Showing papers on "Cooperative binding published in 1978"

Mapping of the residues responsible for the negative cooperativity of the receptor-binding region of insulin

Abstract: Insulin binding to its receptor leads to negatively cooperative interactions among the receptor sites. Studies with 29 insulin analogues (animal insulins and proinsulin, insulin-like growth factor and chemically modified insulins) which vary 1,000-fold in their affinity for the receptor and in their biological potency, suggest that a discrete invariable region on the surface of the insulin monomer is responsible for inducing the negative cooperativity. This domain comprises some of the eight carboxy-terminal residues of the B-chain and the A21 asparagine. Burying of this ‘cooperative site’ in the dimerisation of insulin leads to a loss of negative cooperativity. A revised mapping of the insulin molecule is proposed, featuring distinct bioactive and cooperative sites.

Correlation Among Insulin Binding, Degradation, and Biological Activity in Human Breast Cancer Cells in Long-Term Tissue Culture

Abstract: Insulin interaction with four human breast cancer cell lines in tissue culture was studied with respect to specific binding to receptors, degradation, and biological responsiveness. All four lines bound and degraded 125I-labeled insulin. Binding and degradation were time, temperature, and pH dependent. Unlabeled insulin competed with 125I-labeled insulin for binding to the breast cells with half-maximal inhibition of binding being observed with less than 0.6 ng/ml for three of the four lines. Other peptides competed for insulin binding in proportion to their biological potency. Scatchard analysis of the insulin binding data revealed curvilinear plots consistent with negative cooperativity, and this was confirmed by kinetic studies of dissociation. Quantitative analysis of insulin degradation revealed a similar Km for all four cell lines (1.0 to 2.2 × 10-7m), whereas maximal velocities varied over a 7-fold range. Bacitracin, a polypeptide antibiotic, inhibited insulin degradation by all four lines. In one cell line, typical competitive binding data and Scatchard plots were obtained only after inhibition of degradation with bacitracin. Insulin stimulated precursor incorporation into macromolecules and fatty acids in only two of the four cell lines; with these, significant stimulation was seen with concentrations as low as 0.6 ng/ml. No correlation was found between the amount of specific binding, receptor concentration, or receptor affinity and the ability of insulin to stimulate the cells, suggesting a defect distal to the hormone-receptor interaction in the two unresponsive lines. The two responsive cell lines showed the most insulin degradation. These human cell lines should provide a useful tool for further study of the complex mechanisms of insulin action and for the study of factors that regulate growth of human breast cancer.

Abnormalities of triiodothyronine binding to lymphocyte and fibroblast nuclei from a patient with peripheral tissue resistance to thyroid hormone action.

Abstract: T3 binding to lymphocyte nuclei has been studied in normal individuals and in a patient (MaG) with peripheral resistance to thyroid hormone action. This syndrome is defined by the presence of hypothyroidism or euthyroidism with high plasma levels of thyroid hormone. T3 bound to a single set of binding sites in normal adult lymphocyte nuclei with a mean Ka of 8.9 +/- 7.1 x 109 M-1, and a capacity of 4.4 +/- 2.9 fmol/100 micrograms DNA. A single binding site was also disclosed in MaG's lymphocytes with a Ka of 0.43 x 109 M-1 and a capacity of 10.5 fmol/100 micrograms DNA. This low affinity was not due to the presence of high plasma T3 level in the patient, since administration of 100 micrograms T3 to normal adult volunteers induced the presence of two different binding sites. The mechanism responsible for this phenomenon is unknown. To binding was also studied using cultured fibroblasts which were incubated in serum-less medium before the binding experiments. One single binding site (Ka, 1.9 x 10(10) M-1, capacity, 12.9 fmol/100 micrograms DNA) was detected in normal fibroblast nuclei. In contrast, a curvilinear Scatchard plot was obtained when MaG's fibroblasts were used. This result could be compatible with the presence of either two different binding sites or negative cooperativity. In support of the latter possibility, Hill plots gave a number lower than unity. The results suggest that the syndrome of peripheral tissue resistance to thyroid hormone action due to a defect at the level of the nuclear receptor. The possible existence of similar syndromes due to an alteration at the level of a post-T3-binding mechanism is not eliminated.

