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Results for “ligand binding”

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At least 37 records · Page 2Linked to original sources
PubMed1979

The influence of cell surface receptor clustering on the thermodynamics of ligand binding and the kinetics of its dissociation.

We show that an equilibrium model for the clustering, by divalent ligand, of homogeneous plasma membrane-bound divalent receptors that do not change conformation predicts Scatchard plots with positive second derivatives (concave up). The result is thermodynamically indistinguishable from predictions based upon receptors that are heterogeneous for ligand, or that change conformation in a negatively cooperative way when ligand binds. A more general formulation of the theory, which allows application to dissociation kinetics, predicts that the dissociation of labeled ligand is accelerated in the presence of excess cold ligand and, moreover, that the accelerated dissociation has at least two components. The theory is briefly illustrated by fitting five different sets of data, at several temperatures for two different systems, with parameter values that are well within physically meaningful ranges. The model presented is the simplest and least ad hoc explanation thus far proposed as the basis for the kinetic phenomena, and raises the possibility that the numerous experiments in which such data are observed are primarily minifestations of cell surface clustering.

Insulin↗
PubMed1977

Inactivation and inhibition of Rous sarcoma virus by copper-binding ligands: thiosemicarbazones, 8-hydroxyquinolines, and isonicotinic acid hydrazide.

We have shown that three types of copper-binding ligands, thiosemicarbazones, 8-hydroxyquinolines, and isonicotinic acid hydrazide and their copper complexes, inactivate the transforming ability of RSV and inhibit its RNA-dependent DNA polymerases. Three other compounds, 2-pyridine thiosemicarbazone, 1-formyl isoquinoline thiosemicarbazone, and diphenyl thiocarbazone inhibit transformation by RSV intracellularly. Most but not all of these compounds bind to nucleic acids in the presence of copper, which may be important in their mode of action.

Avian Sarcoma Viruses↗
PubMed1977

New method of quantifying ligand binding based on measurement of an induced response.

A new general method is proposed for quantifying ligand-receptor interactions using the biological response induced by the ligand as an index of ligand binding. With this method the binding of human chorionic gonadotropin (hCG), several hCG derivatives, and luteinizing hormone (LH) to rat Leydig cells was measured by analysis of the ability of these materials to stimulate testosterone formation. As applied here, hormone dose-response curves were generated in the presence of increasing numbers of cells incubated in vitro in a successful attempt to alter the concentrations of bound and free hormone in the incubation mixture. Measurements of testosterone synthesis as a function of the total amounts of hormone and numbers of cells enabled us to evaluate the concentrations of both bound and free hormone at any constant fractional response (i.e. quarter-, half-, or three-quarter-maximal). We were thus able to measure hormone binding at the extremely low hormone concentrations (1 pM) within the steroidogenic dose-response range under conditions that would not have been possible using currently available radioiodinated hCG preparations. The results obrained confirmed the presence of spare functional receptors. Specific quantitative results are discussed in the text.

Animals↗
PubMed2026

Naturally Occurring CodY Variants Alter Ligand Binding, DNA Target Affinity, and Virulence in Clostridioides difficile.

Clostridioides difficile is an important nosocomial pathogen and is the major cause of antibiotic-associated diarrhea and colitis. CodY is a global transcriptional regulator that coordinates metabolism and virulence in Gram-positive pathogens by sensing branched-chain amino acids and GTP. In C. difficile, CodY represses toxin production by inhibiting transcription of tcdR and by influencing c-di-GMP turnover. Here, we characterized two naturally occurring CodY variants, CodY(Y146N) and CodY(V58A), whose substitutions lie near the GTP- and ILV-binding sites, respectively. GTP-binding by CodY(Y146N) was severely compromised, while leucine binding was enhanced; CodY(V58A) showed reduced leucine binding. Both variants exhibited reduced ligand-dependent binding to the tcdR promoter and failed to repress toxin production as effectively as CodY(WT). Expression of virulence-associated genes (tcdR, pdcB) was elevated in strains producing either variant. In a hamster infection model, both variant-producing strains were significantly more virulent than the CodY(WT) strain. These findings demonstrate that single amino acid substitutions in this global regulator can alter ligand affinity and promoter binding, potentially rewiring gene regulatory networks to enhance the pathogenic potential of C. difficile.

