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PubMed1986

Demonstration of specific high affinity receptors for aldosterone in cytosol of rat colon.

Since both aldosterone and glucocorticoids increase cation transport in rat distal colon, and a specific glucocorticoid high affinity cytosolic receptor has been identified in this tissue, it was possible that the action of aldosterone was dependent on interaction with the glucocorticoid receptor. Studies were, therefore, performed to determine whether a specific high affinity receptor for aldosterone was present in rat distal colon. At 4 C, aldosterone binding was saturable and exhibited a high affinity site with an apparent Kd of 6.2 +/- 0.9 X 10(-10) M and a calculated number of binding sites of 57.2 +/- 10.8 fmol/mg cytosol protein. Scatchard plot analysis also revealed a low affinity site with a Kd of 5.9 +/- 1.1 X 10(-8) M and 961 +/- 191 fmol/mg cytosol protein-binding sites. Competitive binding studies demonstrated that the high affinity binding protein was specific for aldosterone, compared to either dexamethasone or RU-28362. Since a specific high affinity receptor protein for aldosterone is present in rat distal colon, these data are consistent with a direct action of aldosterone that is independent of the glucocorticoid receptor system.

Aldosterone↗
PubMed1988

Identification of a protein in adrenal particulates that binds adrenocorticotropin specifically and with high affinity.

This paper is concerned with the identification and isolation in cross-linked form of a protein of bovine adrenal cortical particulates that binds the ACTH probe 125I-[Phe2,Nle4,DTBct25]ACTH-(1-25) amide specifically, reversibly, and with high affinity. This protein may well represent the long sought, adenylate cyclase-linked, low affinity ACTH receptor or a portion thereof. Evaluation of the binding data by Scatchard analysis afforded a linear plot corresponding to a dissociation constant of 2.7 X 10(-9) M with a single class of binding sites. Competitive binding studies using nonradioactive ACTH analogs served to establish the specificity of the binding. ACTH-(1-24), was the most active competitor, followed by [Gln5]ACTH-(1-20) amide, [Gln5,Phe9] ACTH-(1-24), and ACTH-(11-20) amide, the weakest binder of the group. These findings correlate well with the ability of the peptides to stimulate cAMP formation in bovine adrenal cortical cells, i.e. ACTH-(1-24) greater than [Gln5]ACTH-1-20) amide greater than [Gln5,Phe9]ACTH-(1-24). ACTH-(11-20) amide is biologically inactive but inhibits ACTH-(1-24)-stimulated adenylate cyclase with a 50% inhibition ratio of 400:1. Nonspecific binding was suppressed by inclusion in the incubates of the protease inhibitors pepstatin, bacitracin, and benzamidine. The binding protein was cross-linked to the radioactive probe with disuccinimidyl suberate with a high cross-linking yield. The cross-linked material was solubilized with sodium dodecyl sulfate (SDS), and the 100,000 X g supernatant was subjected to SDS-polyacrylamide gel electrophoresis, followed by a autoradiography. The gel showed the presence of a band corresponding to an apparent mol wt of 43,000 (assuming a molecule of ligand bound). This band was absent when cross-linking was performed in the presence of unlabeled ACTH-(1-24). Similar results were obtained when cross-linking was performed with dithiobis (succinimidyl)propionate or ethyleneglycolbis (succinimidyl)succinate. The soluble cross-linked material bound to a column of succinoylavidin Sepharose and could be eluted with guanidinium chloride at pH 1.5. SDS-polyacrylamide gel electrophoresis and autoradiography of the affinity-purified material afforded the same pattern as the unpurified material; however, considerably more radioactivity was present in the high mol wt region of the gels.

Adrenal Cortex↗
PubMed1996

Iodocalcitonin binds to human calcitonin receptors with higher affinity than calcitonin.

The reported binding affinities of cloned human calcitonin receptors (hCTRs) for salmon calcitonin (sCT) vary over a wide range. Because the greatest differences were between values estimated in association binding versus competition binding experiments, we considered the possibility that this variation was due to differences in the affinities of hCTRs for moniodinated sCT (I-sCT) and sCT. We found that I-sCT competed with 125I-sCT for binding with 9.3 +/- 0.58 times the apparent affinity of sCT in COS-1 and Madin Darby canine kidney (MDCK) cells expressing cloned hCTRs. I-sCT was 25 times more potent than sCT in stimulating cAMP formation in MDCK cells expressing hCTRs. These data demonstrate that monoiodinated sCT interacts more avidly with hCTRs than sCT with a consequent enhanced potency in stimulating second messenger formation. Therefore, as I-sCT (or 125I-sCT) binds with different affinity to hCTRs than sCT, affinity measurements must be performed with I-sCT (or 125I-sCT) alone.

Animals↗
PubMed2003

Study on interactions of endocrine disruptors with estrogen receptor-beta using fluorescence polarization.

In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-beta (hrER-beta) binding assay method using fluorescence polarization (FP) by applying a fluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plot and a Hill plot analysis of the saturation binding assay using F-E2 and hrER-beta were studied. F-E2 showed a high affinity for hrER-beta, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-beta binding assay. Competitive binding assays using F-E2, in which the FP values decreased upon the addition of compounds (competitors) were carried out to evaluate the binding characteristics of compounds with and without biological activities to hrER-beta. Twenty-one compounds, such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens, were examined. The obtained sigmoidal inhibition curves were transformed into pseudo-Hill plots and the concentrations at 50% inhibition (IC50) and Hill coefficients, the degree of cooperativity in ER-ligand binding, were obtained from the regression equations. Antagonists exhibited larger Hill coefficients than agonists, showing the correlation between the Hill coefficients and the estrogenic/antiestrogenic activities.

Binding, Competitive↗
PubMed1991

Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibodies against the nucleoprotein of mumps virus: their binding characteristics and cross-reactivity with other paramyxoviruses.

Twelve monoclonal antibodies (MoAbs) directed against the nucleoprotein (NP) of mumps virus were analysed for their binding characteristics. Competitive binding in enzyme-linked immunosorbent assay divided them into eight groups. Two of the MoAbs recognized exclusively 66 kD polypeptide of NP, two recognized 66 kD and 60 kD, and one recognized 66 kD (and 60 kD to a lesser extent) in Western blot assays under either denaturing or partially denaturing conditions. Under partially denaturing condition, another five MoAbs reacted faintly but the remaining two did not react at all. Under denaturing condition, on the other hand, these seven MoAbs showed little reactivity with any polypeptide. Furthermore, denaturation resulted in formation of other polypeptides 55 kD, 50 kD, and 43 kD which all were detected by MoAbs reacting with 66 kD and/or 60 kD. Previously demonstrated antigenic cross-reactivity among the NPs of mumps virus and those of human parainfluenza viruses type 2 and type 4 in radioimmunoprecipitation assay using polyclonal antisera was confirmed by an anti-NP MoAb which showed little reactivity in denaturing Western blot assay.

Animals↗
PubMed1989

Interactions of nicotinamide adenine dinucleotides with varied states and forms of hemoglobin.

Spectrofluorometric techniques were used to quantify NADPH-hemoglobin interactions based on the quenching of NADPH fluorescence upon binding to hemoglobin. Fluorometric titrations were carried out with hemoglobin in varied states and with hemoglobins in which the beta-chain anion site is altered. At pH 6.5 in 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, NADPH binds with high affinity, Kd = 1.03 microM, to deoxy human hemoglobin tetramers. Lower affinity binding of NADPH occurs as the beta-chain anion-binding site is discharged by increasing the pH. Moreover, the cofactor binds in a 1:1 ratio to deoxy tetramers, inositol hexaphosphate binds competitively, and binding is decreased in hemoglobins whose structural alterations result in decreased effects of 2,3-diphosphoglycerate. The cofactor binds to oxidized (met) hemoglobin with an estimated Kd of 33.3 microM but has little or no affinity for the oxy form. These results indicate that NADPH binds at the beta-chain anion-binding site and can be considered as a fluorescent analog of 2,3-diphosphoglycerate. Fluorescence measurements gave no indication of NADPH binding to deoxygenated ferrous or ferric myoglobin. Reductive processes within the erythrocyte, such as reduction of met hemoglobin and hemoglobin-catalyzed enzymatic reactions, may be affected by the significant binding of the reduced cofactor to both deoxygenated and oxidized hemoglobin. Cofactor-hemoglobin interactions predict a shift in redox potential as red cells become oxygenated, which may account for unexplained oxygen-linked shifts in red cell metabolism.

