Showing papers on "Cooperative binding published in 1986"
TL;DR: The binding of factor VII and tissue factor produces a membrane-associated proteolytic complex which may be the primary biological initiator of coagulation and an explicit two-site cooperative binding model is presented which fits these complex data.
Abstract: The binding of factor VII and tissue factor produces a membrane-associated proteolytic complex which may be the primary biological initiator of coagulation Homogeneous tissue factor, a glycoprotein purified from bovine brain, was reconstituted into phospholipid vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (40% phosphatidylserine) with octyl glucoside The vesicles were characterized with respect to size and tissue factor content and orientation Employing data from protease digestion, we deduced that tissue factor is randomly oriented; thus, its effective concentration in these vesicles was half its total concentration In all binding experiments, 1 mol of enzyme was bound per mole of available activator at saturation This stoichiometry was not affected by the form of the enzyme employed or the phospholipid composition of the vesicles With tissue factor incorporated into phosphatidylcholine vesicles, the Kd was 132 +/- 072 nM for factor VII and 454 +/- 137 nM for factor VIIa Thus, the one-chain zymogen binds to the activator with only slightly less affinity than the more active two-chain enzyme Active-site modification of factor VII and factor VIIa with diisopropyl fluorophosphate resulted in tighter binding of the derivatized molecules Inclusion of phosphatidylserine in the vesicles altered the binding both quantitatively and qualitatively With increasing acidic phospholipid, the concentration of enzyme required to occupy half the activator sites was decreased In addition, positive cooperativity was observed, the degree of which depended on the vesicle charge and the form of the enzyme An explicit two-site cooperative binding model is presented which fits these complex data In this model, tissue factor is at least a dimer with two interacting enzyme binding sites
288 citations
161 citations
TL;DR: This work presents individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites, and sets a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.
Abstract: A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cI-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a lambda operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.
141 citations
TL;DR: In this paper, it was shown that lambda repressors at the separated sites touch each other, the DNA bending smoothly so as to accommodate the protein-protein interaction, and electron microscopy was used to visualize the predicted protein-DNA complexes.
Abstract: It has been shown by Hochschild and Ptashne that lambda repressors bind cooperatively to operator sites separated by five or six turns of the helix. Cooperative binding is not observed if the sites are separated by a nonintegral number of turns, unless a four-nucleotide gap is introduced into one of the strands between the two sites. These and other facts suggested that repressors at the separated sites touch each other, the DNA bending smoothly so as to accommodate the protein-protein interaction. Here we use electron microscopy to visualize the predicted protein-DNA complexes.
140 citations
TL;DR: The interaction of the stinging nettle rhizome lectin with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy to reveal two carbohydrate binding sites per molecule consisting of a single polypeptide chain.
77 citations
TL;DR: A two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA, suggesting that clustering may play a role in the physiological activity of drugs.
Abstract: The equilibrium binding of the antitumor compound DHAQ, or mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione], to various DNAs has been examined by optical titration and equilibrium dialysis methods. At low r (bound drug/DNA base pair) values, r less than 0.03, DHAQ binds, in a highly cooperative manner, to calf thymus and Micrococcus lysodeikticus DNAs. The binding isotherms for the interaction of DHAQ with Clostridium perfringens DNA and poly(dA-dT).poly(dA-dT) exhibit a small positive slope at low r values, suggestive of cooperative binding. In contrast, the binding of DHAQ to poly(dG-dC).poly(dG-dC) shows no evidence of cooperative binding even at very low r values. At higher r values (r greater than 0.05), the binding of DHAQ to all the DNAs studied is characterized by a neighbor-exclusion process. A model is proposed to account for the two modes of binding exhibited in the cooperative binding isotherms. The main feature of the proposed model is that local sequence and structural heterogeneity of the DNA give rise to sets of binding sites to which DHAQ binds in a highly cooperative manner, while the majority of the DNA sites bind DHAQ via a neighbor-exclusion process. This two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA. It is suggested that clustering may play a role in the physiological activity of drugs.
