Showing papers on "Cooperative binding published in 1975"
TL;DR: This chapter describes the multiple ligand binding by proteins, binding by multimer proteins, extension of the concept of ligand interaction to covalent bond exchange, cooperativity and ligand correlation, and biological specificity andligand binding.
Abstract: Publisher Summary The interaction of proteins with small ligands embodies the fundamental physicochemical principles that are operative in the physiological regulation of enzyme activity, the specific interactions among proteins and of proteins with nucleic acids and other macromolecules. This chapter describes the multiple ligand binding by proteins, binding by multimer proteins, extension of the concept of ligand interaction to covalent bond exchange, cooperativity and ligand correlation, and biological specificity and ligand binding. Many parts of the compact protein structure can contribute toward the shift in energy and structure taking place on ligand binding. The energies of ligand–ligand interaction, although small are sufficient to shift the covalent equilibria in which proteins take part, to a significant extent, and this may be found responsible for the interconversion of chemical and osmotic energies in metabolism. While, the ligand correlation because of the existence of free-energy coupling among the bound ligands is a molecular property to be explained by a study of the isolated protein, the enhancement of the effects by the simultaneous changes in ligand concentration is a system property that requires consideration of other entities. The consideration of ligand–protein interactions provides with a model for the relative importance to be assigned to molecules in the organic functions. Thus, study of the interactions of proteins and small ligands provides the basis for the understanding of biological specificity at the molecular level.
486 citations
TL;DR: The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4, and results were inconclusive concerning whether premicellar association of unbound fatty acid occurs.
272 citations
TL;DR: Study of interactions of 125 I-insulin with non-tissue materials are presented as examples of non- receptor (“nonspecific”) interactions which share, at least superficially, the criteria commonly attributed to “specific” hormone-receptor interactions.
201 citations
TL;DR: It is evident that the binding of T4 represents a case of negative cooperativity which is presumably due to interaction between ligands.
Abstract: The binding of thyroxine (T4) and 8-anilino-1-naphthalenesulfonic acid (ANS) to human serum prealbumin was measured by equilibrium dialysis at pH 7.4 in 0.05 M phosphate-0.10 M NaCl at 25 degrees. The data were analyzed for the binding constants based on equations for (1) two independent sites and (2) two identical sites with negative interaction. Evaluation by the independent site model gave the following association constants: for T4 binding, KT1 = 1.0 x 10-8 M-1, KT2 = 9.5 x 10-5 M-1; for ANS binding, KA1 = 9.5 x 10-5 M-1, KA2 = 2.1 x 10-5 M-1. The interactive model gave constants kT = 5.5 x 10-7 M-1 and kA = 5.5 x 10-5 M-1. Interaction factors, alpha, defined such that -RT in alpha is the energy of interaction, were: alpha T = 0.041 AND ALPHA A = 0.62 for T4 and ANS, respectively. The "best fit" values for the number of sites were 2.0 and 1.6 for T4 and ANS, respectively. The binding of T4 to human prealbumin was competitive with ANS, and the binding constants evaluated from competition experiments were in agreement with those found for each ligand when studied separately. On the basis of analysis of X-ray data of human prealbumin (Blake et al.) there appear to be two identical T4 sites. It is therefore evident that the binding of T4 represents a case of negative cooperativity which is presumably due to interaction between ligands.
196 citations
TL;DR: The presence of (−) alprenolol increases the rate of [ 3 H] (−)alprenlol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.
183 citations
TL;DR: Data suggest that the heterogeneity in thyrotropin binding constants which is evident in the Scatchard plot reflects a negatively cooperative relationship among the thyrotopin receptor sites, i.e. decreased hormonal affinity as hormone concentrations increase.
116 citations
TL;DR: A mechanism based on interacting sites is proposed that could account for changes in binding energy at one site may be coupled with catalysis at the other to give large rate enhancements in the acylation of tRNA.
