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PubMed1990

Binding of inhibitors to the major glutathione S-transferase from bovine brain. Competitive binding between bilirubin and glutathione.

The binding of non-substrate ligands to the glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) from bovine brain has been investigated kinetically by monitoring the inhibition of the enzyme-catalyzed reaction between glutathione and 1-chloro-2,4-dinitrobenzene. Bilirubin, thyroxine, lithocholic acid, retinoic acid and retinol are competitive inhibitors with respect to glutathione. Cooperative binding effects are observed with lithocholic acid, retinoic acid and retinol while cooperative binding is not observed with thyroxine or bilirubin. Bilirubin is the most potent inhibitor with constants of 0.1 and 110 microM. 50% of the total activity is lost upon binding to the high-affinity site and the remainder is lost at higher bilirubin concentrations. In spite of the apparently favorable binding for bilirubin, it is estimated that the high intracellular concentrations of reduced glutathione will saturate the enzyme and allow only a small fraction of the bilirubin in brain to bind to the enzyme. It is concluded that the binding of these ligands may be of minor importance in vivo.

Animals↗

PubMed1976

Competitive binding of iron by transferrins from different vertebrates.

1. A competitive dialysis technique has been used to study the relative affinities of the two iron-binding sites on transferrin molecules and the relative binding strengths of transferrins isolated from plasma of different species. 2. The comparisons were extended to include desialylated human transferrin, ovotransferrin, and a cyanogen bromide fragment of the latter. 3. Although the results of bilateral experiments could generally be accounted for in terms of the theory of independent sites, there were some exceptions, and cyclic comparisons were inconsistent. 4. All the comparisons made were compatible with a model in which site-interaction occurred, but it was not possible to decide whether the sites were intrinsically identical or not. For most species this corresponded to positive cooperativity, but for rabbit it was negative. 5. The average affinity of transferrin for iron depended on species, but the variation was never more than about one order of magnitude. 6. No effect on the binding constants for human transferrin could be detected when the sialic acid residues were removed. 7. The fragment of ovotransferrin competed fairly effectively with the native molecule for iron, although the average relative affinity was only about 1:15. 8. The relative binding of iron by ovotranferrin and human transferrin was affected little when bicarbonate anion was replaced by oxalate, although the ratio of the two binding constants for ovotranferrin increased.

Animals↗

PubMed1987

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction.

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.

Adenosine Triphosphate↗

PubMed1985

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens.

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.

Antibodies, Monoclonal↗

PubMed1994

Competitive binding experiments reveal differential interactions for dihydropyridine calcium channel activators and antagonists at dihydropyridine receptors on mouse brain membranes.

The binding of the dihydropyridine (+/-)-202-791 and its corresponding calcium channel activating and calcium channel antagonist enantiomers ((+)-S-202-791 and (-)-R-202-791, respectively) to dihydropyridine receptors on mouse brain membranes was studied through competition for [3H]nitrendipine binding and 3H-labelled (+/-)-BAY K8644 ((+/-)-[3H]BAY K8644). Direct binding studies with (+/-)-[3H]BAY K8644 and [3H]nitrendipine revealed high affinity binding to a homogeneous set of dihydropyridine calcium channel activator and antagonist receptors on mouse brain membranes, (+/-)-[3H]BAY K8644 binding to approximately one half as many receptors as did [3H]nitrendipine. Competition binding studies revealed a significant discrimination of both high and low affinity receptors for (-)-R-202-791 and a homogeneous set of receptors for (+)-S-202-791 regardless of whether (+/-)-[3H]BAY K8644 or [3H]nitrendipine was the competing radioligand. Molar ratios (1:1, 5:1, 10:1) of (+)-S-202-792 to (-)-R-202-791 inhibited [3H]nitrendipine binding with displacement binding isotherms substantially different from those predicted on the basis of the binding properties of the individual enantiomers. These data suggest that dihydropyridine calcium channel antagonists and activators bind to different allosterically linked receptors or domains of the dihydropyridine protein associated with the voltage-dependent calcium channels. Furthermore, these results support the concept of multiple binding sites for dihydropyridine ligands.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1999

Directional binding of HMG-I(Y) on four-way junction DNA and the molecular basis for competitive binding with HMG-1 and histone H1.