Clostridioides difficile↗
PubMed1975

Ligand binding and enzymic catalysis coupled through subunits in tyrosyl-tRNA synthetase.

The interaction of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus with its substrates in the aminoacyl adenylation reaction has been studied by stopped-flow fluorescence. The observed changes have been assigned to their chemical and physical processes by comparison with equilibrium dialysis, pyrophosphate exchange kinetics and rapid quenching and sampling techniques to give the rate constants for ligand binding, the formation of tyrosyl adenylate, and the reverse reaction. The stoichiometry of tyrosine and ATP binding in the catalytic process has been determined directly by equilibrium dialysis and equilibrium gel filtration under pyrophosphate exchange conditions, i.e., where a steady state has been set up in which the equilibrium position favors starting materials. It is shown that the rate-determining step in the formation of tyrosyl adenylate involves 1 mole each of tyrosine and ATP. A second mole of tyrosine and ATP bind to the aminoacyl adenylate complex stabilizing the high-energy intermediate. The enzyme tyrosyl adenylate complex that is isolated by gel filtration is in a different conformational state from that in the presence of tyrosine and ATP.

Adenosine Monophosphate↗
PubMed1976

Use of a sequence-specific DNA-binding ligand to probe the environments of EcoRI restriction endonuclease cleavage sites.

The DNAs of bacteriophage lambda and adenovirus were incubated with the sequence-specific DNA-binding ligand 6,4'-diamidino-2-phenylindole. Digestion of the ligand-DNA complexes with EcoRI nuclease and subsequent agarose gel electrophoresis demonstrated that the ligand inhibited nuclease activity at some sites, but not at others. The results suggest that diamidino-2-phynylindole can be used to probe the immediate environments of the EcoRI cleavage sites.

Adenoviridae↗
PubMed1978

Ligand binding sites and subunit interactions of Torpedo californica acetylcholine receptor.

A [3H]bisazido derivative of ethidium bromide was synthesized to identify sites of interaction of ethidium with the acetylcholine receptor from Torpedo californica and to aid in localization of ligand binding sites. For purified solubilized acetylcholine receptor it was shown (a) that the photolabel was competitive with ethidium bromide, (b) dodecyl sulfate--polyacrylamide gel electrophoresis revealed that all four polypeptide components were labeled with [3H]ethidium azide, and (c) alpha-bungarotoxin inhibited the labeling of the 40 000-dalton subunit. Photolabeling of acetylcholine--receptor enriched membrane fragments led to the following conclusions: (a) the photochemical reaction was more selective than for purified acetylcholine receptor, since the 40 000-dalton subunit was preferentially labeled; this result demonstrated differences in the topography of receptor subunits depending on whether the molecule was in detergent solution or in a membrane-bound state, (b) alpha-bungarotoxin inhibited labeling of the 40 000-dalton subunit, (c) ligand-induced conformational changes resulted in different subunit labeling patterns. The results imply that conformational changes generated at the 40 000 molecular weight subunit upon cholinergic ligand interaction cause further intermolecular structural changes that involve subunits of higher molecular weight. These higher molecular weight subunits therefore belong to a supramolecular complex of polypeptides associated with the postsynaptic membrane.

Acetylcholine↗
PubMed1978

Iron-binding ligands in the catalytic site of protocatechuate 3,4-dioxygenase.