Abstract: The dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus which binds (tightly) only one tyrosyl adenylate or tyrosine per dimer is shown from kinetic, equilibrium dialysis, and gel filtration methods to have a second active site. ATP and tyrosine bind strongly and synergistically to the tyrosyl-tRNA synthetase tyrosyl adenylate complex, [E with Tyr similar to AMP], to give the complex [E with Tyr similar to AMP,ATP,Tyr]. This complex probably slowly forms an [E with (Tyr similar to AMP)2] complex which hydrolyses rapidly and does not accumulate. Similarly, the monomeric valyl-enzyme is shown to have two active sites. An [E with Val similar AMP,ATP,Val] complex is formed which probably slowly gives an unstable [E with Val similar AMP)2] complex. In view of this and the recent demonstrations that several aminoacyl-tRNA synthetases are composed of repeating sequences it is suggested that all of these enzymes have at least two active sites. The second site is difficult to detect by normal steady-state kinetic measurements and binding assays as these enzymes exhibit negative cooperativity of substrate binding hand half-of-the sites reactivity. A mechanism based on interacting sites is proposed that could account for these observations: changes in binding energy at one site may be coupled with catalysis at the other to give large rate enhancements. Howeever, this cannot account for the high specificity in the acylation of tRNA, A "VERIFICATION" PROCEDURE SEEMS ESSENTIAL. The proposed mechanism is quite general for catalysis and could be a reason why so many nonregulatory enzymes have subunits.
115 citations
TL;DR: High-affinity Ca2+ binding did not increase actin-activated ATPase activity in the absence of regulatory proteins, but rather inhibited it, and Mg2+ alters Ca2-binding in a complex manner.
97 citations
TL;DR: Separate Ca 2+ binding determinations on each of the two isolated chains of Factor X suggest that the Ca 2+, binding capacity of this protein is a property of the smaller, light polypeptide chain.
91 citations
TL;DR: The morphological observations on ferritin--insulin binding supported the theory that insulin did not need to enter a cell to cause biological effects and was consistent with the negative cooperativity concept of insulin binding to cell receptors.
Abstract: The method for preparing a stable, biologically active, covalently linked ferritin--insulin complex has been modified to provide a 25-fold increase in yield compared to the original procedure while reducing the molar fatio of ferritin to insulin to 1:1 from 40:1. Ultrastructural studies of isolated adipocytes revealed specific binding of ferritin--insulin to the cell surface in irregular clusters associated with the glycocalyx coating. The number of ferritin--insulin molecules observed was consistent with the number of sulin molecules observed was consistent with the number of receptors calculated from 125I-labeled insulin binding studies. The ferritin--insulin was not observed in the cytoplasm of the cell but was found on the convave side of surface connected vesicles. These surface connected vesicles were part of an alveolar-like system of plasma membrane invaginations which project in various directions in the cytoplasm and by thin sectioning can appear as pinocytotic-like microvesicles. The morphological observations on ferritin--insulin binding were supported by the finding that 125I-labeled insulin binding was almost exclusively localized to highly purified plasma membranes isolated by fractionation of adipocytes after incubation with 125I-labeled insulin. These data supported the theory that insulin did not need to enter a cell to cause biological effects and was consistent with the negative cooperativity concept of insulin binding to cell receptors.
78 citations
TL;DR: Studied under elevated salt conditions have shown that the average DNA protein complex length is 50% of the length found at 0.04 or 0.15 M NaCl.
Abstract: The cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA has been studied by electron microscopy. Analysis of the electron microscopic data by means of a simple statistical mechanical model has yielded a value of 3.8-7.6 X 10(10) l./mol for the cooperative binding constant in 0.15 M NaCl. Studied under elevated salt conditions have shown that the average DNA protein complex length is 50% of the length found at 0.04 or 0.15 M NaCl.
TL;DR: The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites which alters the homotropic interaction between fructose 6-phosphate binding sites.
Abstract: Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.
TL;DR: It is concluded from X-ray diffraction studies at low resolution that the binding of sugar and nucleotide substrates to dimeric yeast hexokinase BII crystals exhibits both negative co-operative and positive allosteric co-operativity.
TL;DR: The theory of interactions between isolated ligands is extended to cases of multiple interactions, and the experimental results are tolerably well reproduced for a model in which four anilinonaphthalensulfonate molecules are homogeneously coupled to four molecules of dihydroxybenzoate by free energies of 3.0 and 3.5 thermal units.
