Showing papers on "Cooperative binding published in 1976"

Interaction of sodium decyl sulfate with poly(L‐ornithine) and poly(L‐lysine) in aqueous solution

Abstract: The binding isotherms of sodium decyl sulfate to poly(L-ornithine), poly(D,L-ornithine), and poly(L-lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm–Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L-Lys)n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L-Orn)n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.

Cooperative properties of hormone receptors in cell membranes

Abstract: The binding of many polypeptide hormones to cell surface receptors does not appear to follow the law of mass action. While steady-state binding data are consistent in many cases with either heterogeneous populations of binding sites or interactions of the type known as negative cooperativity, study of the kinetics of dissociation of the type known as negative cooperativity, study of the kinetics of dissociation of the hormone receptor complex allows an unambiguous demonstration of cooperative interactions. Negative cooperativity, which seems to be wide-spread among hormone receptors, provides exquisite sensitivity of the cell at low hormone concentrations while buffering against acutely elevated hormone levels. The molecular mechanisms underlying the cooperativity are still largely unknown. Cooperativity may stem from a conformational transition in individual receptors or involve receptor aggregation in the fluid membrane (clustering) or more extensive membrane phenomena. Thus, new models of hormone action must be considered which integrate the progress in our knowledge of both the complex mechanisms regulating hormone binding to their surface receptors, and the dynamic properties of the cell membrane.

The Role of Negative Cooperativity and Half-of-the-Sites Reactivity in Enzyme Regulation

Abstract: Publisher Summary This chapter discusses the role of negative cooperativity and half-of-the-sites reactivity in enzyme regulation. Negative cooperativity refers to the phenomenon in multisubunit proteins in which the binding affinities of ligands decrease as a function of ligand saturation. In positive cooperativity, the affinity of the protein toward the ligand increases as a function of ligand saturation, thus leading to sigmoidal saturation curves. The chapter analyzes the phenomenon of negative cooperativity from the theoretical point of view, discusses diagnostic tests for the property, and describes some well studied regulatory enzymes. The analysis of the binding of a single ligand to a multisubunit protein is in itself a fairly complex mathematical problem. The problem becomes Herculean when more than one ligand is involved, and yet that is precisely the case for regulatory proteins that usually have more than one substrate, more than one product, several regulatory molecules, and between two and eight subunits, each of which can combine with any of these molecules.

Transition from noncooperative to cooperative and selective binding of histone H1 to DNA.

Abstract: A transition from noncooperative to cooperative binding of DNA and histone h1 occurs between 20 and 40mMNaCI in 5 mM Tris-HCI, pH 7.5. Below 20mM NaCI in mixtures in H1 and excess DNA, H1 binds to all of the DNA molecules, causing them to sediment faster, and does not distinguish between DNA molecules that differ in size or base composition. However, at NaCI concentrations above the narrow transition range, H1 binds to only some of the DNA molecules and leaves the rest free. If the DNA molecules in a mixture are the same size, H1 selectively binds those that have the highest content of adenosine (A)+thymidine (T). By means of competion experiments at salt concentrations spanning the transition range, it is demonstrated that H1 selectivity requires cooperativity. A high degree of selectivity based on A + T content can be produced by cooperative binding: for average DNA sizes of 2 X10(6) daltons, more than ten molecules of calf lymphocyte DNA (57% a+ t) are chosen per molecule of Escherichia coli DNA (50% A+T).

Equilibrium binding of magnesium(II) by Escherichia coli tRNAfMet.

Abstract: Equilibrium dialysis measurements show that tRNAfMet1 in 0.17 M Na+ has one strong Mg2+ binding site, K = 3 X 10(4) M-1, and approximately 26 weak binding sites with K = 4 X 10(2) M-1, with RNA concentration measured in moles of tRNA per liter and T = 4 degrees C. The data fit significantly less well to a model with two strong sites and a large class of weak sites. Binding is noncooperative. Our results differ from previous experiments showing cooperative binding because the binding equilibrium is not coupled to a cooperative conformational change of the macromolecule. Measurements at relatively high Na+ concentrations and low temperature ensure that the tRNA is in the "native" region of the conformational phase diagram for all Mg2+ concentrations.

Insulin Receptors in Cultured Human Fibroblasts

Abstract: In order to study human insulin resistance, we have first characterized the interaction of insulin with specific insulin receptors in cultures of normal human fibroblasts. 125 I-insulin bound rapidly to human fibroblasts in suspension at 15°, achieving steady state between one and three hours. Insulin was not degraded during the binding assays. In competitive binding experiments, 2 ng/ml. (3.3 × 10 −10 M) of unlabeled insulin reduced 125 I-insulin binding by 50 per cent. Insulin analogues competed for binding in proportion to their biologic potencies. A curvilinear Scatchard plot was obtained, suggesting the existence of negatively cooperative site-site interactions among the insulin receptors. This was confirmed directly by studies of the dissociation kinetics. The high affinity, specificity, and negative cooperativity of the fibroblast insulin receptor closely resembles the properties of other human insulin receptors. The cultuted human fibroblast should prove a useful tissue for the study of insulin-resistant states in man.

