>GTX1/4>neoSTX>GTX2/3>dcSTX>GTX5, and this was similar to the order of mouse toxicity of these congeners. Predicted toxin concentrations from observed STXeq values and EC(50) ratios relative to STX were within 20% or better of the actual concentrations used in the assay. In contrast predicted toxin concentrations using mouse toxicity ratios relative to STX did not provide a good match to actual concentrations, except for GTX1/4. This study has shown that the rat brain sodium channel RBA will provide a reliable integration of total toxicity of various PSP toxin congeners present in a sample.

Animals↗
PubMed1983

The determination of maternal and foetal rat plasma corticosterone concentration in late pregnancy by competitive protein binding analysis.

A procedure for the determination of corticosterone in 20 microliters of rat plasma, in maternal and foetal late pregnancy with a protein binding radioassay, is described. The method is suitable in a range of 0.5-3 ng of corticosterone. An amount of 0.5 ng can be detected with a coefficient of variation for duplicate of 13%. This procedure seems to be specific for glucocorticosteroids. The comparison of the results obtained with fluorimetry and the competitive protein binding radioassay show the reliability of both methods although the protein binding radioassay is much more sensitive.

Animals↗
PubMedDate not supplied

Luminal and non-luminal non-competitive inhibitor binding sites on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of the alpha subunit exist the binding sites for agonists such as the neurotransmitter acetylcholine, which upon binding trigger the channel opening, and for competitive antagonists such as d-tubocurarine, which compete for the former inhibiting its pharmacological action. For non-competitive inhibitors, a population of low-affinity binding sites have been found at the lipid-protein interface of the nicotinic acetylcholine receptor. In addition, at the M2 transmembrane domain, several high-affinity binding sites have been found for non-competitive inhibitors such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222 and the hydrophobic probe trifluoromethyl-iodophenyldiazirine. They are known as luminal binding sites. Although the local anaesthetic meproadifen seems to be located between the hydrophobic domains M2-M3, this locus is considered to form part of the channel mouth, thus this site can also be called a luminal binding site. In contraposition, experimental evidences support the hypothesis of the existence of other high-affinity binding sites for non-competitive inhibitors located not at the channel lumen, but at non-luminal binding domains. Among them, we can quote the binding site for quinacrine, which is located at the lipid-protein interface of the alpha M1 domain, and the binding site for ethidium, which is believed to interact with the wall of the vestibule very far away from both the lumen channel and the lipid membrane surface. The aim of this review is to discuss these recent findings relative to both structurally and functionally relevant aspects of non-competitive inhibitors of the nicotinic acetylcholine receptor. We will put special emphasis on the description of the localization of molecules with non-competitive antagonist properties that bind with high-affinity to luminal and non-luminal domains. The information described herein was principally obtained by means of methods such as photolabelling and site-directed mutagenesis in combination with patch-clamp. Our laboratory has contributed with data obtained by using biophysical approaches such as paramagnetic electron spin resonance and quantitative fluorescence spectroscopy.

Affinity Labels↗
PubMed2001

Probing the interaction of dengue virus envelope protein with heparin: assessment of glycosaminoglycan-derived inhibitors.

A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.

Anticoagulants↗
PubMed1975

A competitive protein binding radioassay for 17alpha-ethynylestradiol in human plasma.

A competitive protein binding radioassay system using a rabbit uterine cytosol preparation for the measurement of ethynylestradiol [EE2e] in human plasma has been developed. Sephadex LH-20 column chromatography is used for the separation of the estrogens prior to assay. EE2 levels were measured in 23 normotensive subjects on contraceptive pills containing either 50 mug of EE2 or mestranol. The EE2 values ranged from 144 to 248 pg with a mean of 211 pg (S.D. +/- 37.94). The method is sensitive enough to detect 25 pg of EE2 in plasma. The intra- and inter-assay coefficients of variation were 7.54% and 10.75%, respectively. Water blanks and plasma blanks from subjects not on pills gave negligible readings for EE2.

Animals↗
PubMed1996

Interconverting receptor states at 4 degrees C for the neutrophil N-formyl peptide receptor.