Comparative binding studies with cholinergic ligands and histrionicotoxin at muscarinic receptors of neural cell lines.

Abstract: [3H]Scopolamine and [3H]quinuclidinyl-benzilate (QNB) have been used to study inhibitory acetylcholine receptors of neuroblastoma clone N1E-115 and excitatory acetylcholine receptors of NG108-15 neuroblastoma x glioma hybrid cells. Both [3H]ligands bind with high affinity to muscarinic acetylcholine receptors of the cells. The apparent dissociation constants are 0.4 nM (N1E-115) and 0.5 nM (NG108-15) for [3H]scopolamine, 0.06 nM (N1E-115) and 0.1 nM (NG108-15) for [3H]QNB. The receptor concentration is 25 fmol/mg in N1E-115 and 40 fmol/mg in NG108-15. Binding and release of [3H]-scopolamine are kinetically biphasic processes. [3H]QNB has a similar rate of binding but a much slower rate of release from the receptor. Binding of both [3H]ligands is competitively inhibited by compounds known to interact with muscarinic acetylcholine receptors. With 1 nM [3H]ligand a 50% inhibition is caused by nanomolar concentrations of muscarinic antagonists, by 1 to 100 micromolar concentrations of muscarinic agonists, and by >100 micromolar concentrations of nicotinic cholinergic compounds. Slopes of approximately 1 were found for receptor antagonists and approximately 0.5 for receptor agonists in logit-log plots of competition data. Antagonist binding can therefore be described as interaction with a noncooperative class of receptors, whereas agonist binding exhibits negative cooperativity or heterogeneity in binding sites. The interaction of dihydroiso-histrionicotoxin (H2-HTX) with muscarinic acetylcholine receptors of N1E-115 cells has also been studied by inhibition experiments. H2-HTX inhibits [3H]scopolamine binding in a noncompetitive manner causing a 50% inhibition at an applied concentration of 70 µM. The local anesthetic, tetracaine, shows almost identical characteristics. Dihydro-adaline, granatan-3β-ol and granatan-3α-ol are less strong inhibitors. Structural comparison of these compounds with H2-HTX suggests that the two aliphatic side chains and the proximity of the hydroxyl and amino groups may be important features for the interaction of H2-HTX with the muscarinic acetylcholine receptor.

Interaction of beta1H globulin with cell-bound C3b: quantitative analysis of binding and influence of alternative pathway components on binding.

Abstract: Purified beta1H globulin (beta1H) was shown to bind to C3b coated cells by both immunofluorescent and radioactive tracer techniques. With EAC43, the amount of beta1H bound was directly proportional to the amount of C3 used to prepare the cells; EA, EAC14 and EAC14oxy2 bound very small amounts of beta1H. The C3b binding site on beta1H was labile in that not all of the purified 125I-beta1H was capable of binding to C3b, even when an excess of cell-bound C3b was present. Scatchard analysis of binding of beta1H to C3b-coated cells indicated an equilibrium constant of 10(9) L/M. Deviations from linearity were regularly found on Scatchard analyses. This was consistent with the hypothesis that the beta1H binding sites exhibit negative cooperativity in that as more sites become occupied, it becomes more difficult to fill the remaining sites. The stoichiometry of the reaction between C3b and beta1H was examined using EAC14oxy23 prepared with 131I-C3 and beta1H labeled with 125I. Between 0.5--0.8 beta1H molecules were bound per C3b molecule. Other alternative pathway components influenced the binding of 125I-beta1H to cell bound C3b. Both C3b and native C3 inhibited binding of labeled beta1H at an efficiency approximately 1/1,000 that of unlabeled beta1H. Factor B inhibited binding with 1/280 the efficiency of unlabeled beta1H. Properdin caused a dose-dependent increase in the binding of beta1H; this enhancement was abrogated if B was also present in the reaction mixture. Scatchard analysis indicated that the enhancement of beta1H binding by P resulted in an increased number of available binding sites rather than an increase in the affinity of binding.

