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The influence of iron and gallium carriers on the in vitro and in vivo uptake of 67Ga and 59Fe by tumor cells has been studied. The results appear to indicate that ferritin might be involved in most of the systemic accumulation of 67Ga but that the mechanism of tumor uptake might be different.
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A series of bromo- and silyl-derivatives was studied in experiments on immature female rats in vivo and in vitro. All the estrogens studied were conventionally divided into 4 groups according to their ability for concurrent binding with the rat uterus cytosol receptors and their specific bioeffect: equally concurrent and bioeffective compounds (estrone, ethynyl estradiol); slightly concurrent substances with a pronounced uterotropic action (4-silyl estrogens, mestranol); little effective compounds with 4 to 8% of estradiol concurrent activity (2- and 4-bromo-and silyl estradiols, estriol); compounds, not having concurrent and biological effects (2-silyl estrones, 4-bromoestradiol diacetate).
A competitive binding-protein assay for 25-hydroxycholecalciferol, is described in which serum from vitamin D deficient rats is used as the source of binding protein. Serum samples after extraction are chromatographed using silicic acid. Bovine albumin is used in the buffer as a protein stabilizer and lipid solubilizer, at a concentration shown to enhance the reproducibility of the assay without interfering in the binding properties of 25-hydroxycholecalciferol. The method appears to be sufficiently sensitive for clinical studies even in vitamin D deficiency.
Fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) is greatly enhanced on its binding to bovine serum albumin (BSA). Fluorimetric titration shows that three ANS molecules bind per BSA molecule. The enhanced fluorescence of BSA-ANS is quenched by eosine (EOS); and one EOS physically displaces one ANS bound to BSA. The enhanced fluorescence of free ANS in the hydrophobic environment of the nonionic surfactant Triton X 100 is also quenched by EOS but by an energy transfer mechanism. The dye fluorescene (FLSN) also quenches the fluorescence of BSA-bound ANS, but by the energy transfer mechanism. The binding region of ANS in BSA has been speculated.
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The nucleation step of Escherichia coli RecA filament formation on single-stranded DNA (ssDNA) is strongly inhibited by prebound E. coli ssDNA-binding protein (SSB). The capacity of RecA protein to displace SSB is dramatically enhanced in RecA proteins with C-terminal deletions. The displacement of SSB by RecA protein is progressively improved when 6, 13, and 17 C-terminal amino acids are removed from the RecA protein relative to the full-length protein. The C-terminal deletion mutants also more readily displace yeast replication protein A than does the full-length protein. Thus, the RecA protein has an inherent and robust capacity to displace SSB from ssDNA. However, the displacement function is suppressed by the RecA C terminus, providing another example of a RecA activity with C-terminal modulation. RecADeltaC17 also has an enhanced capacity relative to wild-type RecA protein to bind ssDNA containing secondary structure. Added Mg(2+) enhances the ability of wild-type RecA and the RecA C-terminal deletion mutants to compete with SSB and replication protein A. The overall binding of RecADeltaC17 mutant protein to linear ssDNA is increased further by the mutation E38K, previously shown to enhance SSB displacement from ssDNA. The double mutant RecADeltaC17/E38K displaces SSB somewhat better than either individual mutant protein under some conditions and exhibits a higher steady-state level of binding to linear ssDNA under all conditions.
The competitive binding affinities of thirteen 7-substituted-2,3-dichlorodibenzo-p-dioxins to the human placental cytosolic aryl hydrocarbon (Ah) receptor were determined using [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as the radioligand. Multiple parameter linear regression analysis of the competitive binding C50 values for these compounds gave the following equation: pEC50 (M) = 6.246 + 1.632 pi - 1.764 sigma 0m + 1.282 HB where pi, sigma m and HB are the physiochemical parameters for substituent lipophilicity, meta-directing electronegativity, and hydrogen bonding capacity respectively. The 7-t-butyl- and 7-phenyl-2,3-dichlorodibenzo-p-dioxins were treated as outliers for the derivation of this equation, and these results suggest that only substituents with van der Waals' volumes less than 40 cm3/mol were accommodated in the receptor binding site. The equations previously derived from the binding of the 7-substituted-2,3-dichlorodibenzo-p-dioxins to the rat, mouse, guinea pig, and hamster hepatic cytosolic receptor were different than the correlation obtained using human placental receptor and provide further evidence for the interspecies differences in the molecular and binding properties of the Ah receptor protein.
