Showing papers on "Cooperative binding published in 1977"

The monocyte as a model for the study of insulin receptors in man.

Abstract: We have characterized the cellular composition of preparations isolated from peripheral blood by Ficoll-Isopaque gradient centrifugation.125I-insulin binding to every cell type was measured. A highly significantly positive correlation between specific cell binding fraction and the monocyte concentration of the heterogeneous cell suspension was demonstrated. Depletion of monocytes reduced the insulin binding approximately 80%, which confirms previous findings by other investigators. The granulocytes possessed the second highest binding ability, but only one fourteenth of that of monocytes. Compared to the lymphocyte the monocyte had about 25 times greater insulin binding. Also thrombocytes bound insulin and contamination with these meant that their contribution to the total specific cell binding was not negligible. A reduction in these contaminants is essential. We found that insulin binding to erythrocytes was insignificant. A method of calculating the specific insulin binding to monocytes alone is introduced. The monocyte-insulin-receptor possesses specificity. Only an insignificant degradation of receptor bound insulin could be shown. Evidence of negative cooperativity between receptors was found. Consequently monocytes are considered a useful model for insulin receptor studies in man.

The Effect of Mg2+ on the Ca2+‐Binding Properties of Non‐activated Phosphorylase Kinase

Abstract: The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.

Electrostatic effects in divalent ion binding to tRNA.

Abstract: The binding of Mn2+ to yeast tRNAPhe at 25°C is measured by esr, and found to depend strongly on the concentration of monovalent cations, showing the importance of electrostatic effects. In low sodium ( 50mM/l.), the affinity and the curvature are reduced. In a limited range of sodium concentrations (15–30mM/l.), the folding of tRNA which is induced by the divalent ions results in cooperative binding, leading to upwards convexity of the Scatchard plot. An electrostatic model is developed, based on a single type of binding site which we take to be the phosphates, with a binding constant for Mn2+ in the range of that found for ApA, 10 l./M. We compute the change in the binding constant due to the electrostatic potential of the distant charges (other phosphates and counterions), using a single set of parameters for all sodium concentrations. The model predicts that the plots in low sodium are curved, and a good fit to the experimental results is obtained: it is therefore not legitimate or necessary to interpret these results in terms of two types of binding sites. In high salt, the model gives plots that are only slightly curved, corresponding to weaker electrostatic effects. This shows that a search for sites with a special binding mode should be done in high salt. The computed plots are in good agreement with the data, except for slight differences concerning the first bound ions, which give a possible indication in favor of special binding. Given the observation of one special site for Mg2+ at 4°C in high sodium [Stein, A. & Crothers, D. M. (1976) Biochemistry15, 157–160] in E. coli tRNAfMet, we have measured the binding of Mn2+ at lower temperature. At 12°C, in both yeast tRNAPhe and E. coli tRNAfMet, the plots clearly indicate special binding. A site found in high sodium is on a very different footing from the four to six so-called strong sites unduly derived from low-salt binding plots.

Selective loss of wheat germ agglutinin binding to agglutinin-resistant mutants of Chinese hamster ovary cells.

Abstract: The binding of 125I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 10(6)-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was complex and exhibited positively cooperative binding at the high affinity sites. One of the WGA-resistant mutants (WgaRIII) was apparently not altered in its WGA-binding ability compared with parental cells. However, two of the WGA-resistant lines (WgaRI and WgaRII) had distinct alterations in their WGA-binding properties specific to certain regions of the binding curve. Neither appeared to be affected in either the highest or lowest affinity regions of the binding curve. Thus, lectin-resistant cell mutants altered in specific lectin-binding sites at the cell surface provide a direct approach to analysis of the complex binding parameters that characterize the interaction of WGA with the plasma membrane.

Non-equivalence of the sites of yeast phenylalanyl-tRNA synthetase during catalysis.

Abstract: Yeast phenylalanyl-tRNA synthetase, an enzyme with an α2β2 structure, has two active sites for phenylalanine, tRNAPhe, phenylalanyladenylate and phenylalanyl-tRNAPhe. Determination of phenylalanine binding properties to the free enzyme by equilibrium dialysis shows that only one mole of amino acid binds per mole of enzyme, i.e. absolute negative cooperativity. Binding of the amino acid in the presence of tRNA or of ATP and PPi unmasks the second phenylalanine binding site. The difference between the affinities at the tight and loose binding sites under such conditions is about 10–15. Titration of phenylalanyladenylate sites by the burst of ATP consumption shows the formation of a (enzyme-phenylalanyladenylate)2 complex in the presence of pyrophosphatase; however, the two sites differ widely in their affinity as shown by dialysis experiments. Measurements of hydrolysis rates of enzyme-bound phenylalanyladenylate suggests that when only the high-affinity adenylate site is occupied, the other protomer can still bind phenylalanine and ATP (in the presence of phenylalanine). Two moles of Phe-tRNAPhe bind to the enzyme with a very high affinity (Kd < 48 nM). The presence of millimolar concentrations of ATP, phenylalanine and pyrophosphate triggers negative cooperativity and under these conditions only one mole of Phe-tRNAPhe is bound per mole of enzyme with a Kd value of 0.15 μM. The present results give support to interprotomer catalytic cooperativity in the mechanism of action of yeast phenylalanyl-tRNA synthetase.

Binding Site of Human IgG Subclasses and Their Domains for Fc Receptors of Activated Murine T Cells

Abstract: A subpopulation of activated murine T cells (25%) were found to bear Fc receptors for mouse, rabbit, and human IgG. Studies, by rosette inhibition and indirect immunofluorescence, of their specificity for human IgG subclasses have shown that these receptors bound nonaggregated IgG1, IgG2, and IgG3 in a comparable fashion, but did not bind IgG4. The binding site has been localized exclusively to the Fc region and the binding capacity of this fragment was not affected by mild reduction. Binding of the Fc fragment of IgG4 could not be demonstrated. Using isolated Cγ2 and Cγ3 (pFc′) domains from IgG1 and a Cγ3-derived fragment (Fc′), a major binding site was localized to the Cγ3 domain between residues Gln 342 and His 433 . A 10-fold molar excess of Cγ3 dimer over native Fc was, however, required to show comparable activity. The monomeric Cγ2 domain was 10-fold less active than the Cγ3 fragment. Human urine β2-microglobulin was not cytophilic. These data are discussed and it is suggested that the Cγ2 and Cγ3 domains may contribute to the formation of a cooperative binding site through quaternary interdomain interactions. By redistribution experiments, it was demonstrated that the three human IgG subclasses shown to be cytophilic share common or linked receptors, which were not associated with the H-2 alloantigens.

Opiate receptor: cooperativity of binding observed in brain slices

Abstract: Kinetic constants for binding of 3H-labeled morphine and naloxone were determined from Hill plot analyses and from experiments in which the concentration of tritiated drug was constant and that of nonlabeled drug varied. With brain slices, the binding of either drug exhibited strong positive cooperativity (Hill slope greater than 3); this was not observed in brain homogenates. Thus, in slices the relationship between opiate binding site and ligand may be more relevant physiologically and pharmacologically.

Characteristics of thyrotropin binding to bovine thyroid plasma membranes and the influence of human IgG.

Abstract: The characteristics of binding of 125I-labeled bovine thyrotropin to bovine thyroid plasma membranes were studied. At pH 7.5, 0 C, specific binding was maximal in 3 hr and was progressively inhibited by Na+, Mg++, Ca++ and (NH4)2 SO4 in concentrations of 25 mM and greater. Affinity constants of 1.047 × 108M−1 and 0.57 × 106M−1 were obtained for two binding sites and Hill plots provided a mean slone of 0.86, suggesting negative cooperativity. Addition of 1-thyroxine or 1-triiodothyronine at 0.5 ng to 50 ug per ml had no significant effect but propylthiouracil (10−7 to 10−5M) and KC104 (10−7 to 10−4M) had biphasic effects, first enhancing and then inhibiting binding. Normal human serum IgG, particularly two fractions (3 and 4) obtained by chromatography on columns of cellulose phosphate, and an α-globulin - rich fraction from chromatography of serum proteins on diethylaminoethyl cellulose, inhibited binding at concentrations of 0.5 mg per ml or greater. Thus two possibilities are that normal human serum glo...

Energetics of the cooperative and noncooperative binding of nicotinamide adenine dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase at pH 6.5 and pH 8.5. Equilibrium and calorimetric analysis over a range of temperature.