Quantitative Aspects of Allosteric Mechanisms

Abstract: 1 Basic Concepts of Allosteric Control.- 2 The Structure of Multisubunit Proteins.- I. General Principles.- II. Other Types of Protein Assemblies.- 3 Cooperativity in Multisubunit Proteins - The Basic Concepts.- I. The Hill Equation.- II. The General Adair Equation.- III. The Statistical Correction.- IV. The Hill Coefficient in Terms of Intrinsic Ligand Affinities.- V. The Hill Coefficient at 50% Ligand Saturation.- VI. The Maximal Hill Coefficient.- VII. The Limiting Values of the Hill Slope.- VIII. The Allosteric Dimer.- IX. The Multi-Dimer Case.- X. The Allosteric Tetramer.- XI. The General Tetrameric Case.- 4 The Energy of Subunit Interactions.- I. Determination of Intersubunit Interaction Energy.- II. The Hill Coefficient and the Intersubunit Interaction Energy.- III. The Meaning of Intersubunit Energy of Interaction.- 5 Molecular Models for Cooperativity and Allosteric Interactions.- I. Introduction.- II. The Monod-Wyman-Changeux (MWC) Concerted Model.- 1. Basic Assumptions of the Concerted Model.- 2. The Allosteric Dimer Analyzed by the MWC Model.- 3. Allosteric Inhibition and Allosteric Activation in the MWC Model.- 4. The General Case.- 5. Phenomena Explained by the Concerted Model.- 6. Cooperativity in the Monod-Wyman-Changeux Model.- III. The Koshland-Nemethy-Filmer (KNF) Sequential Model.- 1. Basic Assumptions of the Sequential Model.- 2. The Allosteric Dimer Analyzed by the KNF Model.- 3. Allosteric Activation and Allosteric Inhibition in the KNF Model - the Dimer Case.- 4. The KNF Model - the Tetramer Case.- 5. The Influence of the Intersubunit Binding Domains on the Nature of Subunit Interactions.- IV. The Conformational State of the Protein.- 1. Exclusive Binding in the MWC Model.- 2. The Nonexclusive Binding in the MWC Model.- 3. The Simple Sequential KNF Model.- 4. The General Sequential KNF Model.- 5. Measuring R?.- V. Comparison Between the KNF Model and the MWC Model.- 6 Special Types of Cooperative Systems.- I. Cooperativity Resulting from Ligand-Coupled Protein Association or Dissociation.- 1. Ligand-Coupled Monomer-Dimer Equilibrium.- 2. The General Case.- II, Negative Cooperativity.- III. Protein Association and Dissociation Coupled to Ligand Binding.- 1, Dimerization Coupled to Ligand Binding.- 2. Monomer Multimer Equilibrium Coupled to Ligand Binding.- References.

The taxonomy of binding sites in proteins

Abstract: Conservation of polypeptide fold and mode of ligand binding is frequently found within proteins of related function. Examples illustrating this phenomenon are taken from NAD linked enzymes, nucleotide binding proteins, polysaccharide binding proteins, heme binding proteins and enzymes with essential Fe-S complexes or zinc atoms.

