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PubMed1980

Drug distribution as a function of binding competition. Experiments with the distribution dialysis technique.

In the distribution dialysis technique each of the two dialysis chambers contains a binding system, and a drug is allowed to distribute between them. This technique was tested by using various intracellular and extracellular binder preparations over wide concentration ranges, and model drugs selected for their known binding properties. The drugs were then tested at therapeutic concentrations in standardized systems of liver homogenate (0.5 g ml-1) and whole blood (0.02 ml ml-1). The resulting intracellular/extracellular concentrations ratios were characteristic for the binding properties of the various drugs. Thus, for imipramine, a drug with strong tissue and weaker plasma binding properties, the concentration ratios were 25 for the system homogenate/buffer, 0.8 for buffer/blood, and 15 for the competitive system homogenate/blood. In experiments with homogenates from various tissues (liver, lung, kidney, intestine, brain) and blood in the standard system, the following approximate ratios were obtained: 1 for antipyrine, 2 for phenylbutazone, 14 for imipramine (but only 8 with muscle, skin and adipose tissue). These results reflect both the individual binding to intracellular and extracellular components and the tissue/blood concentration ratios in vivo. It is suggested that distribution dialysis is an in vitro method for characterizing the distribution of the drugs. It is also concluded that drug distribution is largely determined by a binding competition between tissue and blood sites.

Animals↗
PubMed2000

Imaging synaptic neurotransmission with in vivo binding competition techniques: a critical review.

Several groups have provided evidence that positron emission tomography (PET) and single-photon emission computed tomography (SPECT) neuroreceptor imaging techniques might be applied to measure acute fluctuations in dopamine (DA) synaptic concentration in the living human brain. Competition between DA and radioligands for binding to D2 receptor is the principle underlying this approach. This new application of neuroreceptor imaging provides a dynamic measurement of neurotransmission that is likely to be informative to our understanding of neuropsychiatric conditions. This article reviews and discusses the body of data supporting the feasibility and potential of this imaging paradigm. Endogenous competition studies performed in rodents, nonhuman primates, and humans are first summarized. After this overview, the validity of the model underlying the interpretation of these imaging data is critically assessed. The current reference model is defined as the occupancy model, since changes in radiotracer binding potential (BP) are assumed to be directly caused by changes in occupancy of D2 receptors by DA. Experimental data supporting this model are presented. The evidence that manipulation of DA synaptic levels induces change in the BP of several D2 radiotracers (catecholamines and benzamides) is unequivocal. The fact that these changes in BP are mediated by changes in DA synaptic concentration is well documented. The relationship between the magnitude of BP changes measured with PET or SPECT and the magnitude of changes in DA concentration measured by microdialysis supports the use of these noninvasive techniques to measure changes in neurotransmission. On the other hand, several observations remain unexplained. First, the amphetamine-induced changes in the BP of D2 receptor antagonists [123I]IBZM and [11C]raclopride last longer than amphetamine-induced changes in DA extracellular concentration. Second, nonbenzamide D2 receptor antagonists, such as spiperone and pimozide, are not affected by changes in DA release, or are affected in a direction opposite to that predicted by the occupancy model. Similar observations are reported with D1 radiotracers. These results suggest that the changes in BP following changes in DA concentration might not be fully accounted by a simple occupancy model. Specifically, the data are reviewed supporting that agonist-mediated receptor internalization might play an important role in characterizing receptor-ligand interactions. Finally, it is proposed that a better understanding of the mechanism underlying the effects observed with benzamides is essential to develop this imaging technique to other receptor systems.

Animals↗
PubMed1981

Competitive binding studies of compounds that interact with DNA utilizing fluorescence polarization.

The increase in the fluorescence polarization of acridine orange upon binding to DNA molecules is used as the basis of a competitive method to study the interaction of a variety of fluorescent and non-fluorescent compounds with DNA. Test compounds that interact with DNA inhibit both the binding of acridine orange to DNA and the accompanying increase in fluorescence polarization. Actinomycin D exhibits a dose-dependent inhibition of acridine orange-DNA binding with Micrococcus luteus DNA, calf thymus DNA, and poly(dG-dC); no detectable inhibition of acridine orange intercalation into poly(dA-dT) is observed. In contrast, proflavine shows similar acridine orange inhibition for poly(dA-dT), calf thymus DNA and M. luteus DNA.

Acridine Orange↗
PubMed1992

Interaction of lambda cro repressor with synthetic operator OR3 studied by competition binding with minor groove binders.

In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F.

Aminoglycosides↗
PubMed2004

Poly(ADP-ribose) polymerase-1 is a negative regulator of HIV-1 transcription through competitive binding to TAR RNA with Tat.positive transcription elongation factor b (p-TEFb) complex.

Human immunodeficiency virus, type 1 (HIV-1) transcription is regulated by a virus-encoded protein, Tat, which forms a complex with a host cellular factor, positive transcription elongation factor b (P-TEFb). When this complex binds to TAR RNA synthesized from the HIV-1 long terminal repeat promoter element, transcription is trans-activated. In this study we showed that, in host cells, HIV-1 transcription is negatively regulated by competition of poly(ADP-ribose) polymerase-1 (PARP-1) with Tat.P-TEFb for binding to TAR RNA. PARP-1, which has a high affinity for TAR RNA (K(D) = 1.35 x 10(-10) M), binds to the loop region of TAR RNA and displaces Tat or Tat.P-TEFb from the RNA. In vitro transcription assays showed that this displacement leads to suppression of Tat-mediated trans-activation of transcription. Furthermore in vivo expression of luciferase or destabilized enhanced green fluorescent protein genes under the control of the HIV-1 long terminal repeat promoter was suppressed by PARP-1. Thus, these results suggest that PARP-1 acts as a negative regulator of HIV-1 transcription through competitive binding with Tat or the Tat.P-TEFb complex to TAR RNA.

Binding, Competitive↗
PubMed1995

Competitive binding of calcium and magnesium to streptococcal lipoteichoic acid.

Equilibrium dialysis was used to investigate the binding capacity and affinity of lipoteichoic acid (LTA) from the Gram-positive bacterium Streptococcus sanguis for calcium and magnesium by a competitive method. LTA was shown to bind approx. 1 mol of either calcium or magnesium per mole phosphate. Calcium and magnesium dissociation constants were found to be 8.39 +/- 0.31 mmol/l and 15.01 +/- 2.01 mmol/l respectively, indicating that S. sanguis LTA will preferentially bind calcium. LTA may act as a calcium buffer by reducing the free calcium concentration to which the cell is exposed. The capacity to produce large quantities of LTA could thus be as important as aciduricity in selection of species at caries-prone sites.

Binding, Competitive↗
PubMed2005

ERp57 binds competitively to protein disulfide isomerase and calreticulin.

In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS-PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K(D) value of PDI binding to ERp57 was calculated as 5.46x10(-6)M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.

Animals↗
PubMed1996

Evaluation of affinity constants by competitive binding assays: removal of empiricism from the Müller method.

A method is described for the quantitative screening of antigen analogs by competitive radioimmunoassay. This analytical procedure is based on an experimental design developed for the comparison of binding affinities by means of an empirical quantitative expression (Müller, R. (1980) J. Immunol. Methods 92, 589-601). The present analysis, which allows retention of the same general approach without sacrifice of thermodynamic rigor, is illustrated by application to literature results obtained by radioimmunoassay screening of the interactions between antigen analogs and antibodies elicited in response to (i) a scorpion toxin and (ii) 8-hydroxy-2-deoxyguanosine.

Antigen-Antibody Reactions↗
PubMed1995

Stereospecific and competitive binding of drugs to human serum albumin: a difference circular dichroism approach.

Simultaneous binding of two drugs to human serum albumin (HSA) was investigated by difference circular dichroism (delta CD) spectroscopy. Phenylbutazone and diazepam were chosen as specific markers for binding areas I (cumarines) and II (indoles), respectively, and their stereospecific interactions with protein were selectively characterized. Displacers were drugs known to specifically bind to areas I (salicylate) and II (racemic ibuprofen). The results indicate two different interaction mechanisms: a direct competition one (diazepam-ibuprofen and phenylbutazone-salicylate) and an indirect competition one (diazepam-salicylate and phenylbutazone-ibuprofen). The two major binding areas on HSA are distinct, but not independent, entities. Finally, the dissociation constants of marker ligands and competitors complexed to HSA were determined by quantitative analysis of CD data.

