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PubMed1996

Competitive binding of calcium, magnesium and zinc to Streptococcus sanguis and purified S. sanguis cell walls.

Calcium bound by oral bacteria may have important effects on plaque cohesion and mineral dynamics. Equilibrium dialysis was used here to study the effect of magnesium and zinc competition on calcium-binding by Streptococcus sanguis 7863. Experiments were carried out using 45Ca in 0.1-5.0 mmol/l Ca2+ carrier. Increasing [Mg] and [Zn] decreased the apparent Ca-binding affinity, allowing calculation of the affinities for Mg2+ and Zn2+. Dissociation constants were found to be 0.94 +/- 0.23, 1.32 +/- 0.39 and 1.26 +/- 0.41 mmol/l (whole cells) and 1.87 +/- 0.49, 3.05 +/- 1.24 and 4.08 +/- 1.29 mmol/l (cell walls), for Ca, Mg and Zn respectively. Binding capacities for Ca (and, by inference, Mg and Zn) were 27.7 +/- 2.8 mumol/g wet weight (whole cells) and 54.2 +/- 1.6 mumol/g wet weight (cell walls). These data suggest that Mg and Zn act as true competitive inhibitors of calcium binding.

Bacterial Adhesion↗

PubMed2000

Competitive binding to a charged leucine motif represses transformation by a papillomavirus E6 oncoprotein.

E6 oncoproteins from HPV-16 and bovine papillomavirus type 1 (BPV-1) bind to similar leucine-rich peptides termed charged leucine motifs found on the cellular focal adhesion protein paxillin and the E3 ubiquitin ligase E6AP. BPV-1 E6 (BE6) mutants that do not bind to paxillin are defective at inducing cellular transformation. It is possible, however, that BE6 mutants that do not bind paxillin are defective for transformation for an unrelated reason than the ability to bind to charged leucine motifs. To address the role of BE6 interaction with charged leucine motifs, we fused a BE6-binding charged leucine motif to the amino terminus of BE6, thereby creating an autoinhibitory binding domain. We found that the fusion protein failed to bind to paxillin or transform murine C127 cells. Mutation of the amino terminal binding motif in the fusion protein restored both interaction with paxillin and transformation. This demonstrates that BE6 transformation requires binding to charged leucine motifs on particular cellular proteins and that transformation by papillomavirus oncoproteins can be repressed by competitive interactions with charged leucine motifs.

Amino Acid Sequence↗

PubMed2006

Competitive binding of pentraxins and IgM to newly exposed epitopes on late apoptotic cells.

A random distribution of phospholipids among the inner and outer leaflet of the cell membrane occurs during apoptosis and is known as membrane flip-flop. Flip-flopped cells have binding sites for various plasma proteins, such as IgM and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP). In this study, we investigated whether pentraxins and IgM antibodies recognize the same binding sites on apoptotic cells, and whether phospholipids constitute these binding sites. Except for SAP which also bound to early apoptotic cells, pentraxins and IgM preferentially bound to late apoptotic cells. Competition experiments with different phosphatemonoesters revealed that CRP and SAP as well as part of the IgM bound to the phospholipids head groups, SAP mainly to phosphorylethanolamine, CRP to phosphorylcholine and phosphorylethanolamine and to a lesser extent to phosphorylserine, and IgM to phosphorylcholine and phosphorylserine. These results were confirmed in experiments in which proteins were adsorbed from plasma with artificial phospholipids particles. IgM and the pentraxins variably competed for the same binding sites on late apoptotic cells, SAP having the highest and CRP the lowest apparent affinity. We conclude that CRP, SAP, and part of the IgM bind to the phospholipid head groups exposed on apoptotic cells. This shared specificity as well as their shared capability to activate complement, suggest that IgM and the pentraxins CRP and SAP exert similar functions in the removal of apoptotic cells.

Apoptosis↗

PubMed2000

An ultrasensitive competitive binding assay for the detection of toxins affecting protein phosphatases.

An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [125I]microcystin-YR was stable (half-time of dissociation = 1.8 h), allowing non-bound [125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.

Bacterial Toxins↗

PubMed1985

Cationic atmosphere and cation competition binding at negatively charged membranes: pathological implications of aluminum.