To characterize the interrelationship of monoclonal antibodies (MAbs) against respiratory syncytial virus (RSV) and their respective epitopes, we developed a competitive binding assay based on the biotin-avidin system and a tissue culture enzyme-linked immunosorbent assay. The competitive binding assay clearly distinguished between competing and noncompeting MAbs. Eight MAbs against the fusion protein (F protein) demonstrated two blocking patterns consistent with two antigenic sites. MAbs reacting at one site neutralized the virus, while those reacting at the other site did not. Eight MAbs against the large glycoprotein (G protein) demonstrated five blocking patterns consistent with three antigenic sites, one with three epitopes and the other two with one each. None of the MAbs against G protein neutralized the virus. The reaction pattern of the MAbs against three strains of RSV identified three additional epitopes on the F protein and no additional epitopes on the G protein. The epitopes on G protein showed the greatest antigenic diversity among the three strains. These results help us better understand the functional and antigenic structure of the two surface glycoproteins of RSV.
The method of estimating serum thyroxine by competitive binding analysis has been examined stage by stage. The extraction procedures recommended by some authors are shown to extract protein as well as thyroxine, and so produce inaccurate results. A modified method is proposed which increases accuracy and precision.
Binding expressions are derived and analytical procedures developed for the quantitative characterization of inhibitor binding that is only partially competitive with the interaction between an acceptor and the ligand that is being monitored. Two such situations are considered: (i) that in which the partial competition reflects binding of inhibitor to fewer acceptor sites than available to ligand; and (ii) that in which the partial competition reflects binding of inhibitor to acceptor sites in addition to those occupiable by ligand. The potential efficacy of the suggested analyses is then explored by their application to simulated data that span the likely range of experimental behavior. Quantitative analysis of the displacement of [3H]nitrobenzylthioinosine from cultured leukemic cells by an adduct of 5'-S-(2-amino-ethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine with fluorescein-5-isothiocyanate is used to establish that the cells possess 6% fewer sites (150,000 cf. 159,000 sites/cell) for the fluorescent adduct than for the tritiated ligand, and that the binding is 10-fold weaker (binding constant of 0.28 cf. 2.8 nM-1). Corresponding analysis of results obtained with bovine aorta endothelial cells indicates that a 3-fold weaker interaction (binding constant of 1.1 cf. 3.3 nM-1) occurs between the fluorescent adduct and 79% of the cell sites accessible to the tritiated ligand. The present analytical procedures extend the utility of competitive binding assays for the quantitative screening of potential inhibitors by removing the inherent limitation of existing analyses that all acceptor sites be accessible to both the competing solute and the indicator ligand.
A sensitive competitive binding assay for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other ligands of the Ah receptor was developed using a stable preparation of the Ah receptor, the 40-55% ammonium sulfate fraction of liver cytosol from C57BL/6J mice, and the radioligand [125I]2-iodo-7,8-dibromodibenzo-p-dioxin (specific radioactivity, 2176 Ci/mmol, and binding affinity, KD = 6.5 pM). Conditions are described which maximize assay precision and sensitivity, while minimizing sample counting time, ensuring ligand solubility, and permitting attainment of binding equilibrium for competing ligands. Assay conditions were developed to allow calculation of the binding affinity for competing ligands and to ensure that an unknown competitor could be quantified in terms of "TCDD binding equivalents." Standard assay conditions consisted of incubation of 8 pM radioligand and 18-20 pM Ah receptor with 5-1000 pM TCDD, in a 1-ml volume, for 16 hr at 4 degrees. Statistical analysis of the standard curve of bound radioligand versus the log of the concentration of competing TCDD indicated the minimal detectable concentration of TCDD to be 10 pM (3.2 pg in a 1-ml assay alpha less than or equal to 0.01). The simplicity, sensitivity, and reproducibility of this competitive binding assay should prove useful as a screen to detect planar halogenated aromatic hydrocarbons and other ligands of the Ah receptor. The availability of this 125I-labeled dioxin congener also permitted the characterization of Ah receptor-ligand binding over a range of ligand and receptor concentrations not possible with currently available 3H-ligands.
In capillary electrophoresis of primary amine racemates using (+)-(18-crown-6)-tetracarboxylic acid (18C6H4) as a chiral selector, chiral recognition emanates from the differences in the complex formation between 18C6H4 and the two protonated amine enantiomers. The presence of buffer constituents such as tris(hydroxymethyl)aminomethane (Tris) or Na+, capable of forming complexes with 18C6H4, is thus detrimental to the chiral separation of primary amines. Such a competitive binding of buffer constituents was studied by comparing the electrophoretic mobilities of racemic analytes obtained in Tris/citric acid and triethylamine/citric acid buffers. We developed a simple fitting method to determine the competitive binding constant and applied it to the Tris buffer system. The competitive binding constant of Tris with 18C6H4 obtained at pH 3.0 was 27 +/- 4.
The bovine papillomavirus type 1 long control region (LCR) contains DNA sequence elements involved in the regulation of viral transcription and replication. Differences in the levels of transcription have previously been noted between bovine papillomavirus type 1-infected rodent cell lines and bovine cells. To investigate these differences, fragments of the LCR were cloned into an enhancer-deleted chloramphenicol acetyltransferase expression vector and assayed for enhancer activity. A strong constitutive enhancer was found in the 5' portion of the LCR that was most active in primary bovine fibroblasts and had little activity in other cell types. Deletion mapping localized most of the activity to a 113-bp fragment from nucleotides (nt) 7162 to 7275, a region of the viral sequence that also contains the P7185 promoter and an E2-binding site at nt 7203. The enhancer activity of this element could be positively modulated by the full-length E2 transactivator or negatively modulated by the E2 repressor. Site-directed mutagenesis defined two cis elements, CE1 and CE2, which were both necessary for enhancer activity. The CE1 element was required for P7185 activity, whereas the CE2 element was dispensable for P7185 activity. The CE1 and CE2 elements both overlap the E2-binding site at nt 7203. In vitro DNA-binding studies revealed (i) a specific gel retardation complex associated with cellular factor binding at the CE1 element, (ii) a correlation between enhancer activity and the binding of factors to the CE1 element, and (iii) competitive binding between the E2 repressor and the cellular factor at the CE1 element.
A displacement equation describing competitive binding of drugs to protein in solution is derived and examined with four nonsteroidal anti-inflammatory drugs and human serum albumin as model drugs and protein, respectively. Microdialysis/high-performance liquid chromatography was adopted to determine simultaneously the unbound solute and displacing agent in drug-protein solutions. The method is able to locate the binding site and determine affinity constants even up to 10(7) L/mol accurately. A comparison of association constants determined by this method and from capacity factors on HSA-CSP is given.
The competitive binding of chromium, cobalt and nickel chloride salts to murine serum proteins was studied in vitro. Individual metal salt solutions and combinations were incubated with 1:20 dilution of murine serum proteins for 24 h. Then free metal was removed by dialysis. The protein bound metal ions were analysed by graphite furnace atomic absorption spectroscopy. This study determined the saturation binding of cobalt and nickel to serum proteins. Murine serum is mostly saturated when cobalt or nickel is added at the concentration of 2 mol of metal to 1 mol of albumin. Chromium and cobalt have similar protein binding affinity, chromium and cobalt bind to protein in proportion to the added concentration ratio. However, nickel shows significant competition for chromium and cobalt binding moieties. This study provides a reference for future research on the biological role and properties of corrosion products.
Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).