Binding, Competitive↗
PubMed1986

Discrimination of muscle and neuronal Na-channel subtypes by binding competition between [3H]saxitoxin and mu-conotoxins.

The effect of two mu-conotoxin peptides on the specific binding of [3H]saxitoxin was examined in isolated plasma membranes of various excitable tissues. mu-Conotoxins GIIIA and GIIIB inhibit [3H]saxitoxin binding in Electrophorus electric organ membranes with similar KdS of approximately equal to 50 X 10(-9) M in a manner consistent with direct competition for a common binding site. GIIIA and GIIIB similarly compete with the majority (80-95%) of [3H]saxitoxin binding sites in rat skeletal muscle with KdS of approximately 25 and approximately 140 X 10(-9) M, respectively. However, the high-affinity saxitoxin sites in lobster axons, rat brain, and rat heart are virtually insensitive to GIIIA concentrations up to 10 microM. These results and previously published data suggest that three Na-channel subtypes can be distinguished on the basis of toxin pharmacology: Na channels of skeletal muscle and Electrophorus electroplax have high affinity for mu-conotoxins and tetrodotoxin, neuronal Na channels have low affinity for mu-conotoxins and high affinity for tetrodotoxin, while heart Na channels and a similar subtype also found in denervated muscle have low affinity for both mu-conotoxin and tetrodotoxin.

Animals↗
PubMed1997

Non-competitive binding of the nonpeptide antagonist BIBP3226 to rat forebrain neuropeptide Y1 receptors.

[3H]Neuropeptide Y labelled neuropeptide Y receptors in rat forebrain membranes as a homogenous class of high-affinity sites. Between 80 and 85% of these receptors showed high affinity for Y1-selective antagonists such as (R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine amide (BIBP3226). While competitive in functional studies, BIBP3226 produced parallel shifts of the Scatchard plots of [3H]neuropeptide Y saturation binding in rat forebrain membranes. Mechanisms which are routinely invoked to explain non-competitive binding do not apply to BIBP3226. Wash-out experiments, involving successive treatment of the membranes with BIBP3226, buffer (wash-out step) and [3H]neuropeptide Y, argue against irreversible or a pseudo-irreversible binding of the antagonist. Allosteric inhibition is also unlikely since BIBP3226 did not affect the rate of dissociation of [3H]neuropeptide Y in isotope dilution experiments. The non-hydrolyzable guanine nucleotide, 5'-guanylylimidodiphosphate (Gpp(NH)p), abolished the binding of [3H]neuropeptide Y and increased its rate of dissociation in isotope dilution experiments. This suggests that the initial [3H]neuropeptide Y-receptor association is a low affinity process and that the observed binding of [3H]neuropeptide Y is related to the formation of a ternary [3H]neuropeptide Y-receptor-G protein complex. Two- or even multistate models (in which BIBP3226 could potentially behave as an inverse agonist) could therefore be needed to explain the non-competitive antagonism of BIBP3226 in broken cell preparations.

Animals↗
PubMed2004

Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface.

Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding.

Betaine↗
PubMed1985

Competitive binding kinetics in ligand-receptor-competitor systems. Rate parameters for unlabeled ligands for the formyl peptide receptor.

We describe the real-time kinetics of the competition of different ligands for the same receptor and use the computer routine SAAM to simulate this competition. Based on the simulation, we have developed two experimental approaches whereby the parameters of the interaction of nonlabeled ligands with their receptor can be detected; briefly, the analysis of the nonlabeled ligands depends on the perturbation of the kinetics of interaction of labeled ligands with the receptor with which they are in competition. The approach relies primarily upon an analysis of the kinetics of the competition between fluorescent and nonfluorescent ligands using a real-time, homogeneous binding assay in the fluorescence flow cytometer. A secondary approach depends upon an examination of the kinetic impact of antagonists on the responses of cells stimulated by agonists at the same receptor. Experimental verification of these approaches has been obtained using the N-formyl peptides (and their antagonists) which bind to receptors on human neutrophils and produce rapid cell stimulation. We find that agonistic N-formyl peptides have residence times of minutes while nonstimulatory antagonists have residence periods of, at most, a few seconds at these receptors. The limitations and general range of applicability of these procedures are discussed. The main advantage of these approaches is that they permit the evaluation of kinetic parameters of unlabeled ligands, even those which bind weakly or which have brief residence times--properties which make analyses by conventional methods difficult.