Cooperative disordering of single-stranded polynucleotides through copper crosslinking.

Abstract: The thermal transitions of single-stranded polynucleotides are noncooperative. In contrast, Cu(II) cooperatively disorders the single-stranded helical structures of poly(A) and poly(C), as demonstrated by ORD and UV spectral changes as a function of the Cu2+ activity, and by a dramatic chain-length dependence of the spectral changes. Equilibrium dialysis binding studies indicate that the cooperative disordering is paralleled by a somewhat less cooperative binding process. The difference between the thermal- and Cu(II)-induced transition is explained by the following mechanism. (1) Cu(II) initially binds in a noncooperative fashion to phosphate. (2) The Cu(II) so bound forms a second bond to a nonadjacent base site on the same polymer strand or another strand. These intramolecular and intermolecular crosslinks to the bases are responsible for the disordering. (3) The initial crosslinks formed provide nuclei for the cooperative formation of additional crosslinks, producing cooperative spectral changes paralleled by cooperative binding. A comparison of the spectral and binding transitions indicates that there is appreciable noncooperative binding of copper to phosphate, which produces no spectral changes in the presence of added electrolyte. This comparison also indicates that each copper crosslink disorders several bases. The formation of intermolecular crosslinks is demonstrated by a polymer concentration dependence of the disordering. The formation of intramolecular crosslinks can be deduced from the fact that the “cooperative unit” required to explain the differences between the hexamer, which does not readily form intramolecular crosslinks, and the polymer is considerably larger than the cooperative unit determined from the polymer results. The poly(A) disordering transition is less symmetrical than that of poly(C), particularly at low polymer concentrations. These results, together with other phenomena, are explained by a greater flexibility of poly(A), which favors the formation of small intramolecular loops.

Cooperative Binding of Concanavalin A to Thymocytes at 4°C and Micro‐redistribution of Concanavalin A Receptors

Abstract: The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.

Studies on the noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded nucleic acids.

Abstract: The noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA oligomers has been studied by means of equilibrium dialysis. Dialyses were performed under a number of solution and temperature conditions using oligomers of varying length and base compositions. The results of these studies, which include a Scatchard analysis of the binding, have allowed us to propose a model for the cooperative binding of the protein to single-stranded DNA. The results of experiments dealing with the interaction of the protein with single-stranded RNA are also presented.

Yeast Hexokinase: Substrate‐Induced Association‐ Dissociation Reactions in the Binding of Glucose to Hexokinase P‐II

Abstract: A method is described for the purification of native hexokinases P-I and P-II from yeast using preparative isoelectric focussing to separate the isozymes. The binding of glucose to hexokinase P-II, and the effect of this on the monomer-dimer association-dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation. Association constants for the monomer-dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupied in the dimer. At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme glucose complex was 3 × 10−4 mol1 −2 and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I = 0.02 mol1−1, pH < 7.5) was also independent of enzyme concentration (in the range 10–1000.μg ml−1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non-cooperative; values of the intrinsic dissociation constant varied from 2.5 × 10−3 mol 1−1 at pH 7.0 to 6 × 10−3 at pH 6.5. Under intermediate conditions (pH 7.0, ionic strength = 0.15 mol l−1), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperativity (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations. The results show that the phenomenon of half-sites reactivity observed in the binding of glucose to crystalline hexokinase P-II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.

Purine-Nucleoside Phosphorylase from Salmonella typhimurium and Escherichia coli

Abstract: Purine nucleoside phosphorylase from Salmonella typhimurium has been subjected to kinetic analysis, i.e. determination of initial velocity patterns and product inhibition studies. The kinetic results suggest that the enzyme works by a sequential reaction mechanism, where the nucleoside, phosphate, and pentose 1-phosphate are all able to bind to the free enzyme, whereas it appears that the purine base binds after addition of the pentose 1-phosphate. The proposed mechanism is confirmed by substrate binding studies. In addition to the enzymesubstrate complexes suggested by the kinetics, the binding studies revealed a ‘dead end’ complex, consisting of enzyme, phosphate, and purine base. Similar binding experiments were carried out using the enzyme from Escherichia coli. The results suggest that this enzyme works by an identical reaction mechanism. The binding data are in agreement with the presence of six binding sites per native enzyme molecule, one binding site per subunit, for each ligand. Both enzymes show normal Michaelis-Menten kinetics for their substrates with the exception of phosphate, for which the double-reciprocal plots are concave down. This behaviour is seen in both binding and velocity curves, and most likely is a result of negative cooperativity in the binding of phosphate to the enzyme.