The tryptophan fluorescence maximum for holoprotocatechuate 3,4-dioxygenase(holo PCD) is blue-shifted slightly (3 nm) from that of the apoenzyme. In the preparation of apoenzyme, increases in tryptophan fluorescence intensity coincided with decreases in enzyme activity and decreases in iron content. The tryptophan emission intensity of reconstituted enzyme having full enzyme activity was about 90% of that of the holoenzyme. Although apo PCD has similar molecular weight, amino acid content and essentially the same gross quaternary conformation as holo PCD, the absence of iron in apo PCD causes the changes in emission intensity of tryptophan. Findings indicate that some tryptophan residues may be (or may be near) the iron-binding ligands in the catalytic site of protocatechuate 3,4-dioxygenase.

Apoenzymes↗
PubMed2025

The RORγt ligand-binding domain controls the pathogenicity of IL-17A+ T cells differently in autoimmune diseases of the skin and CNS.

The transcription factor RORγt orchestrates Th17 lineage differentiation, thymic T cell development, and the pathogenesis of several autoimmune disorders. Lipid ligands are required for appropriate regulation of RORγt activity, but it is unclear to what extent lipid recognition controls RORγt function in vivo. Here, we show that the mutation of RORγt alanine-304 in the ligand-binding domain (LBD) to isoleucine (A304I) abrogates lipid-dependent Th17 differentiation and selectively ameliorates γδT17 cell-mediated psoriatic skin inflammation. In contrast, there is no improvement in experimental autoimmune encephalomyelitis in RORγtA304I mice. Consistent with this, the expression of genes characteristic of Th17 cells decreases in RORγtA304I mice, along with a compensatory increase of genes characteristic of Th1-like Th17 cells with pathogenic signatures. Thus, RORγt alanine-304 in the LBD is indispensable for generating γδT17 and conventional Th17 cells and for the suppression of the Th1-like Th17 pathogenic population, which decouples the pathogenicity of skin and CNS autoimmune diseases.

Animals↗
PubMed1977

Inhibition of amino acyl tRNA synthetase activity by copper complexes of two metal binding ligands. N-Methyl isatin beta-thiosemicarbazone and 8-hydroxyquinoline.

Copper complexes of N-methyl isatin beta-thiosemicarbazone, 1-formyl isoquinoline thiosemicarbazone and thiosemicarbazide inhibit amino acyl tRNA synthetase activity. Copper complexes of 8-hydroxyquinoline and 8-mercaptoquinoline also inhibit. The 1 : 1 ligand-metal complex is significantly more active than the 2 : 1 complex. The free ligand alone and copper sulfate alone have little, if any, effect. These complexes have no effect on the ATP-PPi exchange reaction and do not cause deacylation of amino acyl tRNAs. This indicates that the process inhibited by these complexes is the amino acylation reaction. This is the first report that these copper binding ligands can inhibit enzymatic processes which involve nucleic acids but which are not viral, bacterial or mammalian cell polymerases.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Ligand binding studies in the mouse olfactory bulb: identification and characterization of a L-[3H]carnosine binding site.

Binding sites for the dipeptide L-carnosine (beta-alanyl-L-histidine) have been detected in membranes prepared from mouse olfactory bulbs. The binding of L-[3H]-carnosine was saturable, reversible and stereospecific and had a Kd of about 770 nM. The stereospecific binding of L-carnosine represented about 30% of the total binding at pH 6.8, and decreased markedly with increasing pH. Binding was stimulated by calcium, unaffected by zinc, magnesium or manganese and inhibited by sodium and potassium. Carnosine binding was sensitive to trypsin and phospholipases A and C, but not to neuraminidase. Nystatin and filipin, which interact with membrane lipids, also interferred with binding. Some peptide analogues of carnosine were potent inhibitors of binding, but a variety of drugs serving as potent inhibitors in other binding systems had no effect on carnosine binding. Carnosine binding to mouse olfactory bulb membranes was 15-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than that seen in membranes prepared from cerebral hemispheres, 5-fold higher than in cerebellum membranes and 3-fold higher than in membranes from spinal medulla and the olfactory tubercle-lateral olfactory tract area. Binding sites for 6 other radiolabeled receptor ligands were also detected in bulb membranes. Peripheral deafferentation of the olfactory bulbs by intranasal irrigation with ZnSO4 led to a loss greater than 90% of the L-[3H]carnosine binding in 4--5 days with much smaller losses in binding of the other 6 ligands over a 180-day observation period. This initial loss of carnosine binding after denervation was due to a loss of binding site stereo-specificity followed by a loss of binding sites. The characteristics of the carnosine binding site in olfactory bulb fulfil 6 of the 7 criteria considered relevant for a functional receptor.