74 citations
TL;DR: The structure of the complex between E. coli (RT500) form I dihydrofolate reductase, the antibacterial trimethoprim and NADPH has been determined by X‐ray crystallography and a flexible chain segment which includes Met 20 is in contact with the inhibitor in the presence of NADPH, but more distant in its absence.
55 citations
TL;DR: Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence, suggesting a greater thermodynamic or kinetic stability of the latter.
Abstract: Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively [Otvos, J.D., & Armitage, I. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. The formation of these oligonuclear complexes designated as Cd4 and Cd3 clusters has now been monitored in MT reconstituted with varying amounts of Cd by using differential chemical modification of Cys with [14C]iodoacetamide. At ratios below 2-3 mol of Cd/mol of MT bound, no differential protection of Cys by the metal, and hence no preferred binding, is detectable. At Cd-to-protein ratios between 3 and 5 mol of Cd/mol of MT, the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain, indicating formation of the Cd4 cluster. At still higher ratios, formation of the Cd3 cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labeling of the different Cys in Cd7-MT indicates that the ligands of the Cd3 cluster are 3 times as accessible to iodoacetamide than those of the Cd4 cluster, suggesting a greater thermodynamic or kinetic stability of the latter.
46 citations
TL;DR: A model is developed for ligand binding to human hemoglobin that describes the detailed cooperative free-energies for each of the ten different ligated (cyanomet) species as observed by Smith and Ackers and is an extension of the simple two-state MWC model.
39 citations
TL;DR: It is demonstrated that cooperative effects mediated by direct contact between bound ligands can be divided into two classes depending on whether dimeric species or aggregates of unrestricted size are formed by bound ligand on the lattice at high levels of occupancy.
38 citations
TL;DR: The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied and two binding affinities of the enzyme for ATP were found.
TL;DR: These binding studies show that at pH 7·0 and 9·0, hydrophobic interactions play a major role, while at pH 5·0 only electrostatic interactions play prominent role in the binding of these detergents.
Abstract: Binding studies of cationic detergents such as cetyl trimethylammonium bromide, Cetylpyridinium bromide and dodecyl trimethylammonium bromide with lysozyme were carried out by equilibrium dialysis, ultraviolet difference and circular dichroism techniques at 25 C. Binding isotherms at pH 5·0, 7·0 and 9·0 show cooperative binding at all concentrations of detergents and the number of available binding sites in lysozyme increases with pH. Gibbs free energy of binding calculated on the basis of Wymans’ binding potential concept increases with pH indicating increased binding strength at higher pH. The ultraviolet difference spectra of the detergent complexes with lysozyme at pH 7·0 and 9·0 in the region of 250–300 nm indicate the involvement of aromatic amino acid residues as probable binding sites and also the carboxylate groups since the binding is cooperative. The circular dichroism spectra also indicate the involvement of aromatic amino acid residues in the binding of these detergents. This is substantiated by the decrease in the intensity of the aromatic positive bands in the near ultraviolet region. The increase in the magnitude of [θ]222 nm values in the far ultraviolet region with the increase in the concentration of the detergent in the complex indicates conformational changes resulting in an increase of α-helical content producing a more ordered structure of lysozyme.These binding studies show that at pH 7·0 and 9·0, hydrophobic interactions play a major role, while at pH 5·0 only electrostatic interactions play prominent role in the binding of these detergents.
TL;DR: The results suggest that N-terminal region of A-chain extending to A3 is important for receptor binding and confirm that A3 does not play an important role for negative cooperativity.
TL;DR: Stable opioid receptor binding activity that retains distinct subtype specificities (μ, 6, and K) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization.
Abstract: Stable opioid receptor binding activity that retains distinct subtype specificities (mu, delta, and kappa) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the delta, mu, and kappa sites (the latter subtype measured by the binding of [3H]dynorphin1-8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii greater than 60 A), whereas both those and several small species present (less than 60 A) bind opiate alkaloids and dynorphin1-8.