Hydrophobic agaroses: basis for a model of multivalent effector-receptor interactions.

Abstract: An increase in the density of butyl residues bound to Sepharose 4B leads to an enhancement of the affinity of these gels for phosphorylase b in the presence of 1.1M ammonium sulfate. A Hill coefficient of 2.9 indicates that a minimum of ca. 3 binding sites is involved in the positive cooperative adsorption of this enzyme. Binding studies of phosphorylase b on butyl-Sepharose of a specific degree of substitution demonstrate that the affinity of the gel for this ligand decreases as a function of fractional saturation. A Hill coefficient of 0.44 indicates negative cooperativity as a result of multivalent binding. From these observations a multivalent, mobile receptor model is derived which can explain such characteristics of effector-receptor interactions as: positive and negative cooperativity, high binding constants and low dissociation rate constants. The application of this model to experiments taken from the literature on the binding of the multivalent effectors concanavalin A and cholera toxin to fat cells shows that the postulated mode of interactions is probably realized in nature.

Direct determination of ligand interactions with beta-adrenergic receptors on intact turkey erythrocytes: correlation of binding with biological activity.

Abstract: Previous studies on the interaction of labeled beta-adrenergic blockers with beta-adrenergic receptors have employed broken cell or membrane preparations. We have now carried out direct binding analysis on intact turkey erythrocytes employing the potent, high specific activity blocker [125I]-hydroxbenzylpindolol (HYP). [125I]HYP binds to a single class of receptor sites with a K of 5.3 X 10(10)M-1 and a binding capacity of 400-500 sites/cell. These results as well as the kinetics of association and dissociation and lack of evidence for negative cooperativity all agree well with studies reported earlier on membrane preparations from the same cells. True dissociation constants (Kd) for agonists and antagonists determined by inhibition of binding of [125I]HYP are in good agreement with results in membrane preparations. These Kd's have been compared directly with activation or inhibition constants for effects on adenylate cyclase using generation of [14C]cAMP from [14C]adenine in intact cells. The close correlation between effects on binding and adenylate cyclase activity in whole cells are similar to results obtained in membrane preparations in the presence of guanine nucleotides, suggesting the presence of an analogous regulatory substance in vivo.

Ligand interactions with the acetylcholine receptor from Torpedo californica. Extensions of the allosteric model for cooperativity to half-of-site activity.

Abstract: The solubilized acetylcholine receptor from Torpedo californica showed positive cooperativity in acetylcholine binding with a dissociation constant of 1.2 X 10(-8) M. Blockade of acetylcholine binding by nicotine was competitive; blockade by d-tubocurarine appeared to result from an allosteric interaction that altered half of the acetylcholine binding sites to a lower affinity form; decamethonium blockade displayed properties of competitive and allosteric inhibition suggesting less specificity for decamethonium binding than seen with either nicotine or d-tubocurarine. The d-tubocurarine inhibition data were evaluated by several possible models involving either differential competitive inhibition or allosteric inhibiton. The data were best described by the allosteric model.

The regulatory control of beta-receptor dependent adenylate cyclase.

Abstract: The characteristics of the β-receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the β-agonists and antagonists to the β-receptor. The regulatory ligands Gpp(NH)p and Ca2+ do not have any direct effect on the β-receptor, but modulate the enzyme activity through the interaction with specific regulatory sites. It was found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide. The regulatory guanyl nucleotide binds to its allosteric site in the absence of hormone, but the activation of the enzyme is slow in the absence of hormone. This role of the hormone can be described by the scheme: Where R is the receptor, E the enzyme, G the guanyl nucleotide, H the hormone, and E′ the activated form of the enzyme. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active form occurs with a k∼1.0 min−1 In the absence of the β-agonist (l-catecholamine) at the β-receptor and at physiological free Mg2+ concentrations, the activation of the enzyme is insignificant. Thus the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. At high (nonphysiological) Mg2+ concentration the conversion of E to E′ occurs slowly in the absence of hormone probably by another pathway. There are two classes of Gpp(NH)p regulatory sites: tight sites and loose sites, both of which can be identified kinetically. We have also identified the tight sites by direct binding studies using 3H-Gpp(NH)p. It is not clear, however, whether these are two distinct classes of sites or whether their existence reflects the presence of negative cooperativity among the guanyl nucleotide regulatory sites. Calcium was found to be a negative allosteric inhibitor of adenylate cyclase. The inhibitory effect of Ca2+ is exerted on the nonactivated enzyme as well as on the Gpp(NH)p preactivated enzyme.