Animals↗
PubMed1979

Anti-V region framework antibodies affect the ligand binding of VL dimer.

The effect of antibodies to the light chain variable region (VL) of protein MOPC-315 (alpha, lambda 2), on the binding of hapten by VL315 dimer or Fv315 (VL + VH) was studied by equilibrium dialysis. Anti-VL did not change the binding properties of Fv but affected the binding properties of VL dimer. At pH 5, the binding properties of VL in the presence or absence of anti-VL were the same, whereas at pH 8, anti-VL reduced the number of ligands bound to VL from two to one. It has previously been shown that VL dimer binds one ligand at pH 5 and two ligands at pH 8, and that VL conformation at pH 5 is tighter. Hence, our results suggest that anti-VL tightens the conformation of VL dimer at pH 8.0 such that it can bind only one ligend. Since Fv is not affected by anti-VL, the results indicate that a combining site made of two identical chains (VL dimer) can undergo a conformational change upon interaction with its antibody. Such conformational change can indirectly affect the binding properties.

Animals↗
PubMed1977

Studies on ligand binding of kidney bean leghemoglobin.

Absorption spectra of different ligand derivative;s of kidney bean leghemoglobin alpha have been recorded. The effect of pH on the absorption spectra of kidney bean leghemoglobin alpha has been studied. The pK of the acid-alkaline transition of the heme-linked water molecule is 8.25 and the pK for the acid dissociation of the heme group is 4.03. Affinities of kidney bean leghemoglobin for two different types of ligands have been studied in comparison with soybean leghemoglobins alpha and c and sperm whale myoglobin. All these leghemoglobins have similar affinities for the small anionic ligand fluoride ion, and they are only slightly more accessible to this ligand than is sperm whale myoglobin. Differences in the primary structure or in conformation of these proteins are reflected in the affinity for the bulky ligand imidazole. The accessibility to imidazole increases in the order sperm whale myoglobin less than soybean Lbalpha less than soybean Lbc less than kidney bean Lbalpha, and also low spin Lbalpha less than high spin Lbalpha. The results are discussed with respect to the amino acid sequences of the leghemoglobins.

Binding Sites↗
PubMed1978

The green hemoproteins of bovine erythrocytes. II. Spectral, ligand-binding, and electrochemical properties.

The two green hemoproteins isolated from bovine erythrocytes (form I and form II) have been characterized as to spectral, electrochemical, and chemical properties. The absorption spectra of the isolated hemoproteins are typical of high spin ferric states. Reduction of the hemoproteins yields high spin ferrohemoproteins. Complexation of the ferrohemoproteins with CO and the ferrihemoproteins with cyanide yields low spin complexes, demonstrating the presence of an exchangeable weak field ligand in both the ferrous and ferric states of the hemoproteins. The differences in position and intensity of the absorption peaks of the visible spectra allow the two forms to be distinguished from one another. The midpoint potential of forms I and II were found to be +0.075 and +0.019 V, respectively, at pH 6.4 and +0.038 and -0.005 V, respectively, at pH 7.0. This is consistent with the gaining of 1 proton/electron during the reduction. The Nernst plot reveals an unusual 0.5-electron transfer, whereas a quantitative titration demonstrates a 1-electron transfer. Form I binds cyanide more tightly than form II (KD of 84 and 252 micrometer, respectively). The observed spectral, electrochemical, and ligand-binding differences between forms I and II can be explained in terms of a greater electron-withdrawing ability of the side chains of the heme of form I relative to the heme of form II.

Animals↗