Abstract: When a ligand X is multiply bound to energetically identical, noninteracting sites of a protein, cooperative binding of this ligand can be induced by the presence of a second ligand Y. This effect should appear whenever multiple interactions exist between the bound X and Y ligands, and vanish when the concentration of Y is made sufficiently large to ensure Y saturation at all concentrations of X. These predictions have been verified for the binding of 8-anilino-1-naphthalenesulfonate to serum albumin, when Y, the effector ion, is 3,5-dihydroxybenzoate. In the presence of 2mM dihydroxybenzoate, the Hill coefficient for anilinonaphthalenesulfonate binding rose steadily from 1 to 1.5 as the number of molecules of ligand bound increased from 1 to 3.3 per albumin molecule. The theory of interactions between isolated ligands, applied in the previous paper (D. A. Kolb and G. Weber (1975), Biochemistry, preceding paper in this issue), is extended to cases of multiple interactions, and applied here to show that the experimental results are tolerably well reproduced for a model in which four anilinonaphthalensulfonate molecules are homogeneously coupled to four molecules of dihydroxybenzoate by free energies of 3.0 and 3.5 thermal units.
TL;DR: Using fluoresceinylthiocarbamoyl-tRNATyr, it could be demonstrated that the binding of two tRNA-molecules is anti-cooperative: the binding sites for tRNA on the synthetase are a priori identical, but when one tRNA is bound, thebinding of the second t RNA is disfavoured.
Abstract: The kinetics of the interaction of tRNATyr and tyrosyl-tRNA synthetase from Escherichia coli have been investigated by stopped-flow experiments. The association rate constants for the formation of the 1:1 and 2:1 complex as well as the rate constants of the dissociation of these complexes have been determined. The kr-values are 2.2 · 108 and 1.4 · 108 M−1 s−1, the kd-values are 1.5 and 53 s−1, measured in 0.03 M potassium phosphate buffer pH 6.
A fluorescent derivative of tRNATyr was prepared by reaction of tRNATyr with fluoresceinisothiocyanate. This labelled tRNA is fully active in the aminoacylation assay; regarding the interaction with the synthetase it shows identical binding parameters as unmodified tRNATyr.
Using fluoresceinylthiocarbamoyl-tRNATyr it could be demonstrated that the binding of two tRNA-molecules is anti-cooperative: the binding sites for tRNA on the synthetase are a priori identical, but when one tRNA is bound, the binding of the second tRNA is disfavoured. In the 2:1 complex, however, the tRNAs are again indistinguishable.
TL;DR: Dissociation experiments established that insulin induced negatively cooperative interactions among JHC-1 insulin receptors, as previously reported in other tissues, providing further support for the concept that negative cooperativity may be a fundamental feature of the interaction of insulin with its receptors.
TL;DR: The spontaneous inactivation of yeast glyceraldehyde-3-phosphate dehydrogenase was found to fit a simple two-state model at pH 8.5 and 25 degrees and methods for distinguishing negatively cooperative binding patterns from mixtures of denatured enzyme and multiple species are discussed.
Abstract: The spontaneous inactivation of yeast glyceraldehyde-3-phosphate dehydrogenase was found to fit a simple two-state model at pH 8.5 and 25 degrees. The first step is a relatively rapid dissociation of the tetramer to dimers with the equilibrium largely in favor of the tetramer. In the absence of NAD+ the dimer inactivates irreversibly. The apoenzyme is quite stable with a half-life for complete activity loss proportional to the square root of the enzyme concentration. Perturbances of the protein structure (by pH, ionic strength, and specific salts), which have no effect on the tetrameric state of the molecule, result in an alteration of the cooperativity of NAD+ binding, the reactivity of the active-site sulfhydryl group, and the catalytic activity of the enzyme. Covalent modification of two of the four active-site sulfhydryl groups has profound effects on the enzymic activity which are mediated by changes in the subunit interactions. Sedimentation analysis and hybridization studies indicate that the interaction between subunits remains strong after covalent modification. Under normal physiological and equilibrium dialysis conditions the protein is a tetramer. Equilibrium dialysis studies of NAD+ binding to the enzyme at pH 8.5 and 25 degrees reveal a mixed cooperativity pattern. A model consistent with these observations and the observed half-of-the-sites reactivity is that of ligand induced sequential conformational changes which are transferred across strongly interacting subunit domains. Methods for distinguishing negatively cooperative binding patterns from mixtures of denatured enzyme and multiple species are discussed.
TL;DR: Equilibrium and covalent binding studies of succinic thiokinase from Escherichia coli indicates that there can be a stoichiometric relationship between coenzyme A binding and the phosphoyrlation capacity of the enzyme.
TL;DR: Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination.
Abstract: Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.
TL;DR: In this article, the authors focused on determination of v and of binding isotherms, in conjunction with changes in cell morphologies and in hypotonic shock behavior as v is varied, and found that the intact erythrocyte engages in two kinds of binding mechanisms, statistical binding and cooperative binding, depending on the compound.