With the aid of high time resolution kinetic data extracted from a flow cytometer, we determined that there are two N-formyl peptide receptor states for human neutrophils at 4 degrees C: a low affinity and a high affinity state. Competitive binding of FMLP, FNLP, and t-BOC with FNLPNTL-FL revealed different kinetic rate constants for two distinct reactions that control the lifetime of the low affinity ligand-receptor complex. For these ligands, the rate constant for dissociation of ligand from the low affinity receptor state (the first reaction) ranges in order of magnitude from 10(-2) to 1 s-1, and the conversion rate constant from the low affinity receptor state to the high affinity receptor state (the second reaction) ranges from 10(-4) to 10(-2) s-1. The antagonist t-BOC differed most significantly from the three agonists by having an association rate constant for the low affinity receptor on the order of 10(5) M-1 s-1; the value for all three agonists was on the order of 10(7) M-1 s-1. Characterization of the receptor conversion at 4 degrees C revealed that it is irreversible (or very slow) and independent of Gi protein and that neither receptor state is a form of receptor precoupled to Gi protein. The affinity conversion and the dissociation characteristics of each receptor state determine the duration of the signaling complex and may contribute to differences in ligand efficacy.

Binding, Competitive↗
PubMed1991

Effect of chronic D-Ala,2 D-Leu5-enkephalin or pertussis toxin treatment on the high-affinity state of delta opioid receptor in neuroblastoma x glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma x glioma NG108-15 hybrid cells with the opioid agonist D-Ala,2 D-Leu5-enkephalin (DADLE) induces a homologous desensitization of the delta opioid receptors present in these cells. Since the Kd value of the delta opioid receptor's high-affinity state reflects the potency of the agonist, we examined the effect of receptor desensitization in NG108-15 cells on the percentage of receptor in the high-affinity state. When NG108-15 hybrid cells were treated with 10 or 100 nM DADLE for 4 hr at 24 degrees C, loss of DADLE's ability to inhibit adenylate cyclase was observed. However, when competition binding experiments were carried out with P2P3 membranes isolated from the delta opioid-desensitized hybrid cells, it was determined that 41.7 +/- 3.4% of the total binding sites remained in the high-affinity state, with no apparent alteration in the Kd value of either high- or low-affinity states. Similarly, when NG108-15 cells were treated with 100 ng/ml of pertussis toxin for 3 hr at 37 degrees C, 39.9 +/- 3.6% of the binding sites remained in the high-affinity state. This reduction in the percentage of receptor in high-affinity state was agonist specific, for chronic treatment of hybrid cells with levorphanol, a partial agonist, or the antagonist naloxone did not alter the percentage of opioid receptors in the high-affinity state. Furthermore, the delta opioid receptors remaining in the high-affinity state after chronic DADLE treatment were still sensitive to both Na+ and guanyldylimidodiphosphate, indicating that opioid ligand binding remained coupled to the G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1996

Interaction of recombinant rat placental lactogen-I with extracellular domains of prolactin receptors from three species.

Rat placental lactogen-I (rPL-I), expressed in rat uteri at midpregnancy, belongs to the GH/PRL/cytokine family of hormones (Wallis, 1992), all members of which exhibit a similar mechanism of receptor activation. Lactogenic activity of rPL-I as determined by Nb2 lymphoma cell bioassay was slightly higher than that of hGH. rPL-I was capable also of stimulating beta-casein production in mouse HC-11 cells. Competitive binding experiments using [125I]hGH or [125I]bPL and purified prolactin receptor extracellular domains (PRLR-ECDs) from rat (r), rabbit (rb), and bovine (b) showed rPL-I to be 12, 40, and 7-fold, respectively, less effective than hGH when competing with [125I]hGH. Displacement of [125I]bPL by rPL-I from rPRLR-ECD and rbPRLR-ECD was also 4-, and 30-fold less effective, respectively, than that by bPL. rPL-I did not compete at all with [125I]bPL for binding to bPRLR-ECD. In contrast, competitive binding experiments with [125I]hGH to a microsomal fraction of Nb2 cells showed rPL-I to be slightly more active than hGH. The stoichiometries of the complexes formed by rPL-I with rbPRLR-ECD, rPRLR-ECD, and bPRLR-ECD were studied by gel filtration. Whereas a 1:1 complex was formed between rPL-I and rPRLR-ECD and rbPRLR-ECD, no complex could be detected between rPL-I and bPRLR-ECD. The present results support the hypothesis that transient forms of a homodimer complex may be sufficient to initiate the biological signal, despite its short half-life.

Animals↗
PubMed1988

Formation of the high-affinity agonist state of the alpha 1-adrenergic receptor at cold temperatures does not require a G-protein.

Two methods were employed to uncouple hepatic alpha 1-adrenergic receptors from their associated G-protein (termed Gp) in order to determine whether locking of the alpha 1-receptor in a high-affinity agonist state at cold temperatures (2 degrees C) represents formation of a ternary complex. Uncoupling is defined as the inability to observe the GppNHp-sensitive, high-affinity agonist state of the receptor in [3H]prazosin competition binding studies performed at 25 degrees C. The first method for achieving uncoupling involved brief alkalinization and resulted in greater than 95% loss of several G-proteins. The second method involved proteolytic cleavage of either part or all of the alpha 1-receptor coupling domain from the binding domain. Following either treatment, receptors were converted to the high-affinity agonist state at 2 degrees C. Thus, while formation of the high-affinity state of the receptor at higher temperatures may require Gp, formation of this state at 2 degrees C does not require Gp or even the entire alpha 1-adrenergic receptor.