Binding of cations to membranes may be the basis for explaining some of the effects of several neurotoxic cations. The binding of Al3+ and the displacement of Ca2+ by Al3+ is studied with the aid of a simple mathematical approach described here and giving the same results when compared to the mathematical formalism described by Nir and Bentz. The method allows the simulation of membranes with low surface charge densities that are relevant for biochemical and pathological implications. Fluorescence quenching of the phospholipid analogue 1-palmitoyl-2-nitrobenzoxadiazol amino caproyl- phosphatidyl choline (C6-NBD-Ptd Cho) embedded in phosphatidyl serine membranes is used to determine the competition between calcium and aluminum for binding. The effect of aluminum in the presence of chelating agents is also studied by quenching experiments. Finally, inhibition of 45Ca2+ binding to phosphatidyl serine has also been investigated in a two-phase system.

Aluminum↗

PubMed2001

Accurate determination of rate constants of very slow, tight-binding competitive inhibitors by numerical solution of differential equations, independently of precise knowledge of the enzyme concentration.

This paper is concerned with the determination of rate constants characterizing the binding and release of a slow binding inhibitor to and from an enzyme, here almond beta-glucosidase. We demonstrate the inability of the conventional method to yield reliable rate constants when one or more of these is less than 1 x 10(-4) per second. Instead one must use the much more accurate fitting of rate constants of the set of simultaneous differential equations characterizing the kinetic model. This procedure has the added advantage, when properly used, that the rate constants found pertaining to the inhibitor are largely insensitive to the particular value used for the enzyme concentration; i.e., the same data set may be fitted using a range of enzyme concentrations with no change in the resulting parameters. Hence the method can be used when little is known about the enzyme, except for the value of K(m), which is readily determined. Also, we report the somewhat unexpected finding that the association rate constant for the substrate (4-nitrophenyl-beta-d-glucopyranoside) is about one-third of the value of the corresponding rate constant for the inhibitor. The method is used to determine rate constants at several temperatures for the strong, slow binding inhibitor 2-phenethylglucoimidazole 1, enabling us to compute standard thermodynamic functions. The identity of these functions with those of isofagomine (2) reported earlier leads us to argue that the two compounds share a common binding mechanism, involving the same groups, whereas the different stabilities of the enzyme-inhibitor complexes must reside in those parts of the molecules that are not identical.

Binding, Competitive↗

PubMed1994

Competitive binding of neutralizing monoclonal and polyclonal IgG to the HA of influenza A virions in solution: only one IgG molecule is bound per HA trimer regardless of the specificity of the competitor.

Two-step solution competition assays were performed in solution with influenza type A virions and hemagglutinin (HA)-specific neutralizing monoclonal antibodies (mabs). These demonstrated that the binding of one molecule of IgG mab per HA trimer prevented the binding of mabs directed against other antigenic sites on the HA (site A, site B, or site D), even though these are topographically separate and antigenically independent. Furthermore the same procedures showed that one molecule of mab per trimer prevented the binding of polyclonal HA-specific IgG obtained from the serum of rabbits immunized with whole virus. This restricted binding is clearly a property of the intact virion, since others using purified HA have shown that up to four IgG molecules of different specificities can bind per trimer. Since the surface area of the globular head of the trimer is equivalent to approximately 10 nonoverlapping antibody footprints, it is not understood how one prebound IgG molecule prevents the binding of other IgG molecules.

Animals↗

PubMed1992

Comparisons of rotavirus VP7-typing monoclonal antibodies by competition binding assay.

Three sets of neutralizing monoclonal antibodies (MAbs) used to type the outer capsid protein VP7 of four group A rotavirus serotypes (1 through 4) were compared in competition immunoassays. Reciprocal competition was observed for each of the VP7 type 2-, 3-, and 4-specific MAbs. The VP7 type 1 MAbs exhibited variable competition patterns with other VP7 type 1 MAbs. MAb RV4:3, which has been used to recognize antigenic variants within VP7 type 1 strains, showed reciprocal competition with the four VP7 type 3 MAbs (RV3:1, YO-1E2, 4F8, and 159) using a VP7 type 3 virus (SA11) as antigen. MAb 2C9, also prepared against VP7 type 1, reacted with VP7 type 3 strains and competed with a VP7 type 3 MAb, 159, using RRV as antigen. Use of the different sets of VP7 type-specific MAbs in the enzyme-linked immunosorbent assay permitted the recognition of six antigenic variants within VP7 types 1, 2, and 3 among specimens whose VP7 type could not be determined previously with only one set of typing MAbs. These results demonstrate differences of typing ability among these VP7-specific MAbs and emphasize the need to improve the sensitivity of typing systems by incorporating panels of MAbs reacting with several neutralizing epitopes.