TL;DR: A pro-equilibrium sequential pathway model involving Mn2+-enzyme and the Mn2-fructose 1,6-bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations.
TL;DR: Using a three-dimensional model of the insulin molecule areas of the protein molecule where binding of glucose can occur were selected and the glucose-binding site very probably involves the area at the insulin surface where most of the invariant and modification-selective residues are present.
TL;DR: It is concluded that the modulation of threonine deaminase activity is wrought by interaction between inhibitor sites and an activator site rather than inhibitor and active sites and that induced transitions rather than concerted transitions more adequately describe the underlying regulatory principle.
TL;DR: It is suggested, by analogy with haemoglobin, that the molecular basis of the negative cooperativity may be the creation of additional intersubunit bonds during the binding of the first two NAD molecules to the tetramer, and a change from a "relaxed" quaternary structure to a "tense" structure at half-saturation.
TL;DR: Sedimentation equilibrium and fluorescence titration data have been analyzed in terms of a monomer-dimer-tetramer model in which the various oligomers have different affinities and/or capacities for the ligand.
Abstract: Human chorionic gonadotropin (hCG) self-associates to form higher molecular weight species in the presence of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). Sedimentation equilibrium and fluorescence titration data have been analyzed in terms of a monomer-dimer-tetramer model in which the various oligomers have different affinities and/or capacities for the ligand. The results indicate that the ligand affinities are in the order tetramer greater than dimer greater than monomer whereas the numbers of ligand binding sites per mole of hCH are in the reverse order. Consequently, addition of ANS first shifts the equilibrium from monomer to tetramer and gives rise to positive cooperativity in the titration curves. At sufficiently high ANS concentration (approximately 0.5 mM), the equilibrium shifts back to the dimer because of its greater binding capacity. This is manifested by a second phase in the titration curve and a decrease in the polarization of ANS fluorescence. The results are discussed in terms of the general problem of ligand controlled protein association and are contrasted to results reported to the previous paper for the homolgous protein, human luteinizing hormone.
TL;DR: Light scattering measurements show a strong aggregation of the polymer-dye complexes depending primarily on the degree of binding to be caused by a spectral flattening as well as an enhanced binding of the dye which both result in a decrease of its absorbance.
TL;DR: The theoretical study of the cooperative binding of a small ligand to a linear homopolymer is extended to systems in which two different complexes can form.
Abstract: The theoretical study of the cooperative binding of a small ligand to a linear homopolymer is extended to systems in which two different complexes can form. The binding isotherms are derived under the assumption that the cooperative interactions exist only between molecules belonging to the same type of binding mode and are limited to nearest neighbors (Ising model). The binding to a single-stranded chain is first considered and two extreme cases are studied: (1) the two complexes can form independently from each other (model of independent classes of binding sites); (2) only one class of binding site exists, each possessing two different states of complexation (three-state model).
Binding to a double-helical chain is also considered. Three simple types of competition between the different modes of binding are distinguished. The corresponding models are defined as: (1) the model of independent classes of binding sites; (2) the model of monoexclusive interactions between the different kinds of complexes (the symmetric and asymmetric cases are both considered); (3) the model of biexclusive interactions. The comparative study of the different cases shows that the binding isotherms are very similar at large polymer-to-ligand concentration ratios, while they can be very different at low polymer-to-ligand ratios. This can be used to obtain information on the mechanism of dye binding to nucleic acids by equilibrium studies as shown in a subsequent paper.
TL;DR: It is shown that, for a self-associating protein system in which monomer and dimer have different affinities and/or capacities for ligand, both the shape and the position of the binding curve depend on protein concentration.
Abstract: The self-association of human luteinizing hormone (hLH) is enhanced in the presence of 8-anilino-1-naphthalenesulfonate (ANS). Sedimentation equilibrium measurements indicate that the hormone exists primarily as a dimer in the presence of excess ANS. It is shown that, for a self-associating protein system in which monomer and dimer have different affinities and/or capacities for ligand, both the shape and the position of the binding curve depend on protein concentration. Gel filtration and fluorescence measurements indicate that the hLH dimer has a single high affintiy site (K greater than 10(6) M-1) for ANS while binding to the monomer is too weak to be observed. This leads to negative cooperativity in the binding and to a shift of the binding curve to lower free ligand concentration with increasing concentration of the hormone.
TL;DR: Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors, andomalous features of the behaviour suggest that the model should be regarded only as a first approximation.
Abstract: Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation.