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At least 343 records · Page 19Linked to original sources
PubMed1992

A model analysis for competitive binding of mexiletine and aprindine to the cardiac sodium channel.

A simulation model was developed to predict complex interaction between antiarrhythmic drugs and cardiac sodium channels. This model has four assumptions: (1) Vmax of the action potential is a linear indicator of available sodium channel conductance; (2) antiarrhythmic drugs block the channel by binding to a single common receptor site associated with the channel; (3) binding and dissociation rate constants differ for the three channel states: activated, inactivated and resting, and (4) both drug-free and drug-bound channels change states far more rapidly than binding and dissociation processes. Binding and dissociation rate constants for the three channel states were calculated from single cell experiments using guinea pig hearts. Vmax changes reflecting tonic and use-dependent sodium channel block in the presence of mexiletine and aprindine were simulated and compared with those obtained in the single cell experiments. The model predicted that 'tonic' Vmax inhibition would be enhanced, whereas 'use-dependent' ones would be attenuated after admixture of mexiletine with aprindine. The mechanisms would involve competitive interaction at the common receptor site. Single-cell experiments supported this prediction. We conclude that our simple two-drug binding model provides a useful tool to predict pharmacological interaction between class I antiarrhythmic drugs given in combination.

Action Potentials↗
PubMed1982

Importance of competitive binding in the detection of antigen specific bovine isotypes and subisotypes by the micro ELISA.

An enzyme-linked immunosorbent assay was developed to detect and quantify the specific bovine immunoglobulin class response to Trypanosoma theileri, Dictyocaulus viviparus and infectious bovine rhinotracheitis virus. Comparative measurement of the specific immunoglobulin classes in whole serum was achieved using monospecific rabbit antibovine IgG1, IgG2 and IgM, followed by a goat anti-rabbit Ig-enzyme conjugate (antiglobulin ELISA). The results obtained in the antiglobulin ELISA compared favourably with the standard (or indirect) ELISA using the purified immunoglobulins. Competitive inhibition between specific immunoglobulins of different isotypes and subisotypes was the major disadvantage of the antiglobulin ELISA. This latter assay failed to detect specific IgG2 against T theileri antigen in both calf and adult whole serum. The inability to detect the specific IgG2 was a result of competitive inhibition by specific IgG1. However, competitive inhibition between specific immunoglobulins was not observed in either of the other test systems using D viviparus and infectious bovine rhinotracheitis virus antigens.

Animals↗
PubMed1997

Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.

To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9-epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB-binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.

Aflatoxin B1↗
PubMed1997

A new class of biochemical oscillator models based on competitive binding.

It has been noted that single-enzyme systems can undergo strongly damped transient oscillations. In this paper, we present a nonlinear dynamics analysis of oscillations in undriven chemical systems. This analysis allows us to classify transient oscillations into two groups. In the first group, oscillations arise from rapid oscillatory relaxation to a slower transient relaxation mode. These oscillations are always strongly damped. In the second group, it is the slowest relaxation mode which is implicated in the oscillations so these can be very lightly damped. This second class of oscillations has not previously been studied in enzymology. We show that a remarkably simple single-enzyme system, namely competitive inhibition with substrate flow, generates transient oscillations which belong to the second class. In an attempt to design an experimentally realizable version of this model, we then discovered a system which is capable of sustained oscillations. In this experimentally realizable model, two substrates compete to bind to a macromolecule. The flow of one substrate is controlled by a simple feedback device. Sustained oscillations are observed over a very wide range of parameters. In both models, oscillations are favored by a wide disparity in rates of binding and dissociation of the two substrates to the macromolecule.

Binding, Competitive↗
PubMedDate not supplied

The steroid-responsive hiccup reflex arc: competitive binding to the corticosteroid-receptor?

Hiccups occurring secondary to high-doses of corticosteroids are a well-recognized problem in the field of neurosurgery. Numerous reports of oral, intravenous and intraarticular corticosteroids inducing hiccups exist in the literature. To date, there is only one case of anabolic steroids inducing hiccups. We now present a case of a patient who underwent a suboccipital craniotomy for resection of a cerebellar pontine angle meningioma. Postoperatively the patient was on high doses of Decadron and Oxandrin, an anabolic-anticatabolic agent used to combat the deleterious effects of corticosteroids. The patient suffered intractable hiccups postoperative day one, resistant to Thorazine. Oxandrin was discontinued to assess the possibility of a anabolic steroid-induced singultus. The hiccups resolved within 24 hours. This report validates the previous report on anabolic steroids inducing hiccups and exemplifies the ability for steroids as a class, due to there backbone structural homology, to induce function even as competitive inhibitors.