Antibodies, Monoclonal↗

PubMed1986

Urinary immunoglobulin G: a routine and highly sensitive technique based on competitive binding.

The evaluation of both small amounts of urinary immunoglobulins and the albumin/immunoglobulin ratio are important indices during the early stages of diabetic nephropathy. For this reason there is at present a need for highly-sensitive and specific routine tests for urinary immunoglobulins. A technique has been designed based on the competition between radiolabelled immunoglobulin G and immunoglobulins in the urine to be tested in binding to an anti-immunoglobulin antibody in solid phase. The immunoglobulin fraction of a rabbit anti-human immunoglobulin antiserum in a basic Ca-carbonate buffer is left to coat highly adsorbent polystyrene microtitre tubes. After the antibody-coated tubes have been saturated with gelatine, a 1/5 dilution of the urine to be tested and an equal volume of purified and radiolabelled human immunoglobulin G are left to incubate and are then added to the antibody-coated tubes. After incubation and repeated washings, radioactivity is counted. This technique has the characteristics of a routine assay for the accurate detection of immunoglobulin G in the urine. It requires only a few microliters of urine, takes only 3 hr to complete (after having coated the tubes) and detects about 120 ng of immunoglobulin G/ml, well below what is needed to assess physiological or pathological values.

Binding, Competitive↗

PubMed2006

Differences in hydration coupled to specific and nonspecific competitive binding and to specific DNA Binding of the restriction endonuclease BamHI.

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.

Binding Sites↗

PubMed1994

Monoclonal antibodies to human pancreatic procolipase: production and characterization by competitive binding studies.

Hybridomas secreting monoclonal antibodies (MAbs) specific for human pancreatic colipase were established and 11 clones were selected by using a dot immunobinding assay. Characterization of the MAbs was carried out by using direct and competitive epitope mapping methods, including ELISA and inactivation of colipase-dependent pancreatic lipase. Monoclonal antibodies showed four distinct patterns of reactivity. Monoclonal antibody 5.30 (group I) inhibited colipase-dependent lipase activity. The dissociation constant of the inactive antibody-antigen complex was 10(-9) M. Monoclonal antibodies 48.30, 66.24, and 153.23 (group II) had no effect on activity although they bound competitively with MAb 5.30 to antigen as shown by their capacity to displace MAb 5.30 from the antibody-antigen complex and by ELISA additivity test. Dissociation constants calculated from the displacement curves were 0.9 10(-9) M, 0.6 10(-9) M, and 2 10(-9) M, respectively. Noninhibitory MAbs 13.29, 16.25, and 33.30 bound competitively with MAbs of group II but not with MAb 5.30 (group I). Monoclonal antibodies of group IV (MAbs 17.6, 18.1, 37.39, and 169.29) had no effect on activity and did not react with immobilized antigen. None of the MAbs reacted in ELISA with reduced and carboxymethylated human procolipase, indicating that epitopes involved conformationally dependent determinants on protein antigen. Anti-human colipase MAbs showed no cross-reactivity with porcine or equine procolipases. Monoclonal antibodies described here appear to be useful tools for studying surface hydrophobic domain of colipase and/or interaction between colipase and lipase in its active conformation (open lid).

Animals↗

PubMed1985

Protein quantitation of as low as 10-ng/ml concentrations by competitive binding to polystyrene latexes.

A protein quantitation method which offers protein detection as low as 10 ng protein/ml and accurate quantitation as low as 30-100 ng protein/ml, depending on the protein, has been designed. The assay, which is relatively quick and simple to perform, utilizes the strong, nonspecific adsorption of proteins onto polystyrene latexes. A competition is created between a marker enzyme and the analyte protein for a limited amount of latex surface area. Due to inactivation of the enzyme upon binding to a hydrophobic latex surface, measurement of enzyme activity allows determination of the bound/free enzyme ratio and thus the competing protein concentration. Considerations of sensitivity and simplicity are suggested to make this assay superior to others presently available.