TL;DR: The effect of guanine nucleotides on the binding of [3H] Quinuclidinyl benzilate to heart muscarinic receptors was studied and it was observed that GTP and Gpp (NH)p increased antagonist binding.
TL;DR: The mixed-sequence self-complementary oligomer d(A6-T6) has an unusual low-temperature binding isotherm which suggests a single strong binding site and a larger number of weaker binding sites which bind propidium cooperatively.
Abstract: To evaluate the length and sequence dependence of the unusual interaction properties observed for nonalternating A/T sequences in deoxyribonucleic acid (DNA) [Wilson, W. D., Wang, Y. H., Krishnamoorthy, C. R., & Smith, J. C. (1985) Biochemistry 24, 3991-3999], we have synthesized the oligomers d(A-T)6, dA10 X dT10, and d(A6-T6) and evaluated their interaction with the intercalator propidium. Propidium visible spectral shifts on adding all three oligomers are quite similar. Low-temperature spectrophotometric binding measurements indicate that d(A-T)6 has a significantly larger binding constant for propidium than dA10.dT10, as with the analogous alternating and nonalternating DNA polymers. The oligomer dA10.dT10 displays positive cooperativity in its propidium binding isotherm, and its binding constant increases with increasing temperature while d(A-T)6 does not display positive cooperativity, and its binding constant decreases with temperature, again as with the analogous polymers. van't Hoff plots indicate that the propidium binding enthalpies are approximately -9 and +6 kcal/mol for the alternating and nonalternating DNA samples, respectively. The mixed-sequence self-complementary oligomer d(A6-T6) has an unusual low-temperature binding isotherm which suggests a single strong binding site and a larger number of weaker binding sites which bind propidium cooperatively. A van't Hoff plot indicates that the cooperative sites d(A-T)6 have binding constants and binding enthalpies similar to dA10.dT10. Similar rate constants are observed in the sodium dodecyl sulfate driven dissociation reaction of propidium from d(A-T)6 and d(A6-T6), but the association reaction of propidium is significantly slower with d(A6-T6) than with d(A-T)6.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Observations suggest that interactions among the subunits of the oligomeric cytoskeletal calmodulin-dependent protein kinase regulate enzyme activation, enhancing the sensitivity of the enzyme to small changes in the intracellular calcium levels that may be particularly relevant to signaling at the synapse.
Abstract: The kinetics of autophosphorylation of the cytoskeletal form of the neuronal calmodulin-dependent protein kinase type II were studied as a function of calmodulin binding under the same conditions. Whereas calmodulin binding was noncooperative with respect to calmodulin concentration (Hill coefficient = 1), the activation of autophosphorylation and the phosphorylation of exogenous substrates showed marked positive cooperativity (Hill coefficient greater than or equal to 1.6). Reduction of the active calmodulin concentration by the addition of the calmodulin antagonist trifluoperazine confirmed the cooperative nature of enzyme activation, because autophosphorylation was more sensitive to the drug than was binding at high concentrations of calmodulin. At intracellular levels of calmodulin the binding and activation of autophosphorylation were cooperative functions of magnesium and calcium concentration. The calmodulin-dependent cooperative activation seems to be a unique feature of the cytoskeletal, but not the soluble, form of the protein kinase and may result from the supramolecular organization of the cytoskeletal enzyme. These observations suggest that interactions among the subunits of the oligomeric cytoskeletal calmodulin-dependent protein kinase regulate enzyme activation, enhancing the sensitivity of the enzyme to small changes in the intracellular calcium levels that may be particularly relevant to signaling at the synapse.
TL;DR: A more complex model than that of Hill et al. may account for the observed fluorescence changes, and it seems equally likely that at least in the absence of Ca2+, the change in fluorescence may be reflecting a more complex behavior than only the transition of tropomyosin-actin units between the weak and strong S-1-binding forms.
TL;DR: An ion-exchange chromatography procedure was used to resolve apometallothionein from the metallo- form in a study of metal-thiolate cluster formation and the results of metal and protein yields are inconsistent with random, noninteractive binding.