Adrenal Cortex Hormones↗
PubMed1985

Solid-phase enzyme-receptor assay (SPERA): a competitive-binding assay for glycopeptide antibiotics of the vancomycin class.

A solid-phase enzyme-receptor assay (SPERA) has been developed for glycopeptide antibiotics of the vancomycin class such as teicoplanin, vancomycin, ristocetin, avoparcin, actaplanin, A-47934, A-41030, and A-35512-B. The assay exploits the mechanism of most action of these antibiotics, which is based on their interaction with acyl-D-alanyl-D-alanine, a constituent of the walls of most growing bacterial cells. The antibiotics and enzyme-labeled teicoplanin compete for a synthetic analog of the biological receptor, albumin-epsilon-aminocaproyl-D-alanyl-D-alanine. The various antibiotics produced different competition curves, 50% displacement being obtained with antibiotic concentrations ranging from 0.04 to 4 mg/L, vancomycin and actaplanin being the weakest and strongest competitors, respectively. For teicoplanin in human serum the intra-assay CV was 7.2%, the interassay CV was 11.2%, and the analytical recovery 94%. Teicoplanin concentrations obtained by SPERA (chi) correlated well with those obtained by microbiological assay (y): y = 1.03 chi + 0.053 (r = 0.943; n = 60). We conclude that SPERA is a powerful tool for identification and quantitative detection of glycopeptide antibiotics, even in complex media.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Determination of hydrocortisone in human blood plasma using competitive binding with endogenous transcortin].

A direct express-method is developed for estimation of hydrocortisone, based on the use of individual human blood plasma endogenous transcortine as a binding system. The method is designed for non-serial estimation of hydrocortisone in human blood plasma and may be used for an express-analysis, since extraction of the hormone from blood plasma by organic solvents is omitted. The dissociation constant of the transcortine-hydrocortisone complex was calculated using Scatchard plot. On the basis of this procedure and considering the rule of acting masses the initial concentration of receptor sites was measured for transcortine at a given blood plasma dilution and theoretical curves were constructed for the reaction of competitive substitution of labelled hydrocortisone by the unlabelled hormone. Estimation of hydrocortisone, carried out by means of these theoretical curves of substitution, closely correlated with the data of experimental standard curves of substitution (CEA-IRE-Sorin). The express-method of hydrocortisone estimation was used in clinical research.

Binding, Competitive↗
PubMed1987

Transcription of the ADH2 gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids.

Transcription of the ADH2 gene in the yeast Saccharomyces cerevisiae was inhibited by excess copies of its own promoter region. This competition effect was promoter specific and required the upstream activation sequence of ADH2 as well as sequences 3' to the TATA box. Introducing excess copies of ADR1, an ADH2-specific regulatory gene, did not alleviate the competition that was observed in these circumstances during both constitutive and derepressed ADH2 expression. Excess copies of the upstream region did not release ADH2 from glucose repression, consistent with the view that ADH2 is regulated by positive trans-acting factors.

Alcohol Dehydrogenase↗
PubMed2002

Study of competitive binding of enantiomers to protein by affinity capillary electrochromatography.

Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule.

Binding, Competitive↗
PubMedDate not supplied

On the analysis of competitive binding of various ligands to cooperative and independent binding sites of macromolecules.

The paper deals with the practical evaluation of multiple binding equilibria of macromolecules and different ligands competing for the same binding sites. The necessary formalism is reviewed and set up for the equilibria involving a macromolecule with various classes of independent binding sites and/or a class of cooperative sites and up to three different ligands in competition for them. In particular, it was necessary to extend the Hill approximation to treat simultaneous competition for cooperative as well as independent binding sites, while earlier attempts are shown to be inadequate. Criteria are developed for a qualitative analysis of complex binding patterns using the Scatchard-plot of the experimental data in order to obtain a model of the binding structure and an adequate set of input parameters for the numerical analysis. Numerical examples refer to the binding of calcium and magnesium to the sarcoplasmic reticulum as studied by competitive replacement of manganese ions [3].

Binding Sites↗
PubMed2006

Competitive binding of cholesterol and ergosterol to the polyene antibiotic nystatin. A fluorescence study.