Abstract: The binding of nicotinamide adenine dinucleotide (NAD+) to yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) has been studied at pH 6.5 and 8.5, at 5,25, and 40 degrees C, by calorimetry, fluorometry, spectrophotometry, equilibrium dialysis, and flow dialysis. As reported earlier for pH 7.3 (Velick S.F., Baggott, J.P., and Sturtevant, J.M. (1971), Biochemistry 10, 779), the binding is accompanied by enthalpy changes which become rapidly more negative as the temperature increases, with delta Cp = -500 to -750 cal deg-1 (mole of NAD+ bound)-1, and by entropy changes which also, as required by the large negative delta Cp, become rapidly more negative with increasing temperature. The binding data at pH 6.5 can be fitted on the basis of either four identical noninteracting sites, or of four sites showing a small degree of negative cooperativity. The data at pH 8.5, particularly at 40 degrees C, require the introduction of positive cooperativity, as was previously shown by Kirschner et al. (Kirschner, K., Eigen, M., Bittman, R., and Voigt, B. (1966), Proc. Natl. Acad. Sci. U.S.A. 56, 1661), and can be equally well fitted on the basis of a sequential model (Adair, G.S. (1925), J. Biol. Chem. 63, 529) or a concerted model (Monod, J., Wyman, J., and Changeux, J.P. (1965), J. Mol. Biol. 12, 88). It is proposed that the observed thermodynamic changes are largely the result of a hydrophobic effect due to a decrease in the exposure of nonpolar groups to the solvent, and of a tightening of the protein structure when the coenzyme is bound with concomitant decrease in the number of easily excitable internal degrees of freedom.

Binding studies of a spin-labelled oxidized coenzyme to bovine-liver glutamate dehydrogenase.

Abstract: NAD+ with a nitroxide piperidine ring linked to the NH2 group of the adenine possesses full coenzymatic activity with glutamate dehydrogenase. Electron spin resonance spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin-label. Binding studies in phosphate buffer demonstrate: (a) weak binary binding to the enzyme with a dissociation constant in the order of 2mM;(b) an indication for negative cooperativity or different sites for binding to enzyme-2-oxoglutarate, with dissociation constants in the order of 20--250muM; (c) similar but much weaker binding to enzyme-2-oxoglutarate-ADP; (d) a strong positive cooperative binding to enzyme-2-oxoglutarate-GTP, dependent on the enzyme concentration. Binding of phosphate to the enzyme with a Kd of about 20 mM or binding of pyrophosphate or tripolyphosphate with a Dd of about 2.5 mM enhances the binding of spin-labelled NAD+ in the presence of 2-oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.

Thermodynamics of mucopolysaccharide-dye binding. II. Binding constant and cooperativity parameters of acridine orange-dermatan sulfate system.

Abstract: In the acridine orange–dermatan sulfate system, free and bound dye can be distinguished from each other spectroscopically. This permits the use of fluorometric methods to study the binding of acridine orange to the acid mucopolysaccharide dermatan sulfate. Experiments were conducted at 24°C in 10−3M citrate/phosphate buffer at pH = 7.0. The binding of the dye is highly cooperative, as evidenced by considerable interaction between adjacent bound dye molecules. Analysis of the data indicates that dermatan sulfate binds 2.3 ± 0.3 mol of acridine orange per dermatan sulfate uronic acid residue with a cooperative binding constant, Kq ranging from 4.9 to 6.0 × 105M−1 which corresponds to a free energy of 7.74 ⩽ ΔG° ⩽ 7.86. The cooperativity parameter q apparently increases with increasing polymer-to-dye ratio.

Hapten-induced allosteric transition in the light chain dimer of an immunoglobulin.