TL;DR: In this article, the authors investigated whether the slowly dissociating component of insulin and human growth hormone binding and the homologous down-regulation of their receptors in IM-9 cultured human lymphocytes are due to distinct conformations of the receptor or, rather, to a redistribution within the cell.
Abstract: We have investigated whether the slowly dissociating component of insulin and human growth hormone (hGH) binding and the homologous down-regulation of their receptors in IM-9 cultured human lymphocytes are due to distinct conformations of the receptor or, rather, to a redistribution within the cell. To do so, we used intracellular K+ depletion, which has been shown to inhibit reversibly coated-pit formation and ligand internalization in some cell lines. IM-9 cells incubated in K+-free buffer after a hypotonic shock rapidly lost their K+, which was stabilized at +/- 50% of control by incubation in K+-free binding assay buffer. In K+-depleted cells, the hGH dissociation kinetics became monoexponential and, in contrast with control cells, compatible with the equilibrium constant derived from saturation and association data using a simple model. The loss of hGH receptors during competition studies was abolished. The down-regulation by unlabeled hGH was decreased by 80%. In contrast, insulin receptor kinetics remained unchanged (non-first-order) in the K+-depleted cells; the negative cooperativity and the down-regulation (60%) were identical to those of control cells. Quantitative electron microscopic autoradiography showed a decrease of +/- 50% in the fraction of 125I-labeled hGH internalized. The number of visible coated pits in the membrane was reduced by 80%. Thus, in IM-9 cells, association with coated pits and endocytosis appear to play a major role in the kinetics of hGH binding and in the down-regulation of its receptors, but not in insulin-receptor binding kinetics and down-regulation.
01 Jan 1986
TL;DR: In this paper, a large difference is observed between the affinities of sites III and IV (009 X 10(6) M-1) and sites I and II (00007 X 10 (6)-M-1 ) for calcium, and this difference, together with positive cooperativity previously observed, explains the stepwise conformational changes induced by calcium.
Abstract: Stimulus-response coupling mediated by calmodulin involves several steps: a transitory increase in calcium concentration from 01 to 10 microM, induced by external stimuli; interaction of calcium with calmodulin, accompanied by stepwise structural transitions; the coordinated interaction with and activation of the many calmodulin-regulated enzymes and proteins The binding of calcium to calmodulin is a cooperative and selective process that is modulated by magnesium At physiological ionic strength, and only in the presence of magnesium, a large difference is seen between the affinities of sites III and IV (009 X 10(6) M-1) and sites I and II (00007 X 10(6) M-1) for calcium This difference, together with the positive cooperativity previously observed, explains the stepwise conformational changes induced by calcium The interaction of calmodulin with its target proteins requires the integrity of different portions of the calmodulin molecule Calmodulin-regulated enzymes can be divided into three classes according to their abilities to bind with and to be activated by calmodulin fragments: enzymes which are activated by the C-terminal fragment, such as the Ca2+-ATPase and phosphorylase kinase; enzymes which require both halves of the molecule, such as cyclic AMP phosphodiesterase and myosin light chain kinase; and enzymes whose interaction with calmodulin fragments is too weak to be detected by activation, such as calcineurin and the multiprotein kinase Thus different enzymes may be activated by different calmodulin conformers and the stepwise changes exhibited by calmodulin at different calcium levels can be used to regulate different metabolic pathways
TL;DR: The binding of trifluoperazine to calmodulin and the two adducts was determined, and binding of the phenothiazine was found to be characterized by apparent positive cooperativity and an apparent limiting stoichiometry of about seven binding sites per protein molecule.
TL;DR: Calcium binding to thermolysin is enhanced in the presence of an active site directed inhibitor, suggesting that there may be positive cooperativity between substrate and calcium binding.
TL;DR: Results suggest the existence of a binding domain/receptor for 24,25(OH)2D3 in the chick intestine which is independent of the 1,25-dihydroxyvitamin D3 receptor.
TL;DR: The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity and the binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis.