Animals↗
PubMed1984

Idiotypic analysis of anti-I-Ak monoclonal antibodies. II. Detection of shared idiotopes on syngeneic BALB/c and allogeneic A.TH-derived anti-I-Ak mAb by BALB/c-derived anti-I-Ak anti-idiotypic mAb.

The IA2, IIID1, and VC6 BALB/c-derived anti-11-5 anti-idiotypic (anti-Id) monoclonal antibodies (mAb) described in the companion paper were used to study the idiotopes expressed by a panel of 13 anti-I-Ak and 10 anti-I-Ek mAb. IA2 and IIID1, which detect binding-site-related idiotopes of the syngeneic BALB/c-derived 11-5 mAb, each react with public idiotope(s) (IdX) of two allogeneic A.TH-derived anti-I-Ak mAb (8B and 39J). VC6, which detects an 11-5 idiotope that differs from the IA2 and IIID1 idiotopes, binds to the 8B but not the 39J mAb. The three anti-Id mAb examined do not bind to idiotopes on 11 other anti-I-Ak or to 10 other anti-I-Ek mAb tested. These data demonstrate that mice immunized with a murine anti-I-Ak mAb probably produce syngeneic anti-IdX serum antibodies. Furthermore, they suggest that the 11-5 IdX markers identified are shared by alloantibodies of the BALB/c (Igh1a) and A.TH (Igh1e) mouse strains and may be expressed independently of Igh-C allotype markers. The sharing of idiotopes between the 11-5, 8B (anti-Ia.2), and 39J (anti-Ia.19) mAb led us to reinvestigate the antigenic specificity of the 11-5 mAb and the genetic complexity of the serologically detectable Ia.2 and Ia.19 epitopes present on I-Ak molecules. Two main points emerge from these studies. First, the 11-5 BALB/c-derived anti-I-Ak mAb, previously considered to have an anti-Ia.2 specificity, was shown by direct binding and competition binding assays to possess instead a reactivity pattern more compatible with that of an anti-Ia.19 mAb. Second, the anti-Ia.2 and anti-Ia.19 mAb, which were provisionally considered to bind epitopes in a single region of an I-Ak molecule, termed epitope cluster IV, actually define at least four different subgroups of this cluster. These subgroups are designated as IV A-D, and IV D is detected by the 11-5 and 39J anti-Ia.19 mAb. This tentative subdivision of I-Ak epitope cluster IV suggests there exists a minimum of seven topologically distinguishable regions of an I-Ak molecule that give rise to polymorphic allodeterminants, some or all of which may mediate lymphocyte interaction.

Animals↗
PubMed1994

2,3,7,8-Tetrachlorodibenzo-p-dioxin versus 3-methylcholanthrene: comparative studies of Ah receptor binding, transformation, and induction of CYP1A1.

Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC) cause transcriptional activation of the CYP1A1 gene via their interaction with the aromatic hydrocarbon (Ah) receptor. Direct radioligand binding and competitive binding studies demonstrated that the cytosolic Ah receptor from the mouse hepatoma cell line Hepa-1 bound TCDD with an affinity approximately 3-4-fold greater than that for MC. However, TCDD was approximately 1,000-fold more potent than MC as an inducer of CYP1A1-mediated aryl hydrocarbon hydroxylase activity in cultured Hepa-1 cells as assessed at 14 h following exposure to inducer. To understand the basis for this quantitative discrepancy between Ah receptor binding affinity and CYP1A1 induction potency, we systematically compared TCDD and MC for their abilities to activate sequential events in the CYP1A1 induction mechanism that occur subsequent to initial binding to the cytosolic Ah receptor. Using a gel retardation assay, TCDD and MC were shown to be equipotent in causing in vitro transformation of the cytosolic Ah receptor to its DNA-binding form. In addition, the transformed Ah receptor bound to a specific dioxin-responsive enhancer sequence with the same apparent affinity when MC was the ligand as when TCDD was the ligand. At an early time point (i.e. 2 h) in the CYP1A1 induction process, TCDD was only approximately 4-25-fold more potent than MC in stimulating the nuclear uptake of the ligand-Ah receptor complex, and the two ligands displayed a relatively small difference (> or = 10-fold) in CYP1A1 mRNA induction potency. When assessed at 4 h following ligand treatment, TCDD was only approximately 10-fold more potent than MC as an aryl hydrocarbon hydroxylase inducer, suggesting a time-dependent reduction in the potency of MC in intact cells. Exposure of Hepa-1 cells to MC over a 16-h time course resulted in an increased ability of these cells to convert [3H]MC to alkali-extractable metabolites. Our data are consistent with the idea that TCDD and MC display relatively small differences in their intrinsic abilities to activate Ah receptor-mediated events. The reduced biological potency of MC observed in intact cells and whole animals is at least partially due to the more rapid metabolic inactivation of this ligand compared with the poorly metabolized TCDD. By extension, the extraordinary toxicity of TCDD may not be explained solely by its high affinity for the cytosolic Ah receptor.