Animals↗
PubMed2000

ORC1 interacts with c-Myc to inhibit E-box-dependent transcription by abrogating c-Myc-SNF5/INI1 interaction.

BACKGROUND: The c-myc oncogene product (c-Myc) is a transcription factor that forms a complex with Max and recognizes the E-box sequence. c-Myc plays key functions in cell proliferation, differentiation and apoptosis. As for its activity towards cell proliferation, it is generally thought that c-Myc transactivates the E-box-containing genes that encode proteins essential to cell-cycle progression. Despite the characterization of candidate genes regulated by c-Myc in culture cells, these have still not been firmly recognized as real target genes for c-Myc. RESULTS: We found that c-Myc directly bound to the N-terminal region of origin recognition complex-1 (ORC1), a region that is responsible for gene silencing, in a state of complex containing other ORC subunits and Max in vivo and in vitro. Furthermore, ORC1 inhibited E-box-dependent transcription activity of c-Myc by competitive binding to the C-terminal region of c-Myc with SNF5, a component of chromatin remodelling complex SNF/Swi1. CONCLUSIONS: These results suggest that ORC1 suppresses the transcription activity of c-Myc by its recruitment into an inactive form of chromatin during some stage of the cell cycle.

Binding, Competitive↗
PubMed1993

Age-related changes in pulmonary muscarinic receptor binding properties.

The goal of this study was to elucidate mechanisms responsible for age-related reductions in responsiveness to cholinergic muscarinic stimulation in guinea pigs, by examining the binding properties of muscarinic receptors and their coupling to guanine nucleotide regulatory proteins as a function of animal age. In addition, the binding constants of three selective muscarinic receptor antagonists pirenzepine, [11-((2-[(diethylamino)methyl]-1-piperidinyl)-acetyl)-5, 11-dihydro-6H-pyrido(2,3)(1,4)benzodiazepine-6-on], and 4-diphenylacetoxy-N-methylpiperidine methobromide were examined. We found that there were no changes in either the receptor density or the affinity of the muscarinic receptor with age. There was a significant reduction in the affinity constant for the high-affinity agonist binding site in the old tissues (7.63 +/- 0.08) compared with the young tissues (8.31 +/- 0.10). Guanine nucleotides lowered agonist affinity for the receptor in young lungs, however, they had no effect on agonist binding in old tissues. Antagonist competition binding curves in young tissues revealed that 73% of the receptors are of the M2 type, with 27% being of the M3 subtype. In contrast, antagonist competition binding curves in the old tissues revealed that 37% of the receptors were of the M2 subtype, 30% were M3, and 33% were of the M1 subtype. Our studies provide evidence that the loss of sensitivity to cholinergic muscarinic stimulation in the senescent lung may be due to changes in both muscarinic receptor subtypes and receptor coupling to G proteins.

Aging↗
PubMed1978

Interaction of ovalbumin and its asparaginyl-carbohydrate fractions with concanavalin A.

The interaction of ovalbumin and its asparaginyl-carbohydrate fractions with concanavalin A was studied. Relative affinities were obtained by competitive binding studies using p-nitrophenyl alpha-D-mannopyranoside. Ovalbumin was separated into two fractions, I and II, by chromatography on concanavalin A-Sepharose. Ovalbumin and its fractions I and II interacted with concanavalin A in solution with binding affinities at 10 degrees C of 2 . 10(5) M-1, 3 . 10(4) M-1 and 2 . 10(6) M-1, respectively. The seven asparaginyl-carbohydrate fractions, obtained by fractionation on Dowex 50W-X2 (H+) and Durrum DA-4 (borate)columns, bound to concanavalin A with approximately the same affinity as native ovalbumin, suggesting that the sugar residues for binding in the isolated carbohydrates are exposed in the native protein. The binding of ovalbumin to concanavalin A was minimal after treatment with alpha-D-mannosidase in spite of the fact that only one half of the available mannose residues were hydrolyzed when compared to those removed by similar treatment of the asparaginyl-carbohydrate before fractionation. It is concluded that those alpha-D-mannosyl residues in ovalbumin that are required for binding to concanavalin A are accessible to alpha-D-mannosidase while the residual mannosyl groups are "buried" from interaction with concanavalin A and the enzyme.

Asparagine↗
PubMed2003

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling.

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.