Abstract: THE dimer of immunoglobulin light chains (L2) was shown by X-ray crystallography to be structurally homologous to the native immunoglobulin Fab fragment (HL) (refs 1–3). This led to the suggestion that L2 is a model for a primitive antibody1 and for the T-cell receptor22,23. In L2 315 (from the mouse myeloma protein MOPC 215) functional homology was also implicated in that both L2 and HL bind the same nitroaromatic haptens with similar fine specificity4,5. We have considered the possibility that this homology extends also to hapten-induced conformational changes and that studies with L2 will yield information on the nature of such processes in immunoglobulins. L2 315 constitutes a particularly good model system for such studies as it binds two hapten molecules per dimer at symmetry-related sites4,5. It is thus possible to monitor the conformational effects of the first binding hapten through the mode of interaction with the second, as revealed by the shape of the saturation curve6. We report here that dinitrophenyl-lysine (DNPL) and its fluorescent analogue nitrobenzoxadiazole-alanine (NBDA) (ref. 7) both display sigmoidal binding curves, implying positive cooperativity in their interaction with L2 315. This observed cooperativity is shown to arise from a hapten-induced conformational transition which may be described by the allosteric model6. We propose that this allosteric transition involves changes in the relative position of the chains in L2, lending support to suggestions8,9 that a similar process occurs in the structurally homologous intact immunoglobulin. These findings are, therefore, relevant to the question of antigen-triggered effector functions of antibodies through an allosteric mechanism8,10–12.

The anatomy of cooperative binding between protamines and DNA.

Abstract: DNA was titrated with protamine under equilibrium conditions while binding was simultaneously followed by electron microscopy and the fluorescamine technique. Positive cooperativity concluded from the binding assays was shown to be correlated with an aligned association of DNA fibres. Subsequent structural modifications, dependent upon the protamine/DNA ratio, suggests a mechanism for DNA compaction by protamines. In the framework of this model, protamine phosphorylation appears to promote formation of DNA interstrand links yielding a very pronounced filigree structure.

Control of Density and Microredistribution of Concanavalin‐A Receptors in Rat Thymocytes at 4°C

Abstract: In a previous paper, we reported that at 4°C, a cooperative binding of concanavalin A to rat thymocytes is observed which corresponds to a modification of the membrane leading to the recruitment of receptors and their immobilization. In this paper, we report that both phenomena are modulated at 4°C by drugs such as colchicine and cytochalasin B; cooperative binding of concanavalin A, which reflects receptor recruitment is only slightly modified by each drug alone. When the two drugs are used simultaneously, the binding of concanavalin A to rat thymocytes at low concentrations of the lectin is decreased, while at high concentrations it remains unchanged. The binding of succinyl-concanavalin A to drug-treated cells is lowered at all concentrations of lectin. Also, we have studied the effects of colchicine and cytochalacine B on the binding of horseradish peroxidase to cell-bound concanavalin A, or succinyl-concanavalin A. We have found a decreased amount of horseradish peroxidase binding to concanavalin A bound to cells treated with colchicine or cytochalasine B. In the presence of the two drugs the decrease of peroxidase binding suggested a synergistic action of colchicine and cytochalasin B.

Dual-mode cooperative binding of adenosine 5'-triphosphate to poly(L-lysine)

Abstract: Equilibrium dialysis of adenosine triphosphate interacting with polylysine at low polymer concentration yield Scatchard exhibiting an apparent non-cooperative binding mode superimposed on a positively cooperative one. The same behavior has been reported in the literature for various mononucleotides and basic poly(amino acids). It is pointed out here that both modes must not be considered as independent but to be mutually exclusive. Applying a pertinent theoretical approach yields higher degrees of cooperativity than obtained for mutual independence. Another necessary modification in the analysis of data takes into account that one ligand interacts with more than one equivalent binding contact (i.e., an elementary charge here). This leads to a further enhancement of the actual cooperativity. Increasing the polymer concentration in the binding experiments reveals a cooperative effect also in the first binding mode. This is physically interpreted and theoretically analyzed in terms of bound dimers stabilized by base stacking.

Distinctions between the two-state and sequential models for cooperative ligand binding.

Abstract: The two-state and sequential models for positive cooperativity in ligand binding can produce significantly different theoretical binding curves when presented in a Scatchard plot. The conditions that produce the greatest differences have been examined. The theoretical differences have been used to select the two-state model as the best model for describing the binding of acetylcholine to acetylcholine receptors that have been solubilized by Triton X-100 and sodium cholate.

The interactive binding of two ligands by an allosteric protein.