Animals↗
PubMedDate not supplied

[Cortisol determination in human plasma by a modified method of competitive protein binding].

The authors described a simple and economic modification of the competitive protein binding method of cortisol determination in human blood plasma satisfying the current requirements presented to the saturation analysis. Blood plasma of women given combined contraceptive the preparation was used as a binding system. Analysis of experimental results using a programmed computer was carried out on the basis of standard curve linearization. Preeision sensitivity and specificity of this modified method are assessed. This modification was approved in measuring the cortisol level in the blood plasma of various groups of patients. Comparison with routine competitive binding methods and their analysis is presented.

Dexamethasone↗
PubMed2006

Competitive metal binding to a silicate-immobilized humic material.

The investigation of the competitive binding of metal ions to a biogenic material comprised of organic peat immobilized in a polysilicate matrix was undertaken. This material was packed into 5.0mL bed-volume columns using 40-60mesh size particles. Two separate mixtures of metal ions were studied by monitoring the solution pH and the concentration of each metal in the effluent as a function of the volume of influent introduced to the material. These mixtures contained either the metal ions Ca(2+), Mg(2+), and Cu(2+) or the ions Cu(2+), Hg(2+), and Pb(2+). A general order of binding affinities was determined to be Mg(2+)<Ca(2+)<<Cu(2+)<Pb(2+)<Hg(2+). Comparisons of amounts of metal ions bound and protons released indicated the initial release of two protons for each divalent metal ion bound. However, prolonged exposure of the material to the metal solutions yielded a molar ratio of 1:1. This suggests the involvement of ion exchange sites followed by that of sites using an alternate binding mechanism. The presence of binding sites with varied mechanisms and metal ion affinities was further illustrated through the initial binding and subsequent release of lower affinity metal ions (e.g., Mg(2+) and Ca(2+)).

Adsorption↗
PubMed2000

Native N-methyl-D-aspartate receptors containing NR2A and NR2B subunits have pharmacologically distinct competitive antagonist binding sites.

The pharmacological properties of native N-methyl-D-aspartate (NMDA) receptors were determined in rat brain sections with quantitative autoradiography of [(3)H](E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding. With five competitive antagonists as displacers, two subpopulations of binding sites were observed in the horizontal plane of section examined. These two populations corresponded anatomically to NR2A and NR2B subunits. Quantitative analysis of NR2A-like and NR2B-like binding sites was enabled by examining the cerebellar granule cell layer, which expresses NR2A and NR2C subunits, and the medial striatum, which predominately expresses NR2B subunits. The antagonists (R)-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid and (R)-2-amino-5-phosphonopentanoate (D-AP5) displayed similar affinities at cerebellar NMDA receptors and medial striatal NMDA receptors. In contrast, the NMDA receptor antagonists (+/-)-6-(1H-Tetrazol-5-ylmethyl)decahydroisoquinoline- 3-carboxylic acid, (S)-alpha-amino-5-(phosphonomethyl)[1,1'-biphenyl]-3-propanoic acid, and (+/-)-cis-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid displayed varied, higher affinities at medial striatal NMDA receptors than at cerebellar NMDA receptors. For the five antagonists, there was a strong correlation (r = 0.9) between the cerebellar K(i)/medial striatum K(i) ratio and the NR2A K(i)/NR2B K(i) ratio for recombinant receptors. Thus, [(3)H]CGP39653 labels two pharmacologically distinct populations of NMDA receptors that have pharmacological and anatomical properties consistent with NR2A and NR2B subunits. Because native NR2A- and NR2B-containing receptors are pharmacologically distinct, it should be possible to develop NR2A- and NR2B-selective glutamate site antagonists.

2-Amino-5-phosphonovalerate↗
PubMed1975

Assay of cortisol in serum by competitive protein binding without solvent extraction.

A method for the determination of serum cortisol by competitive protein binding is proposed in which solvent extraction is replaced by heat inactivation. Results obtained by the new technique correlate well with those obtained by solvent extraction. A saving in manipulative time and glassware is effected when the method is applied in a routine assay.

Animals↗
PubMed1978

A simple and specific competitive protein binding assay for 24,25-dihydroxyvitamin D in human serum.

A sensitive and specific competitive protein binding assay for 24,25-dihydroxyvitamin D was developed using rat kidney cytosol as the binding protein. It also included a chromatographic step on a Sephadex LH 20 column to separate the metabolite specifically. The average value for 24, 25-dihydroxyvitamin D in serum in 19 male and female healthy subjects aged from 20 to 40 was found to be 8.24 +/- 0.34 ng/ml (1.98 +/- 0.08 . 10(-8) mol/l) mean +/- S.D. In hepatic insufficiencies the levels are higher (14.15 +/- 3.59 ng/ml) than in normal subjects. A comparison between 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D levels was made.

Adult↗
PubMed1978

Automated measurement of serum thyroxine with the "ARIA II," as compared with competitive protein binding and radioimmunoassay.

Two conventional serum thyroxine assays, run in separate laboratories, one by competitive protein binding and one by radioimmunoassay, were used to evaluate the automated ARIA II (Becton Dickinson Immunodiagnostics) serum thyroxine assay. Competitive protein binding as compared to ARIA II with 111 clinical serum samples gave a slope of 1.04 and a correlation coefficient of 0.94. The radioimmunoassay comparison to ARIA II with 53 clinical serum samples gave a slope of 1.05 and a correlation coefficient of 0.92. The ARIA II inter-assay coefficient of variation for 10 replicates of low, medium, and high thyroxine serum samples was 6.2, 6.0, and 2.9%, respectively, with an inter-assay coefficient of variation among 15 different assays of 15.5, 10.1, and 7.9%. The automated ARIA II, with a 2.2-min cycle per sample, gives results that compare well with those by manual methodology.

Antibody Specificity↗
PubMed1987

A competitive receptor binding radioassay for beta-1 and beta-2 adrenergic agents.

A rapid and sensitive competitive receptor binding assay for beta-1 and beta-2 adrenergic binding for adrenergic agents has been developed. The steps that are critical for the success of the assay are given in detail so that the assay can be set up in any routine laboratory with relative ease. The rationale behind the use of specific reagents is discussed. The assay requires microgram quantities of test compound, a radiolabeled specific beta adrenergic antagonist [3H]dihydroalprenolol (DHA), and turkey erythrocyte beta-1 and rat erythrocyte beta-2 receptor membranes. Serial dilutions of sample are incubated with appropriate receptor membranes and DHA for 1 hr at room temperature. After equilibrium is attained, the bound radioligand is separated by rapid filtration under vacuum through Whatman GF/B filters. The amount of bound DHA trapped on the filter is inversely proportional to the degree of beta-1 or beta-2 adrenergic binding of the sample. Separation of bound from free radioligand by filtration permits rapid determination of a large number of samples. This assay quantitates and differentiates beta-1 and beta-2 adrenergic binding of synthetic adrenergic agents.

Adrenergic beta-Agonists↗
PubMed1975

[A competitive radioisotope binding technic for the determination of prednisolone and of a new synthetic corticoid].

A competitive protein binding technique for the determination of plasma prednisolone in dexamethasone-treated men was found suitable for the estimation of plasma levels of a new non-steroidal compound having a chemical structure related to that of prednisolone. The method used allowed us to determine the time of appearance of this compound in the peripheral plasma after oral administration, the time of the peak and the apparent disappearance half-life which were comparable to that of prednisolone.

Administration, Oral↗
PubMed1977

Linearization of data for saturation-type competitive protein binding assay and radioimmunoassay.

Most of the commonly-performed competitive protein-binding radioassay methods utilized in the clinical laboratory are based on the principle of saturation analysis. Although many different methods for linearization of saturation-type assays have been proposed, the algebraic equivalency of all of these methods has not been adequately documented. In this manuscript we have shown the physical and mathematical basis for various methods for linearlization of saturation type assays and the algebraic equivalency of these linearization methods. We have also shown that key parameters such as slope and intercept may be dependent on different components of the assays system with differnt linearization methods. An understanding of these key parameters can help the analyst to evaluate changes in these key parameters and to integrate these parameters in a complete quality control system.

Binding, Competitive↗