Competition studies between cholesterol and ergosterol were carried out to gain insight into the binding interactions between nystatin and these sterols. Lipid vesicles were prepared with mixtures of palmitoyloleoylphosphocholine/ergosterol/cholesterol, and both sterol molar ratio and total content were varied. The inhibitory effect of cholesterol toward the ergosterol ability to induce the formation of long-lived fluorescent antibiotic species was used to detect nystatin-cholesterol interactions. It was found that the key factor controlling nystatin photophysical properties in the ternary lipid mixtures was their ergosterol/cholesterol molar ratio and not their overall sterol content. Moreover, permeabilization studies showed that nystatin was able to form pores in all the mixed vesicles, but the initial rate of pore formation was also dependent on the ergosterol/cholesterol molar ratio. Our data show that ergosterol is displaced by competing cholesterol, indirectly confirming cholesterol's ability to coassemble with nystatin. The distinct spectroscopic properties emphasize the different molecular architecture adopted by nystatin-cholesterol and -ergosterol complexes, and therefore are relevant to understanding the interaction of the antibiotic with membranes.

Anti-Bacterial Agents↗
PubMed1993

Comparison between direct binding, competition and agglutination assays in the characterization of polyclonal anti-idiotypes against anti-HBs human monoclonal antibodies.

Polyclonal anti-idiotypic antibodies to human monoclonal anti-HBs antibodies (MoAb1) were raised in rabbits and designated Ab2-H1 and Ab2-H2. These Ab2 were characterized using three assays. A direct binding ELISA evaluated specificity towards a panel of human monoclonal antibodies and gamma globulins. Competition radioimmunoassay (CRIA) revealed Ab2 specificities towards Ab1 antigen binding sites by inhibition of HBsAg/Ab1 binding. Ab2-H1 and Ab2-H2 had comparable reactivities in ELISA and CRIA, whereas, using affinity purified Ab2, a fast (10 min) agglutination test (Spherotest) revealed different Ab1/Ab2 binding properties. Ab2-H1 reacted in this Spherotest with the Ab1 against which it was known to be specific (Ab1-H1), whereas in the same assay Ab2-H2 showed no activity towards the variable regions of the Ab1 used for its production (Ab1-H2). When injected into rabbits Ab2-H1 induced anti-HBs Ab3 antibodies but Ab2-H2 did not, thereby confirming the assay results.

Agglutination Tests↗
PubMed2002

Interferons inhibit tumor necrosis factor-alpha-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition with NF-kappa B.

Enhanced expression of matrix metalloproteinase-9 (MMP-9) correlates with invasion during tumor progression. Interferons (IFNs) inhibit MMP-9 activation in response to tumor necrosis factor-alpha (TNF-alpha), and the latter activates the MMP-9 gene through NF-kappaB. Understanding the molecular basis for MMP-9 inhibition may provide tools to control cell invasion. The data reported here show the critical role of interferon regulatory factor-1 (IRF1) in the inhibition of MMP-9. (i) IFN treatment suppresses TNF-alpha-induced MMP-9 reporter activity in STAT1(+/+) cells but not in STAT1(-/-) cells. (ii) IRF1 transfection blocks TNF-alpha-mediated MMP-9 activation. (iii) IFNs phosphorylate STAT1 and induce IRF1 but do not affect Ikappa-B degradation nor NF-kappaB nuclear translocation. (iv) Nuclear NF-kappaB (p50/p65) and IRF1, but not STAT1, bind to the MMP-9 promoter region containing an IFN-responsive-like element overlapping the NF-kappaB-binding site. (v) Recombinant IRF1, although unable to bind to an NF-kappaB consensus sequence, competes with NF-kappaB proteins for binding to the MMP-9 promoter. (vi) Conversely recombinant p50/p65 proteins reduce IRF1-DNA binding. (vii) In cells cotransfected with IRF1 and/or p65 expression vectors, an excess of IRF1 reduces MMP-9 reporter activity, whereas an excess of p65 blocks the inhibitory effect of IFN-gamma. Thus, in contrast to the known synergism between IRF1 and NF-kappaB, our data identify a novel role for IRF1 as a competitive inhibitor of NF-kappaB binding to the particular MMP-9 promoter context.