Alkaline Phosphatase↗

PubMedDate not supplied

Do general anaesthetics act by competitive binding to specific receptors?

Most proteins are insensitive to the presence of anaesthetics at concentrations which induce general anaesthesia, while some are inhibited by certain agents but not others. Here we show that, over a 100,000-fold range of potencies, the activity of a pure soluble protein (firefly luciferase) can be inhibited by 50% at anaesthetic concentrations which are essentially identical to those which anaesthetize animals. This identity holds for inhalational agents (such as halothane, methoxyflurane and chloroform), aliphatic and aromatic alcohols, ketones, ethers and alkanes. This finding is all the more striking in view of the fact that the inhibition is shown to be competitive in nature, with anaesthetic molecules competing with substrate (luciferin) molecules for binding to the protein. We show that the anaesthetic-binding site can accommodate only one large, but more than one small, anaesthetic molecule. The obvious mechanism suggested by our results is that general anaesthetics, despite their chemical and structural diversity, act by competing with endogenous ligands for binding to specific receptors.

Anesthesia, General↗

PubMed1994

Competitive binding analysis of monoclonal antibodies reactive with porcine alveolar macrophages using anti-CD14 and anti-CD18.

Four monoclonal antibodies (mAb) from the myeloid subset panel of the First International Swine CD Workshop (74-22-15, DH59B, PM16-6, and MUC21A) were analyzed using competitive inhibition studies with anti-human CD14 (My4) and anti-human/anti-porcine CD18 (MHM23) on porcine alveolar macrophages. Results suggested that none of the mAb tested recognized CD14 or CD18 on porcine alveolar macrophages. Additionally, the cross-reactivity of My4 with porcine alveolar macrophages was established.

Animals↗

PubMed1995

The response regulators CheB and CheY exhibit competitive binding to the kinase CheA.

The autophosphorylating kinase CheA of the bacterial chemosensory signaling pathway donates a phosphoryl group to either of two regulator proteins, CheY or the receptor methylesterase (CheB). With isothermal titration calorimetry, it was demonstrated that CheA and CheA fragment composed of amino acid residues 1-233 (CheA1-233) bound to CheY with similar dissociation constants of 2.0 and 1.2 microM at 298 K, respectively, indicating that the CheY binding site is wholly within the 1-233 amino acid locus. CheB bound to CheA1-233 with a KD of 3.2 microM, and also bound to intact CheA with the same affinity. CheY was found to complete with CheB for binding to CheA1-233, in spite of the low level of sequence identity between CheY and the regulatory domain of CheB. The competitive nature of CheY and CheB binding was determined in two independent sets of experiments: titration experiments in which either a CheB-CheA1-233 complex was titrated with CheY or CheB was titrated with a CheY-CheA1-233 complex, and competitive affinity chromatography experiments that used a Ni-NTA-chelating resin as an affinity matrix for complexes of the histidine-tagged CheA1-233 fragment and CheY or CheB. The effects of phosphorylation, binding-site mutations, and active-site mutations were also studied to probe the influence of conformational changes in CheY as a regulatory mechanism of CheY-CheA Interactions. Phosphorylated CheY, in the presence of excess EDTA, was found to have a 2-fold lower affinity for CheA1-233, and 6 mM Mg2+ further reduced the affinity of phosphorylated CheY for CheA1-233 (ca. 3-fold), although Mg2+ on its own had no effect on the interactions of either CheB or CheY with CheA1-233. The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA under physiological conditions. The idea that phosphorylation may induce a significant conformational change, reducing the strength of the CheY-CheA interaction, is supported by the relative values of the association constants measured for CheY active-site and binding-site mutants. A binding-site mutation (A103V) in CheY, which is remote from the site of phosphorylation produced a 10-fold reduction in Ka, whereas active-site mutations produced a modest (2-fold) reduction.

Bacterial Proteins↗

PubMed2003

Competitive binding of fatty acids and the fluorescent probe 1-8-anilinonaphthalene sulfonate to bovine beta-lactoglobulin.

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Anilino Naphthalenesulfonates↗

PubMed1994

Quantitative analysis of mutually competitive binding of human Raf-1 and yeast adenylyl cyclase to Ras proteins.

