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At least 73 records · Page 4Linked to original sources
PubMed1989

[Competitive binding of various beta-lactam compounds to penicillin-binding proteins of Escherichia coli].

Competing interaction of two novel N-acyl derivatives of ampicillin i.e. N'-benzylchlorbenzimidazole (No. 48) and N-pyrazolytiazole (No. 72) derivatives and 14C-benzylpenicillin with penicillin-binding proteins (PBP) of E. coli was studied. It was shown that ampicillin and its derivative No. 48 markedly differed in their affinity to various PBPs. Derivative No. 72 did not prevent binding of the labeled benzylpenicillin to any PBP which corresponded to its low antimicrobial activity. Analogous experiments with new cephalosporin structures i.e. active and inactive N-acyl derivatives of cephalosporin showed that the active derivative No. 94 i.e. N-methyltiobenzimidazole derivative had the highest affinity to PBP-2 and PBP-5. The inactive derivative No. 68 i.e. N-chlorbenzimidazole derivative also had high affinity to PBP-1b, PBP-2 and PBP-3 essential for the cell. No activity of the latter compound against intact cells of E. coli was probably due to its low penetration through the outer membrane of the bacterial cell. Estimation of affinity of the beta-lactam structures to various PBPs not only provided data on the mechanism of their action but also made it possible to explain in some cases the peculiarities of their antimicrobial spectrum.

Anti-Bacterial Agents↗
PubMed2000

Competitive binding to the oligopeptide binding protein, OppA: in-trap cleanup in an Fourier transform ion cyclotron resonance mass spectrometer.

This communication demonstrates that gentle infrared laser heating can remove unwanted buffer adducts from a gas-phase protein complex without dissociating the complex itself. Specifically, noncovalent complexes of the oligopeptide-binding protein, OppA, bound to either (Ala)3 or LysTrpLys were electrosprayed from aqueous buffer solution into a 9.4 tesla Fourier transform ion cyclotron resonance mass spectrometer. In addition to the intact complexes, several additional buffer adduct species were produced under the conditions of the experiment. Irradiation of the trapped ion population with a continuous-wave infrared CO2 laser at relatively low power (2.5 W) for 1 s dissociated the buffer adducts but retained the intact protein:peptide complexes. Adduct-free complex(es) were then readily identified, and signal-to-noise ratio also increased by an order of magnitude because the same number of protein ions are distributed over fewer species. Higher IR power (5 W for 1 s) dissociated the adduct-free complex(es) without internal fragmentation. The present in-trap clean-up technique may prove especially useful for identifying and screening the combinatorial library ligands most strongly bound to a receptor in the gas phase.

Bacterial Proteins↗
PubMedDate not supplied

[Estimation of 25-hydroxyvitamin D in blood serum using competitive binding with protein].

A modified procedure is described for estimation of 25-hydroxyvitamin D (25OHD) in blood serum, which involves a principle of the saturating analysis. Blood serum from rats, maintained on a ration free of vitamin D, was used as a binding system. Evaluation of the procedure was carried out by conventional criteria (accuracy, reproducibility, sensitivity). Content of 25OHD was estimated in blood of rats, maintained on rations containing various amount of vitamin D, as well as in blood of women in childbirth and in newborns from different climatogeographical zones.

Animals↗
PubMed1987

Improvement on the competitive binding assay for the measurement of cyclic AMP by using ammonium sulphate precipitation.

The protein-binding assay developed by Brown, Albano, Ekins, Sgherzi & Tampion [(1971) Biochem. J. 121, 561-562] and Brown, Ekins & Albano [(1972) Adv. Cyclic Nucleotide Res. 2, 25-40] was modified by using precipitation with (NH4)2SO4 of the protein-cyclic AMP complex instead of adsorption of the free nucleotide on charcoal. The half-life of the protein-cyclic AMP complex obtained in the presence of charcoal was lower than that of the (NH4)2SO4-precipitated complex. In consequence, owing to the great stability of the precipitated protein-cyclic AMP complex, this method allows more accurate and reproducible determinations.

Ammonium Sulfate↗
PubMed1991

Competitive binding radioassay for the determination of 5-fluorodeoxyuridine and 5-fluorodeoxyuridine-5'-monophosphate levels in plasma and tumor tissue.