Base Sequence↗
PubMed1996

Tl+/Na+ Competitive Binding at the Surface of Dodecylsulfate Micelles in Water-Urea Mixtures

The effect of urea on the competition between Tl+ and Na+ ions to bind with the surface of dodecylsulfate micelles in aqueous solution has been studied by fluorescence techniques, employing methods based on fluorescence quenching data (FQD) and critical micelle concentration measurements (CMCM). The results obtained are interpreted in terms of selectivity coefficients K (Tl+/Na+) defined within the framework of the pseudophase ion-exchange formalism. It is found that the ion specificity observed in the absence of urea [K(Tl+/Na+) = 3.8 +/- 0.3 (from FQD), K(Tl+/Na+) = 2.5 +/- 0.3 (from CMCM] is progressively lost in the presence of increasing concentrations of urea. Typically, values of K (Tl+/Na+) = 1.4 +/- 0.2 (from FQD) and K (Tl+/Na+) = 1.2 +/- 0.2 (from CMCM) in a water-urea mixture at 8 M urea, were obtained. The results are discussed in terms of urea association to the micellar interface and changes in the characteristics of the external medium.

Journal Article↗
PubMed1997

Cleavage of collagen RNA transcripts by hammerhead ribozymes in vitro is mutation-specific and shows competitive binding effects.

We report here the in vitro use of hammerhead ribozymes as an approach to the gene therapy of osteogenesis imperfecta (OI). Our strategy for the treatment of this dominant genetic disorder is based on selective reduction of the level of the mRNA transcripts from the mutant allele. We studied the in vitro cleavage activity of five different hammerhead ribozymes targeted against synthetic transcripts of two naturally occurring human collagen mutations and against a point mutation introduced into a construct containing a portion of the mouse COL1A1 gene. This is the first demonstration that ribozyme cleavage is absolutely dependent on the presence of the ribozyme cleavage site introduced by the disease-causing mutation. Cleavage specificity and activity were unchanged when the cleavage site was located in transcripts of progressively longer length. Cleavage efficiency depended directly on the ratio of ribozyme/substrate, as well as on the time and temperature of incubation. We investigated the competitive effects of both total RNA and normal synthetic transcripts on ribozyme cleavage activity. The ribozyme was able to localize and cleave its specific target even in the presence of a vast excess of total RNA. However, cleavage efficiency was linearly inhibited by the presence of a non- cleavable competitor substrate which contained a ribozyme binding site identical to the site present in the cleavable target. Although this competition could be eliminated by introducing a mismatch into one ribozyme binding arm, the presence of the mismatch decreased ribozyme cleavage efficiency. The mutation- specificity of ribozyme cleavage demonstrated in this work provides support for in vivo studies aimed at ribozyme development as a treatment for dominant negative genetic disorders.

Animals↗
PubMed1999

Flavone antagonists bind competitively with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation.

Previous analyses suggested that potent aryl hydrocarbon receptor (AhR) antagonists were planar, with a lateral electron-rich center. To further define structural requirements and mechanism for antagonism, ten additional flavone derivatives were synthesized. Based on their ability to 1) compete with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the AhR; 2) inhibit TCDD-elicited binding of AhR to dioxin-responsive elements (DRE) in vitro; and 3) inhibit TCDD-induced transcription of DRE-dependent luciferase in stably transfected hepatoma cells, the most potent flavones contained a 3'-methoxy group and a 4'-substituent having one or more terminal atoms of high electron density (-N3, -NO2, or -NCS). Furthermore, these had low agonist activity as assessed by their inability to elicit AhR. DRE binding or to induce luciferase. Compounds containing bulkier 3' or 4'-substituents, or a 3'-OH group were less potent antagonists, and some were partial agonists. In rat liver cytosol, 3'-methoxy-4'-azido- and 3'-methoxy-4'-nitroflavones bound competitively (with TCDD) to the AhR, indicating that they bind to the TCDD-binding site. When hepatoma cells were exposed to these flavones, AhR complexes were primarily immunoprecipitable from the cytosol and contained 90 kDa heat shock protein. In contrast, AhR in TCDD-treated cells was primarily immunoprecipitated from nuclear extracts and was associated with Arnt but not 90 kDa heat shock protein. Immunocytofluorescence analysis in intact cells further indicated that the potent antagonist inhibited nuclear uptake of AhR and blocked TCDD-dependent down-regulation of AhR. Together, these data indicate that the most potent antagonists bind the AhR with high affinity but cannot initiate receptor transformation and nuclear localization.

Animals↗