Ras proteins appear to have two distinct downstream effectors, adenylyl cyclase in Saccharomyces cerevisiae and a product of raf-1 protooncogene in higher organisms. We found that in vitro activation of adenylyl cyclase by yeast Ras2 and human H-Ras proteins is subject to competitive inhibition by its leucine-rich repeats domain and by the N-terminal regulatory domain of human Raf-1 protein. Kinetic analyses of the inhibition patterns enabled us to determine exact dissociation constants (Kd) of the two polypeptides for Ras2 and H-Ras. The leucine-rich repeats domain bound to the posttranslationally modified Ras2 with the Kd of approximately 13 nM, which was close to the value (7 nM) of the whole adenylyl cyclase. The Kd of Raf-1 for the modified H-Ras, 3.5 nM, was significantly lower than that for Ras2, 24 nM, whereas adenylyl cyclase bound preferentially to Ras2. Similar inhibition was also observed in vivo by suppression of RAS2Val-19-dependent heat shock sensitivity and of Ras-dependent cAMP response to glucose upon overexpression of Raf-1 in yeast. These results indicate that the leucine-rich repeats domain contains the Ras protein-binding site and that Raf-1 and adenylyl cyclase, sharing no structural homology with each other, bind to a similar, if not identical, region of Ras with comparable affinities.

Adenylyl Cyclase Inhibitors↗

PubMed1999

Stimulation of aromatase P450 promoter (II) activity in endometriosis and its inhibition in endometrium are regulated by competitive binding of steroidogenic factor-1 and chicken ovalbumin upstream promoter transcription factor to the same cis-acting element.

In stromal cells of endometriosis, marked levels of aromatase P450 (P450arom) mRNA and activity are present and can be vigorously stimulated by (Bu)2cAMP or PGE2 to give rise to physiologically significant estrogen biosynthesis. Since eutopic endometrial tissue or stromal cells lack P450arom expression, we studied the molecular basis for differential P450arom expression in endometriosis and eutopic endometrium. First, we demonstrated by rapid amplification of cDNA 5'-ends that P450arom expression in pelvic endometriotic lesions is regulated almost exclusively via the alternative promoter II. Then, luciferase reporter plasmids containing deletion mutations of the 5'-flanking region of promoter II were transfected into endometriotic stromal cells. We identified two critical regulatory regions for cAMP induction of promoter II activity: 1) a-214/-100 bp proximal region responsible for a 3.7-fold induction, and 2) a -517/ -214 distal region responsible for potentiation of cAMP response up to 13-fold. In the -214/-100 region, we studied eutopic endometrial and endometriotic nuclear protein binding to a nuclear receptor half-site (NRHS, AGGTCA) and an imperfect cAMP response element (TGCACGTCA). Using electrophoretic mobility shift assay, cAMP response element-binding activity in nuclear proteins from both endometriotic and eutopic endometrial cells gave rise to formation of identical DNA-protein complexes. The NRHS probe, on the other hand, formed a distinct complex with nuclear proteins from endometriotic cells, which migrated at a much faster rate compared with the complex formed with nuclear proteins from eutopic endometrial cells. Employing recombinant proteins and antibodies against steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter transcription factor (COUP-TF), we demonstrated that COUP-TF but not SF-1 bound to NRHS in eutopic endometrial cells, whereas SF-1 was the primary NRHS-binding protein in endometriotic cells. In fact, COUP-TF transcripts were present in both eutopic endometrial (n = 12) and endometriotic tissues (n = 8), whereas SF-1 transcripts were detected in all endometriotic tissues (n = 12), but in only 3 of 15 eutopic endometrial tissues. Moreover, we demonstrated a dose-dependent direct competition between SF-1 and COUP-TF for occupancy of the NRHS, to which SF-1 bound with a higher affinity. Finally, overexpression of SF-1 in eutopic endometrial and endometriotic cells strikingly potentiated baseline and cAMP-induced activities of -517 promoter II construct, whereas overexpression of COUP-TF almost completely abolished these activities. In conclusion, COUP-TF might be one of the factors responsible for the inhibition of P450arom expression in eutopic endometrial stromal cells, which lack SF-1 expression in the majority (80%) of the samples; in contrast, aberrant SF-1 expression in endometriotic stromal cells can override this inhibition by competing for the same DNA-binding site, which is likely to account for high levels of baseline and cAMP-induced aromatase activity.

Animals↗