Abstract: The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity [Hill coefficient = 1.65 +/- 0.1; total number of binding sites at pH 6.6 in 5 mM sodium phosphate buffer = 32 500 +/- 940 pmol/mg; Ka = (3.0 +/- 0.5) X 10(5) M-1]. The binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis. The ratio of the interaction of hemoglobin to band 3 protein per se was 1:1, whereas the band 3 preparation as a whole (protein + lipids) was 3:1. Saturating levels of glyceraldehyde-3-phosphate dehydrogenase blocked only 33% of the total binding sites which were localized at the cytoplasmic segment; the remaining 67% was localized in lipids by their extraction with acetone. Reconstitution of acetone-extracted band 3 with phospholipid liposomes indicated phosphatidylserine as the binding site. The positive cooperativity in binding to acetone-extracted band 3 was increased (Hill constant = 2.1 +/- 0.1) compared to the band 3 preparation. After separation of the alpha and beta chains of hemoglobin, only the alpha chain binds to band 3 with positive cooperativity to an extent of 45-50% of native hemoglobin with similar affinity. The binding capacity of p-(hydroxymercuri)benzoate (HMB) derivatives of hemoglobin and its alpha chain was less than that of native hemoglobin, whereas HMB-beta chain or beta chain did not bind.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Comparative studies show that rabbit skeletal muscle FSR, in contrast to the frog one, shows negative cooperativity in its interactions with Ca2+ and AMPOPCP under some conditions and that the ratio of Ca-binding sites to AMPopCP- binding sites is 2, corresponding to the well-known stoichiometry with ATP.
Abstract: In order to obtain a better understanding of the mechanism of the function of fragmented sarcoplasmic reticulum (FSR), we examined the binding of beta,gamma-methylene [3H]adenosine triphosphate (AMPOPCP), an unhydrolyzable ATP analogue, and 45Ca to FSR from bullfrog skeletal muscle. In medium containing 100 mM KCl and 20 mM Tris-maleate (pH 6.80) on ice, FSR has a single class of [3H]AMPOPCP-binding sites which amount to 4.4-8.6 nmol/mg protein (usually about 7 nmol/mg protein). The affinity was in the range of 6.2-12.3 X 10(3) M-1 in the absence of Ca2+. Ca2+ increased the affinity for AMPOPCP without changing the total number of binding sites, whereas Mg2+ decreased it. The change of the affinity is due to the direct effect of Ca2+ and Mg2+ on FSR. The possibility that Mg-AMPOPCP, Ca-AMPOPCP, and free AMPOPCP might have different affinities to FSR is excluded. The extent of Ca2+-induced enhancement in AMPOPCP binding is dependent not only on Ca2+ concentration but also on the concentration of AMPOPCP. The binding sites for AMPOPCP are likely to be the ATP-binding sites on Ca2+-ATPase protein on the basis of several lines of evidence, including competition between ATP, ADP, or AMP. FSR also binds 7-13 nmol Ca/mg protein (usually about 8 nmol/mg protein) with the affinity of 4-14 X 10(4) M-1 in the absence of the nucleotide in a similar medium containing 4 mM MgCl2. The ratio of Ca-binding sites to AMPOPCP-binding sites is mostly 1, but occasionally 2, corresponding to the ratio of Ca accumulated to ATP hydrolyzed by frog FSR. In the presence of a sufficient amount of the nucleotide, the affinity for Ca2+ was also increased. These findings are well explained by the random sequence binding model of Ca2+ and AMPOPCP, which bind to FSR with positive cooperative interaction between them. However, high concentrations of the nucleotide result in a negative cooperative interaction in the nucleotide binding in the presence of Ca2+, whereas no cooperativity is observed in the absence of Ca2+. Stimulation of Ca binding by AMPOPCP is also correspondingly affected. Comparative studies show that rabbit skeletal muscle FSR, in contrast to the frog one, shows negative cooperativity in its interactions with Ca2+ and AMPOPCP under some conditions and that the ratio of Ca-binding sites to AMPOPCP-binding sites is 2, corresponding to the well-known stoichiometry with ATP.