Histone H1, HMG-1 and HMG-I(Y) are mammalian nuclear proteins possessing distinctive DNA-binding domain structures that share the common property of preferentially binding to four-way junction (4H) DNA, an in vitro mimic of the in vivo genetic recombination intermediate known as the Holliday junction. Nevertheless, these three proteins bind to 4H DNA in vitro with very different affinities and in a mutually exclusive manner. To investigate the molecular basis for these distinctive binding characteristics, we employed base pair resolution hydroxyl radical footprinting to determine the precise sites of nucleotide interactions of both HMG-1 and histone H1 on 4H DNA and compared these contacts with those previously described for HMG-I(Y) on the same substrate. Each of these proteins had a unique binding pattern on 4H DNA and yet shared certain common nucleotide contacts on the arms of the 4H DNA molecule near the branch point. Both the HMG-I(Y) and HMG-1 proteins made specific contacts across the 4H DNA branch point, as well as interacting at discrete sites on the arms, whereas the globular domain of histone H1 bound exclusively to the arms of the 4H DNA substrate without contacting nucleotides at the crossover region. Experiments employing the chemical cleavage reagent 1, 10-orthophenanthroline copper(II) attached to the C-terminal end of a site-specifically mutagenized HMG-I(Y) protein molecule demonstrated that this protein binds to 4H DNA in a distinctly polar, direction-specific manner. Together these results provide an attractive molecular explanation for the observed mutually exclusive 4H DNA-binding characteristics of these proteins and also allow for critical assessment of proposed models for their interaction with 4H DNA substrates. The results also have important implications concerning the possible in vivo roles of HMG-I(Y), histone H1 and HMG-1 in biological processes such as genetic recombination and retroviral integration.

Animals↗

PubMed1997

Characterization of smooth lipopolysaccharides and O polysaccharides of Brucella species by competition binding assays with monoclonal antibodies.

Previously, four epitope specificities on the O chain of Brucella species were reported: M, A, C, and C/Y. In this work, according to monoclonal antibody binding to smooth lipopolysaccharides of Yersinia enterocolitica 0:9, Brucella abortus W99 (A-dominant strain), and B. melitensis Rev1 (M-dominant strain), seven O-chain epitope specificities were defined: M, A, C (M > A), C (M = A), C/Y (M > A), C/Y (M = A) and C/Y (A > M). Competitive binding assays between these monoclonal antibodies suggested that these different epitopes are probably overlapping structures.

Antibodies, Monoclonal↗

PubMed1977

Solid-phase competitive-binding radioimmunoassay for detecting antibody to the M antigen of histoplasmin.

A radioimmunoassay (RIA) was designed and compared with complement fixation and immunodiffusion tests for their relative ability to detect antibodies in sera of histoplasmosis patients. M antigen, purified from histoplasmin, was fixed to microtiter wells as the solid phase, and specific rabbit 125I-labeled anti-M globulin was the source of indicator antibodies. The optimal concentrations for the competitive-binding assay were 1.6 ng per well for M antigen and 650 ng per well for the 125I-labeled anti-M globulin. A panel of sera from 29 histoplasmosis patients and from patients with other mycoses was screened for RIA activity and in complement fixation and immunodiffusion tests that used histoplasmin and Histoplasma capsulatum yeast-form antigens. The sera of 22 histoplasmosis patients reacted in the RIA, 21 in the complement fixation, and 16 in the immunodiffusion tests. Sera of patients with other mycotic infections did not react in the RIA, with the exception of those of one blastomycosis patient and one candidiasis patient. The RIA could be modified to quantitate M antigen; as little as 125 pg could be detected. The evaluation of this panel of histoplasmosis patients' sera showed that the RIA was about equivalent in sensitivity to the complement fixation test. Some advantages of the RIA over the complement fixation test were that RIA was less prone to cross-reactions and gave better quantitation of low-titered sera. The RIA was a 1-day test, was not hindered by the anti-complementary activity of some sera, and could be modified to quantitate minute amounts of M antigen.

Antibodies, Fungal↗

PubMedDate not supplied

[Experience in the use of competitive binding of hormones with proteins for the determination of non-metabolized glucocorticoids in the blood and urine in some pituitary-adrenal diseases].

A method of competitive protein-binding analysis was applied to the study of nonmetabolized glucocorticoids (NMG) in the plasma and urine of healthy persons and those suffering from hyper- and hypocorticism. NMG determination by this method reflected adequately the funcitonal condition of the adrenal cortex under normal conditions and in hypophyseo-adrenal diseases, including tests with ACTH and dexametazan, and coordinated with the results obtained by standard chemical methods.

Adrenal Insufficiency↗

PubMed2006

Voltammetric detection of single base-pair mismatches and quantification of label-free target ssDNA using a competitive binding assay.

The application of electrochemical techniques for DNA detection is motivated by their potential to detect hybridisation events in a more rapid, simplistic and cost-effective manner compared to conventional optical assays. Here, we present an electrochemical DNA sensor for the specific and quantitative detection of single-stranded DNA (ssDNA). Probe oligonucleotides were immobilised onto thin gold film electrodes by a 5'-thiol-linker. Hybridisation was detected by means of the electroactive redox-marker methylene blue (MB) covalently attached to the 5'-end of the target ssDNA and voltammetric techniques. MB-labeled target ssDNA was recognised down to 30 pmol. By application of a competitive binding assay, non-labeled ssDNA was detected down to 3 pmol. In addition, the DNA-modified electrodes were capable of sensing single base-pair mismatches at different positions within the sequence of the hybridised double-stranded DNA (dsDNA).