Abstract: The equilibrium constants describing the simultaneous binding of the ligands AMP and 1-anilinonapthalene-8-sulfonate (ansyl) by phosphorylase b have been evaluated. The binding of a single molecule of AMP per dimer unit causes only a moderate reduction in the binding constant for a single molecule of ansyl, but reduces that for a second molecule of ansyl by 10-fold. The binding of two molecules of AMP per dimer unit almost abolishes the binding of a single molecule of ansyl.

Analytical and graphical examination of strong binding by half-of-sites in proteins: illustration with aspartate transcarbamylase.

Abstract: In multiple binding of ligands to a protein, the binding sites may seem to behave as if they are partitioned equally between two modalities. This paper analyzes three different molecular situations in which two actual assemblages appear: (i) two classes of sites exist at the outset in the ligand-free macromolecule; (ii) all sites are initially identical but after half are occupied, the affinity of the residual ones is altered; (iii) all sites are initially identical but they interact in a pairwise manner. The contours of affinity profiles—graphs of normalized stoichiometric binding constants (iKi) versus stoichiometric step number i—are examined for each situation to provide a basis for discriminating among them. Proper procedures for evaluating the site binding constants are then described. To illustrate these procedures, published experimental data for two real systems, binding of substrate or modifier by the enzyme aspartate transcarbamylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2), are scrutinized and the meaning of the calculated binding parameters is examined. The results demonstrate concretely that site binding constants cannot be specified without assuming a particular molecular model, but the stoichiometric constants can be assigned unambiguously without regard to the type of behavior at the individual sites.

An effect of enzyme and ligand concentration on the state of aggregation of aspartate transcarbamylase of E. coli: I. The binding of CTP and ATP to the enzyme

Abstract: Detailed binding studies of the inhibitor, cytidine triphosphate (CTP), to native Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) reveal significant changes in subunit interaction when enzyme concentration is altered. In contrast, similar binding studies of the activator, adenosine triphosphate (ATP), do not reveal such changes, but do indicate more complex subunit interactions than previously reported. Equilibrium dialysis studies at 4 °C are consistent with six binding sites for CTP and ATP per enzyme molecule of molecular weight 310 000, at all enzyme concentrations. CTP binding studies reveal a progressive change from apparent positive to negative cooperativity as the enzyme concentration is decreased. ATP binding studies reveal complex subunit interactions involving a mixture of apparent negative and positive cooperativity. Sucrose gradient studies indicate the presence of at least three enzymatically active polymeric forms of the enzyme. The preliminary sedimentation studies indicate possib...

A Kinetic Study of the Cooperative Binding of Toluidine Blue to Poly(acrylic acid)

Abstract: Kinetic studies have been carried out on the cooperative binding of toluidine blue to poly(acrylic acid) by the electric-field pulse method in order to compare the binding property with that for poly(α-L-glutamic acid). Based on the results of our experiments in the coupling of the aggregation process of the dye to the nucleation process, we were able to arrive at a mechanism which we introduce in this paper. The association rate constant of the dye aggregation process was found to be about one order of magnitude smaller than that for the poly(α-L-grutamic acid). The reason for this is the difference in the polymer structures following hydration. Also the effect of the degree of ionization of a polymer on the various properties of the binding of a dye was also examined. It was found consequently that the bound dyes became more stable and compact with an increase in the degree of ionization.

Origins of specificity in the binding of small molecules to dihydrofolate reductase.

Abstract: Dihydrofolate reductase is the target for the therapeutically important 'anti-folate' drugs such as methotrexate and trimethoprim. Methotrexate is a powerful inhibitor of the enzyme, binding up to 10,000 times more tightly than the structurally similar substrate, folate. Two contributions to this striking difference in affinity have been identified: the two ligands bind in different charge states, and there are conformational differences between the two complexes. The origins of the tight binding of methotrexate have been explored further by studying the binding of 2,4-diaminopyrimidine and p-aminobenzoyl-L-glutamate, which may be considered as 'fragments' of methotrexate. These two compounds bind simultaneously but also cooperatively, the binding of one 'fragment' leading to a 50-fold increase in the affinity for the other. Studies of structural analogues of these fragments show that the specificity as well as the strength of binding can be altered by the presence of the other 'fragment'; both positive and negative cooperativity are observed. The relation of these observations to methotrexate binding, and the notion of intramolecular cooperativity in ligand binding are discussed.