Insulin Binding to Adipocytes: Evidence for Functionally Distinct Receptors

Abstract: Dissociation of 125 I-insulin from adipocyte insulin receptors was studied as a function of receptor occupancy. When cells were equilibrated with a tracer amount of 125 I-insulin and the insulin-receptor complexes allowed to dissociate in diluted 125 I-insulin–free buffer, in the presence or absence of unlabeled insulin, the unlabeled insulin led to acceleration of 125 I-insulin dissociation rates. Thus, the unlabeled insulin (dilution + insulin) led to higher fractional receptor occupancy and a clear-cut acceleration of 125 I-insulin dissociation rates, a phenomenon consistent with negatively cooperative site-site interactions. This effect was dependent on the total insulin concentration employed and was also influenced by the temperature of the incubations. The insulin derivatives, desalanine desasparagine and desoctapeptide insulin, did not accelerate dissociation of 125 I-insulin. Therefore, we find evidence that adipocyte insulin receptors exhibit similar properties to those originally described by De Meyts et al. (J. Biol. Chem. 251 : 1877, 1976) for other insulin receptor systems. However, we also find that, under conditions where the accelerating effect of native insulin is either not appreciable or is maximal, 125 I-insulin does not dissociate from its receptors as a first order process. This suggests that, even in the absence of negatively cooperative effects, adipocyte insulin receptors do not behave as a kinetically homogeneous population. Furthermore, when 125 I-insulin was allowed to associate with cells at high initial levels of receptor occupancy, the subsequent dissociation of the 125 I-insulin was faster than when only the negatively cooperative effect was determined. Therefore, something in addition to the cooperative effect led to further acceleration of insulin dissociation. If receptors exist with functionally distinct binding characteristics such that one group of receptors has a high affinity and a low capacity while another group has a lower affinity and a high capacity, then, when cells are associated with 125 I-insulin plus high concentrations of unlabeled insulin, most of the 125 I-insulin binds to the low affinity (fast-dissociating) sites. Therefore, dissociation rates are faster when high insulin concentrations are used in the association phase, since negative cooperativity as well as rapid dissociation from a functionally lower affinity receptor are being determined. When the material that dissociates from the cells was examined, it was found that the rapidly dissociating material was intact insulin while the more slowly dissociating material contained a significant proportion of degraded material. The rate of dissociation of intact insulin could be accelerated, whereas the rate of dissociation of degraded insulin could not be accelerated in the presence of unlabeled insulin in the buffer during the dissociation phase. Thus, negatively cooperative site-site interactions do not account for all the kinetic characteristics that were observed, and the data are best explained by a model consisting of functionally heterogeneous binding sites, i.e. low affinity, high capacity sites, which are susceptible to the cooperative effect but don9t degrade insulin, and high affinity, low capacity sites, which participate in the degradative process but are not susceptible to the cooperative effect.

Insulin binding sites on rat liver nuclear membranes: biochemical and immunofluorescent studies.

Abstract: Preliminary investigations (Horvat et al., '75) indicated the nucleus of rat liver as a site for specific binding of insulin. In this report these observations are confirmed. Nuclei from rat liver were isolated in a highly purified state as verified by interference contrast and electron microscopy and by chemical analysis. Extensive scanning of the preparations did not reveal the presence of structures resembling plasma membranes. The nuclear envelope was isolated by a modification of the method of Kay et al. ('72). Electron micrographs showed the presence of nuclear "ghosts" and few other recognizable nuclear elements, but no plasma membranes (60--80 A thick) were detected. The preparation was found to contain specific insulin binding activity. Specificity of the binding sites for insulin was demonstrated in competition studies with other polypeptide hormones and a synthetic insulin analog. Scatchard analysis of the binding data indicates the presence of a single class of high affinity receptors. In contrast to findings with plasma membranes the hormone-receptor complex is very stable and the kinetics of the dissociation of bound [125I]-insulin do not indicate negative cooperativity of the binding sites. Immunofluorescent labeling of intact, unfixed nuclei showed a specific fluorescent halo only around those nuclei that have been preincubated with insulin. All other controls were negative.

The Binding of Calcium and Magnesium to Sarcoplasmic Reticulum Vesicles as Studied by Manganese Electron Paramagnetic Resonance

Abstract: The binding pattern of the biologically relevant ions calcium and magnesium has been investigated via the binding of the ion-analogue manganese. The binding parameters of manganese are obtained conveniently by standard electron paramagnetic resonance techniques, the binding of calcium and magnesium is inferred from competition experiments. The quantitative analysis was carried out with a computer program which was able to treat the competition of three kinds of ions for three classes of independent and one class of cooperative binding sites. It was possible to correlate specific binding classes with biological functions of the sarcoplasmic reticulum membrane. The magnesium and manganese specific binding class of medium affinity is related to the catalytic function of these ions in the ATP-splitting. For the first time a manganese and calcium specific class of cooperative binding sites has been observed and it can be related to the inhibition of the calcium transport at high concentrations of manganese or calcium. From the binding results a model can be developed which allows a full description of the role of the divalent ions and their specific binding sites during calcium transport.

The interaction of caerulein with the rat pancreas. 1. Specific binding of [3H]caerulein on plasma membranes and evidence for negative cooperativity.