A competitive binding radioassay was developed to measure 5-fluoro-2'-deoxyuridine (FUDR) as well as 5-fluoro-2'-deoxyuridine monophosphate (FdUMP), FdUMP has been measured by a competitive binding radioassay with thymidylate synthase as the binding enzyme (TS assay). FUDR was enzymatically converted to FdUMP by thymidine kinase, and then the converted FdUMP was measured by the competitive binding assay to determine the concentration of FUDR in plasma and tumor tissue. As little as 100 pg/ml of FUDR or 50 pg/ml of FdUMP can be detected quantitatively by this method. When TS assay and high-performance liquid chromatography were compared for the measurement of FUDR and FdUMP levels in plasma and tumor tissue of Ehrlich carcinoma (EC)-bearing mice following administration of FUDR, a close agreement was observed for FUDR levels, though low FdUMP levels were detectable only by the TS assay method. The examination of intracellular metabolism of FUDR in EC cells by this method showed that metabolic conversion of FUDR into FdUMP or 5-fluorouracil is rapid. Thus, we have established a highly sensitive method for measuring not only FdUMP but also FUDR with TS assay. This should be very useful for experimental and clinical studies on fluoropyrimidines.

Animals↗
PubMed2004

Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion measurements.

It is important to characterize drug-albumin binding during drug discovery and lead optimization as strong binding may reduce bioavailability and/or increase the drug's in vivo half-life. Despite knowing about the location of human serum albumin (HSA) drug binding sites and the residues important for binding, less is understood about the binding dynamics between exogenous drugs and endogenous fatty acids. In contrast to highly specific antibody-antigen interactions, the conformational flexibility of albumin allows the protein to adopt multiple conformations of approximately equal energy in order to accommodate a variety of ligands. Nuclear magnetic resonance (NMR) diffusion measurements are a simple way to quantitatively describe ligand-protein interactions without prior knowledge of the number of binding sites or the binding stoichiometry. This method can also provide information about ligand orientation at the binding site due to buildup of exchange-transferred NOE (trNOE) on the diffusion time scale of the experiment. The results of NMR diffusion and NOE experiments reveal multiple binding interactions of HSA with dansylglycine, a drug site II probe, and caprylate, a medium-chain fatty acid that also has primary affinity for HSA's drug site II. Interligand NOE (ilNOE) detected in the diffusion analysis of a protein solution containing both ligands provides insight into the conformations adopted by these ligands while bound in common HSA binding pockets. The results demonstrate the ability of NMR diffusion experiments to identify ternary complex formation and show the potential of this method for characterizing other biologically important ternary structures, such as enzyme-cofactor-inhibitor complexes.

Binding, Competitive↗
PubMed2002

Competition binding experiments for rapidly ranking lead molecules for their binding affinity to human serum albumin.

Many lead molecules that have high affinity for a therapeutic target in vitro exhibit a reduced efficacy in vivo. Drug binding to human serum albumin is a major contributor to this reduction in potency, and many drug discovery programs expand significant resources preparing compounds that have decreased albumin binding. As rational and structure-based approaches have already been demonstrated to design compounds that have reduced affinity for albumin, the ability to rapidly and accurately assess protein binding will be valuable in lead optimization. This review will summarize some of the NMR-based efforts towards developing universal, rapid, accurate, and site-specific assays for estimating protein binding.

Binding Sites↗
PubMed2001

Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands.

BACKGROUND: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N-formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands. METHODS: Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO-NLFNYK-tetramethylrhodamine. RESULTS: Calibration parameters were determined for six fluorescently-labeled N-formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO-NLFNYK-tetramethylrhodamine determined directly or in competition with CHO-NLFNYK-fluorescein were statistically identical. CONCLUSIONS: Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated.

Binding, Competitive↗
PubMed2006

In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique.

The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients.

Algorithms↗
PubMed1994

Inhibition of hematopoiesis by competitive binding of transcription factor PU.1.

Transcription factors have been shown to play a role as "master switch" factors in the programming of hematopoietic cell commitment and differentiation. PU.1 is a hematopoietic-specific member of the Ets family of transcription factors. In human bone marrow CD34-enriched progenitor cells, PU.1 expression was upregulated during the early phases of granulocytic/monocytic differentiation, preceding expression of its target genes encoding CD11b and the macrophage-colony-stimulating factor receptor, whereas PU.1 was expressed at stable levels throughout erythroid differentiation. To study PU.1 function, we synthesized double-stranded phosphorothioate oligonucleotides containing a characterized PU.1 site and demonstrated their ability to specifically compete for PU.1 DNA binding. When added to CD34+ cells in vitro, wild-type PU.1-binding oligonucleotides significantly blocked hematopoietic colony formation, whereas mutated PU.1 oligonucleotides which no longer bind PU.1 had no specific inhibitory effect. These results demonstrate that PU.1 is developmentally upregulated during normal human myelopoiesis and that the function of PU.1 is critical for the development of in vitro hematopoiesis.

Antigens, CD↗