Binding, Competitive↗
PubMed1992

Competitive binding of a monoclonal antibody SZ-21 with anti-PLA1 antibodies and its potential for clinical application.

Both intact IgG and Fab fragments of a monoclonal antibody SZ-21, directed against platelet glycoprotein (GP) IIIa, inhibited binding of anti-PLA1 antibodies to PLA1-positive platelets in a dose dependent manner. Conversely, the binding capacity of the platelets for SZ-21 was decreased in the presence of anti-PLA1 antibodies. In Western blots, the determinant for SZ-21 was present on GPIIIa from either PLA1 or PLA2 homozygotes, but SZ-21 did not affect the interaction of anti-Yukb alloantibodies with their target antigen on GPIIIa. These results suggest that SZ-21 reacts with an epitope on the GPIIIa molecule very close to but not identical with that for anti-PLA1. The existence of PLA1-reactive alloantibodies in serum could be demonstrated by their competitive effect on the binding of SZ-21.

Antibodies, Monoclonal↗
PubMed1988

Solid phase enzyme-linked competitive binding assay for riboflavin.

A new solid-phase enzyme-linked assay for riboflavin (vitamin B2) is described. The assay is based on the competition between analyte vitamin molecules and a glucose-6-phosphate dehydrogenase-3-carboxymethylriboflavin conjugate for a limited number of riboflavin-binding protein sites immobilized on Sepharose particles. Significant improvements in conjugate catalytic activity and thus detectability are achieved by optimizing the reaction conditions used to covalently link 3-carboxymethylriboflavin to the enzyme. Optimization experiments include studying the effects of reaction pH and organic solvent composition. Final assay detection limits and the sensitivity of the dose-response curves are dependent on the ratio of conjugate to binding protein sites utilized in an equilibrium assay protocol. Selectivity of the method correlates well with that predicted based on the known association constants of riboflavin-binding protein with flavin analogs. The assay is shown to offer adequate detection limits and selectivity for direct measurement of riboflavin in urine, infant formula, and vitamin capsules.

Binding Sites↗
PubMed1990

Binding competition of toxic shock syndrome toxin 1 and other staphylococcal exoproteins for receptors on human peripheral blood mononuclear cells.

Binding of toxic shock toxin 1 (TSST-1) and staphylococcal enterotoxin A (SEA) to human peripheral blood mononuclear cells (PBMC) was investigated by using 125I-labeled ligands. Scatchard analyses revealed similar numbers of receptors (approximately 5,000 to 8,000) and similar dissociation constants (Kd, approximately 20 to 25 nM) per PBMC. SEA but not enterotoxin B, C1, C2, C3, D, or E significantly inhibited binding of 125I-TSST-1 to PBMC. Cross-competition of TSST-1 and SEA in binding assays suggests that they may bind to overlapping or separate epitopes on the same receptor.

Bacterial Toxins↗
PubMed1991

Binding competition of okadaic acid derivatives to anti-okadaic acid antibody.

The serologic activities of structurally related okadaic acid derivatives have been determined. Binding of [3H]okadaic acid to rabbit anti-okadaic acid is inhibited with equal effectiveness by okadaic acid, dinophysistoxin-1, acanthifolicin, okadaic acid tetramethyl ether, and okadaic acid spiroketal II. Okadaic acid spiroketal I, which lacks the F- and G-rings of okadaic acid, inhibits serologic binding about 60 times less effectively. The F- and G-rings of okadaic acid may comprise part of the epitopes recognized by some of the polyclonal antibodies.

Animals↗