Abstract: 1. The binding of [3H]caerulein (a stable, biologically active labeled analog of cholecystokinin-pancreozymin) to semi-purified rat pancreatic plasma membranes was investigated. The binding was dependent on time and temperature, as well as saturable, specific and reversible. This process was pH-dependent and optimal at pH 7.0. Cysteine and serine residues in plasma membranes were of importance for binding. Mg2+ favored the binding. 2. The acceleration of the dissociation of [3H]caerulein in the presence of an excess of native caerulein suggests that binding was characterized by a negative cooperativity. The fast dissociation state evoked by a high degree of occupancy by caerulein was inhibited by lowering the temperature, by decreasing the pH, or by the presence of wheat germ agglutinin.

Anionic control of hemoglobin function

Abstract: Hemoglobin is in a state of dynamic equilibrium between high and low affinity forms As in the case of many other enzymes, the equilibrium can be shifted by binding effector molecules at sites remote from the protein's active site We do not yet know the rules by which nature has chosen anionic or cationic effectors as control molecules for specific enzyme forms, but it appears clear that in the case of human hemoglobin the oxygen affinity is primarily modulated by the concentration of anions in the external medium In many laboratories around the world the question of how this anionic control is achieved is under investigation This investigation began many years ago and many of the questions that faced early investigators of hemoglobin function are still facing researchers today Detailed studies of the structural and functional properties of human hemoglobin variants have provided greater insight into the mechanism by which cofactor binding is linked to oxygen binding Of particular interest in this regard are the substitutions which involve the eight positively charged residues of the 2,3-diphosphoglycerate binding site The characteristics of these hemoglobin mutants and their response to organic and inorganic anions suggest that the equilibrium between high and low affinity conformations of hemoglobin is strongly affected by the net positive charge in the central cavity Thus it appears that in normal human hemoglobin the binding of anionic cofactors directly influences the oxygen affinity by neutralizing the charged groups of the diphosphoglycerate binding site and thereby stabilizing the deoxy conformation

The specific interaction of s-100 protein with synaptosomal particulate fractions. site-site interactions among s-100 binding sites1

Abstract: — The Scatchard plot of the specific binding of the brain-specific S-100 protein to synaptosomal particulate fractions (SYN) is curvilinear, concave upwards. This could indicate the existence either of multiple classes of sites with different but fixed affinities, or of site-site interactions of the type defined as negative cooperativity among a single class of sites. To discriminate between these possibilities, the dissociation test described by De Meyts et al. (1976) for demonstrating negative cooperativity among insulin binding sites of human lymphocytes or liver membranes, was applied to the interaction of S-100 with SYN. The results show that the dissociation of the 125I-labelled S-100-site complex is faster due to an ‘infinite’(100-fold) dilution of the complex plus an excess of unlabelled S-100 than due to dilution only, the effect of unlabelled S-100 being specific and dose-dependent. 125I-IabeIIed S-100 dissociation is time, temperature, and Ca2 +-dependent. The effect of unlabelled S-100 is more evident at a low site occupancy than at a high one, suggesting that at high site occupancies 125I-labelled S-100 binding sites could be already negatively cooperating. It can be reasonably excluded that the effect of unlabelled S-100 is due to inhibition of rebinding of the dissociated tracer. Na+ and K+ stimulate the dissociation even at physiological concentrations. At low pH 125I-labelled S-100 dissociates very little, while at high pH dissociation is greatly stimulated. Finally, the protein denaturating reagent urea accelerates the dissociation even at concentrations as low as 1m. These data suggest that negative cooperativity occurs among S-100 binding sites, but do not exclude other possibilities. Together with previously reported findings, they further support the view that S-100 binds to highly specific sites in nervous membranes.

Studies of ligand binding to cholera toxin, III. Cooperativity of oligosaccharide binding.

Abstract: Binding parameters for the interaction of cholera toxin and choleragenoid with monosialo-gangliotetraose, the oligosaccharide moiety of ganglioside II3 NeuAc-GgOse4-Cer, have been measured by equilibrium displacement dialysis. The experimental data were evaluated by a curve fitting computer program. The binding curves obtained at 6 degrees C reflected positive cooperativity with average Hill coefficients in the range of 1.16 to 1.25. The cholera toxin as well as its ganglioside-binding protomer B-protein bind 4 mol of monosialogangliotetraitol per mol of protein. The presence of disulfide-cleaving agents like 2-mercaptoethanol did not eliminate the cooperativity of the binding characteristics, but increased the Hill coefficient.