Binding, Competitive↗
PubMed1975

[Radioimmunoassay of n-butylbiguanide in human serum].

A radioimmuno assay for butylbiguanide in human serum is described. The detection limit is 1.5 ng per assay tube, the intra assay variance 3 percent, and the inter assay variability 12 percent. Antibodies to the biguanide group were obtained by immunizing rabbits with p-carboxy-phenethyl-biguanide coupled to bovine serum albumin. The affinity of the antibodies for tyrosine, urea, guanidine, butylbiguanide, phenethyl-biguanide, and p-hydroxy-phenethyl-biguanide is shown by competitive binding studies. The recovery rate is 100 percent.

Animals↗
PubMed2005

Identification of all alpha1-adrenoceptor subtypes in rat lung.

The function of lung tissue is regulated via a release of neurotransmitters from autonomic nerves. The neurotransmitters of sympathetic nervous system, adrenaline and noradrenaline, activate both alpha-adrenoceptors and beta-adrenoceptors. Although the function and expression of beta-adrenoceptors can be considered major, some doubts exist about the function and expression of alpha1-adrenoceptor subtypes in the lung tissue. Therefore a set of competition binding experiments was employed in order to discriminate between the alpha1-adrenoceptor binding site subtypes in the rat lung. We identified three subpopulations of alpha1-adrenoceptor binding sites in the rat lung (alpha1A, alpha1B and alpha1D).

Animals↗
PubMed2004

The influence of G protein subtype on agonist action at D2 dopamine receptors.

In previous studies, we have shown that agonists influence the ability of D2 dopamine receptors to couple to G proteins and here we extend this work. The human D2Short dopamine receptor and a natural polymorphism of this D(2Short)(Ser311Cys), have been studied by co-expressing the receptors in insect cells with Gbeta1gamma2 and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[3H]spiperone competition binding experiments and through stimulation of [35S]GTPgammaS binding. Although the Ser311Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D2 receptor, agonist stimulation of [35S]GTPgammaS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Galpha(o) compared to the Galpha(i) subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins.

Animals↗
PubMed2003

Gender comparison of muscarinic receptor expression and function in rat and human urinary bladder: differential regulation of M2 and M3 receptors?

Since symptoms of bladder dysfunction occur more frequently in women than in men and since muscarinic receptors are the physiologically most important system to mediate bladder contraction, we have compared the number, subtype distribution and function of muscarinic receptors in bladders from male and female rats. Muscarinic receptor function was also assessed in bladder strips from male and female human bladder. Male and female rats expressed a similar number of muscarinic receptors (144+/-5 vs. 140+/-6 fmol/mg protein in saturation radioligand binding). While competition binding curves for the moderately M(2)-selective methoctramine were not consistently better fitted by a two-site model, most competition curves for the M(3)-selective darifenacin were biphasic and yielded 29+/-10% and 31+/-7% high affinity sites (corresponding to M(3) receptors) in male and females, respectively. Immunoreactivity of alpha-subunits of the G-proteins G(q/11), G(i1/2), G(i3) and G(s) did not significantly differ between both genders. The muscarinic receptor agonist carbachol similarly stimulated inositol phosphate accumulation in bladder slices from male and female rats with calculated maximum responses of 69+/-17 and 77+/-18% over basal and pEC(50) values of 4.90+/-0.45 and 4.40+/-0.46, respectively. While darifenacin inhibited carbachol-stimulated inositol phosphate formation approximately 100-fold more potently than methoctramine, each antagonist was similarly potent in both genders. Carbachol concentration-dependently contracted bladder strips with a pEC(50) of 5.66+/-0.05 and 5.72+/-0.06 and maximum effects of 4.3+/-0.1 and 4.2+/-0.2 mN/mg wet weight in male and female rats, respectively. The contractile effect of carbachol was concentration-dependently antagonised by the non-selective atropine (1-30 nM), the M(1)-selective pirenzepine (1-30 M), the M(2)-selective methoctramine (1-10 microM) and the M(3)-selective darifenacin (10-100 nM), with the latter exhibiting a partly unsurmountable antagonism. The overall potency of all four antagonists suggested that contraction was mediated predominantly if not exclusively by M(3) receptors with no appreciable differences between both male and female rats. Similarly, the maximum effects (4.4+/-0.6 vs. 4.4+/-2.4 mN/mg) and pEC(50) (6.07+/-0.05 vs. 6.32+/-0.14) of carbachol did not differ between genders in bladder samples from 25 consecutive patients. We conclude that number und function of muscarinic receptors and the relative roles of their M(2) and M(3) subtypes do not differ between urinary bladders of male and female rats; at least with regard to overall muscarinic responsiveness this situation appears to be similar in humans.

Animals↗