TL;DR: Experiments using equilibrium dialysis and fluorescence quenching provided direct evidence that L-aspartic acid binds not only to the active site but also a second site with lower intrinsic affinity for it, suggesting the existence of a covalent beta-asparaginase intermediate.
Abstract: Experiments using equilibrium dialysis and fluorescence quenching provided direct evidence that approximately four moles of L-aspartic acid were bound per mole of tetrameric L-asparaginase from Escherichia coli, with a dissociation constant on the order of 60-160 microM. In addition, a set of weaker binding sites with a dissociation constant in the millimolar range were detected. Kinetic studies also revealed that L-aspartic acid inhibited L-asparaginase competitively, with an inhibition constant of 80 microM at micromolar concentrations of L-asparagine; at millimolar concentrations of the amide, an increase in maximal velocity but a decrease in affinity for L-asparagine were observed. L-Aspartic acid at millimolar levels again displayed competitive inhibition. These and other observations suggest that L-aspartic acid binds not only to the active site but also a second site with lower intrinsic affinity for it. The observed "substrate activation" is most likely attributable to the binding of a second molecule of L-asparagine rather than negative cooperativity among the tight sites of the subunits of this tetrameric enzyme. Further support for L-aspartic acid binding to the active site comes from experiments in which the enzyme, when exposed to various group-specific reagents suffered parallel loss of catalytic activity and in its ability to bind L-aspartic acid. Different commercial preparations of Escherichia coli L-asparaginase were found to contain approximately 2-4 moles of L-aspartic acid; these were incompletely removed by dialysis, but could be removed by transamination or decarboxylation. Efficiency of dialysis increased with increasing pH. Taken together, this set of results is consistent with the existence of a covalent beta-aspartyl enzyme intermediate.
Journal Article•
TL;DR: Four insulin-specific hybridoma antibodies with respect to their kinetic properties as well as the binding behaviour of some combinations of them were investigated, and all but one antibodies were shown to bear homogeneous binding sites, thus with high probability being monoclonal.
Abstract: We investigated four insulin-specific hybridoma antibodies with respect to their kinetic properties as well as the binding behaviour of some combinations of them. From equilibrium binding data all but one antibodies were shown to bear homogeneous binding sites. They revealed homogeneity of binding sites also by kinetic experiments, thus with high probability being monoclonal. At 0 degree C, two of them showed discrepancies between kinetic and steady state binding data in as much as, at steady state, the measured bound-to-free ratio of tracer insulin was 3-4 times lower than calculated from kinetic data. Thus a simple bimolecular reaction mechanism could possibly not be applicable. Mixing two monoclonal insulin antibodies, neither cooperative nor additive binding to the insulin molecule could be observed but only competitive effects. Especially, no positive cooperativity between two or more antibodies could be detected, which would be able to account for the higher affinity usually observed for polyclonal vs. monoclonal antibodies.
TL;DR: Binding data for calf thymus DNA did not fit the site-exclusion model with the cooperative binding parameter as a variable nor the specific site, negative-cooperative binding model.
Abstract: Chartreusin binds cooperatively to poly(dA-dT)•poly(dA-dT)and poly(Dg-dC)•poly(dG-dC). Both the site-exclusion model and the specific site model yield cooperative binding constants of about 5×105M-1 and 3×105 M-1 for the AT and GC polymers, respectively, and the same stoichiometry and intrinsic binding constant for both polymers of 5 nucleotides per binding site and 3.1×104M-1, The Scatchard plot for calf thymus DNA is curved in the opposite sense from that of cooperative binding. These binding data did not fit the site-exclusion model with the cooperative binding parameter as a variable nor the specific site, negative-cooperative binding model The site-exclusion model with a cooperative binding parameter of unity yielded a binding constant of about 4×104M-1 and a stoichiometry of about 5 nucleotides per binding site. The same model for transfer and ribosomal RNA yielded binding constants of 5×103 M-1 and 7×103 M-1 and stoichiometries of about 13 and 6 nucleotides per binding site, respectively.