Base Pair Mismatch↗

PubMedDate not supplied

The use of two fluorescent dyes to identify sperm in a competitive binding assay to oocytes.

The relationship of most sperm laboratory assays to male fertility is inconsistent. Assays that measure traits required to fertilize oocytes are expected to have the most predictive value. A new assay that measures the competitive ability of two sperm samples to bind to oocytes was developed. Two populations of sperm were labeled using a pair of lipophilic dyes. A concentration of 75 microM of the two dyes, DiQ (4-[4-(dihexadecylamino)styryl]-N-methylquinolinium iodide; an orange-red dye) and DiOC16 (3,3'-dihexadecyloxacarbocyanine perchlorate; a yellow-green dye), intensely stained 66 and 73% of sperm, respectively, without affecting sperm motility or oocyte-binding ability. Because sperm could be stained with fluorescent dyes, sperm from two semen samples were mixed together in a droplet, and oocytes were added to allow sperm to bind oocytes competitively. Oocyte-bound sperm from each sample were counted. Binding was specific; nonspecific sperm binding was estimated by sperm bound to two-cell mouse embryos and averaged one to three sperm per embryo. Staining with DiQ or DiOC16 did not affect oocyte-binding ability since more than 80% of the sperm bound were stained with either dye. Furthermore, if different ratios of DiQ- or DiOC16-stained sperm from the same ejaculate were prepared in droplets and oocytes were added, the percentage of sperm bound to the oocytes reflected the percentage of sperm in the droplet; there was no differential effect of either dye. This assay used fixed oocytes because sperm bound equally to fixed and fresh bovine oocytes. This competitive oocyte-binding assay allows one to make a series of pairwise comparisons between a group of males or to include an internal control sample in sperm-oocyte binding assays. This assay may allow more accurate prediction of the oocyte-binding ability of sperm.

Animals↗

PubMed1999

Analysis of competition binding between soluble and membrane-bound ligands for cell surface receptors.

Binding of the Fc portion of IgG coated on targets to Fcgamma receptors (e.g., CD16) expressed on leukocytes (i.e., 2D binding) is an initiating step for immune responses such as phagocytosis or antibody-dependent cellular cytotoxicity. In vivo, circulating leukocytes are exposed to plasma IgG. The competition from soluble IgG (i.e., 3D binding) may affect the 2D binding. Many cell surface receptors, CD16 included, have soluble counterparts. While their physiological significance is not clear, receptor-based competitive inhibition therapy, in which soluble receptors, ligands, and their analogs are employed to compete with surface-bound receptors and ligands to prevent unwanted adhesion, is widely used to treat various diseases. To provide a quantitative basis for design of these therapeutic approaches, we developed a mathematical model for 2D and 3D competition binding. The model relates cell-surface adhesion (in the presence and absence of dislodging forces) to the concentration of the soluble competitor, the densities of the surface-bound receptors and ligands, as well as the binding affinities of the 2D and 3D interactions. Binding of CD16-expressing cells to an IgG-coated surface in the presence of a soluble competitor (IgG or anti-CD16 antibody) was quantified by a centrifugation assay. The agreement between experiment and theory supports the validity of the model, which could be useful in predicting the efficacy of the competitor.

Animals↗

PubMed1986

Determination of calmodulin by competitive binding assay.

Calmodulin levels in tissue or cellular extracts can be determined by competition with 125I-calmodulin in a filtration-based direct binding assay. The method is rapid, uses readily available stable components, and possesses a selectivity and sensitivity comparable to that observed with immunoassay and phosphodiesterase activation. This assay provides a tool to readily probe changes in calmodulin levels in cells and tissues as a function of pathophysiologic state.

Animals↗

PubMed1997

The competitive binding of STAT3 and NF-kappaB on an overlapping DNA binding site.

Interleukin-1 (IL-1) and interleukin-6 (IL-6), two early-response cytokines expressed during an acute inflammatory reaction, regulate the expression of several acute phase proteins (APP) in the liver. IL-1 relays its signal to specific genes via NF-kappaB, whereas IL-6 sends its signal to the nucleus via STAT1alpha and STAT3. Interestingly, overlapping binding sites for STAT3 and NF-kappaB can be found on promoters of several APP genes. We show here that both STAT3 and NF-kappaB are active during inflammation and are capable of binding to a STAT3/NF-kappaB overlapping DNA motif derived from the alpha2-macroglobulin gene promoter. In vitro binding assays demonstrated that NF-kappaB competes with STAT3 binding on this probe. Our results suggest that these transcription factors regulate each others' function through competition for overlapping DNA binding sites.

Acute-Phase Proteins↗

PubMed2006

Competitive binding of protein kinase Calpha to membranes and Rho GTPases.

Previously, we have shown that protein kinase C (PKC) forms a direct high-affinity, isozyme-specific and membrane lipid-independent interaction with Rho GTPases [Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445]. Since the cellular activation of PKCalpha involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein-protein interaction of PKCalpha with the Rho GTPase, Cdc42, and the protein-lipid interactions of PKCalpha with membranes. It was hypothesized that the interaction of PKCalpha with membrane-bound Cdc42 would contribute to the overall membrane-binding affinity of the kinase by providing an additional anchor. However, it was found that the incorporation of isoprenylated Cdc42 into membranes resulted in an apparent decrease in the membrane-binding affinity of PKCalpha, whereas the association of PKCbetaI, PKCdelta, PKCepsilon, and PKCzeta was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCalpha membrane association and for the ensuing activation, whereas the maximal levels of binding and activation attained at saturating PS and Ca2+ concentrations were in each case unaffected. Overall, these findings suggest that PKCalpha undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCalpha-Cdc42 complex, which possesses a membrane-binding affinity that is reduced relative to that of the individual components due to competition between Cdc42 and PS/Ca2+ for binding to PKCalpha. Consistent with this, it was found that the interaction of PKCalpha with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCalpha-Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCalpha and Cdc42 may be regulated by competing protein-protein and protein-lipid interactions.

Animals↗

PubMed1998

Competitive binding of xenobiotic oestrogens to rat alpha-fetoprotein and to sex steroid binding proteins in human and rainbow trout (Oncorhynchus mykiss) plasma.

The ability of a variety of "environmental oestrogens" to compete with radiolabelled steroids to rat alpha-fetoprotein (AFP) and to sex steroid binding proteins was investigated in human and rainbow trout (Oncorhynchus mykiss) plasma. For [3H]oestradiol binding to AFP, diethylstilbestrol and 4-nonylphenoxyacetic acid showed significant competition at concentrations about 100-fold greater than oestradiol (relative binding affinities approximately 1% c.f. oestradiol). All other compounds (phytooestrogens: coumestrol, daidzein, genistein; others: 4-nonylphenol, 4-tert-octylphenol, 4-nonylphenoldiethoxylate, 4-tert-butylphenol, bisphenol-A (Bis-A), bis(2- ethylhexl)phthalate, dioctylphthalate, dibutyl phthalate, 2, 4'DDT (op' enantiomer), 2,4'-DDE (mixed enantiomers), kepone) showed only very weak or no competition (relative binding affinities <<0.1% c.f. oestradiol). The situation for both human and fish plasma was very similar, with only very high concentrations (>>1000 fold more than the natural ligand) of a few of the compounds showing any ability to displace the natural ligand. These results suggest that environmental oestrogenic agents are unlikely to produce biological effects by displacing endogenous steroids from plasma steroid binding proteins unless they are present in very high concentrations.

Animals↗

PubMed1977

Competitive binding among oral strptococci to hydroxyapatite.

The relative affinity of various oral streptococci for hydroxyapatite (HA) differed widely. Streptococcus mutans affinity for dextran-coated HA was the highest of all the streptococci to any HA coating. S salivarius had the lowest affinity, and S sanguis affnity was higher then S miteor for saliva-coated HA. Competition for binding sites on saliva-dextran-coated, dextran-coated HA. Hhwever, competition between this pair was not for the same binding site but for closely associated sites.

Binding, Competitive↗

PubMed1995

An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule.

The dissociation constant for the binding of a spectroscopically invisible or non-radioactive ligand to its protein receptor can be determined in a competition experiment by using a structural analog that contains a reporter group. Many plotting and numerical analysis methods have been developed to calculate the binding constant of unlabeled ligand from the displacement experiments. However, a common problem with these plotting methods is that the equation transformations inevitably result in non-standard error distribution, and thus simple linear regression can not be used to extract correct values for the parameters. In the case of the numerical analysis methods, one would be faced with the possible existence of multiple solutions. In this paper, the exact mathematical expression for describing competitive binding of two different ligands to a protein molecule is presented in terms of the total concentrations of species in the system. Thus, using a commercially available non-linear regression program, all unknown parameters for describing this system can be determined by fitting the experimental data to the algebraically explicit equation without any data transformations. The distribution curves of all the species in the system can also be constructed with this equation. It is particularly useful for the cases in which the concentrations of all the species in the system are comparable to each other.

Binding, Competitive↗