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Showing papers on "Cooperative binding published in 1984"


Journal ArticleDOI
TL;DR: The initially simple model of reversible, bimolecular, and noncooperative interaction between receptor and insulin has been revised to include the existence of at least three affinity states that may be linked to modulation of the biological response induced by the insulin-receptor complex.
Abstract: During the last decade, earlier suggestions that insulin acts at the plasma membrane level via combination with receptors have been amply confirmed in studies of 125I-labeled insulin binding kinetics. Efforts have been devoted to the development of homogeneous, stable, and bioactive tracers, and a preparation of monoiodo[TyrA14]insulin showed 100-125% biological activity. The initially simple model of reversible, bimolecular, and noncooperative interaction between receptor and insulin has been revised to include the existence of at least three affinity states that may be linked to modulation of the biological response induced by the insulin-receptor complex. Thus negative cooperativity seems important in reducing oscillations of insulin action with variations in plasma insulin concentration, and formation of a high-affinity state or positive cooperativity may lead to desensitization of receptors. The kinetic phenomena suggest that receptor-binding affinity and function are actively regulated by insulin itself. At present the receptor model is purely functional and does not imply molecular mechanisms. However, recent advances in the analysis of receptor structure and biochemistry promise that the molecular equivalents of the kinetic phenomena may be elucidated in the near future. Furthermore the reaction between receptor and insulin is irreversible because of degradation of receptor-bound insulin, which may result in termination of the metabolic activation. Morphological and biochemical work suggests that internalization of the receptor-insulin complex from the plasma membrane transfers insulin to intracellular organelles like the lysosomes, the Golgi apparatus, or nucleus, where degradation by insulin protease takes place, whereas the receptor is recycled back to the membrane. Recent advances in the studies of biosynthesis and cellular dynamics of receptors indicate that intracellular processing and redistribution of binding sites may play a role in the mechanism of insulin action. Insulin receptors are widely distributed in all cell types, but evidence has accumulated that receptors show tissue and species variations in their functional properties regarding binding affinity, insulin specificity, cooperativity, and insulin degradation and in structural properties such as antigenic determinants and glycosidic composition. Perhaps these differences reflect cellular adaptations and variations in the physiological role of insulin.(ABSTRACT TRUNCATED AT 400 WORDS)

197 citations


Journal ArticleDOI
TL;DR: The dimer of the 6-isomer of carboxytetramethylrhodamine, in which the two carboxyl groups are in para positions on the phenyl moiety, proved to be an effective site-filling ligand and led to an explanation for isomeric discrimination in the binding site.

82 citations


Journal ArticleDOI
TL;DR: The present study demonstrates that human fetal membranes bind 125I-epidermal growth factor (125I-EGF), with chorion binding more than amnion, and suggests that they are target tissues for EGF.
Abstract: The present study demonstrates that human fetal membranes bind 125I-epidermal growth factor (125I-EGF), with chorion binding more than amnion. The chorion binding was also higher than that by decidua, but lower than in placenta. The lower binding by chorion compared to placenta was entirely attributable to a lower number of available EGF receptors and not to lower affinity. Chorion and placenta , but not amnion or decidua, from Cesarean section bound significantly more 125I-EGF when compared to tissues obtained after vaginal delivery. The binding of 125I-EGF to chorion exhibited dependency on time, temperature of incubation, pH of the incubation media, and amount of chorion protein. The 125I-EGF was not degraded during the binding reaction with chorion. The binding was specific in that unlabeled EGF inhibited 125I-EGF binding in a dose-dependent manner, whereas very high concentrations of other unlabeled hormones and growth factors had minimal effects on 125I-EGF binding. When some of these other hormones were tested for binding as tracers (125I-hCG, [3H]prostaglandin E1 and F2 alpha), neither chorion, amnion, decidua, nor placenta specifically bound these ligands. The 125I-EGF specific binding to chorion was saturable and a Scatchard plot of this data was curvilinear, which appears to be due to negative cooperativity. The apparent dissociation constant calculated from the initial slope of the plot was 0.20 nM, which is in excellent agreement with the concentration of unlabeled EGF required for half maximal inhibition of 125I-EGF binding, 0.26 nM. Autoradiography at the light microscope level revealed the presence of silver grains in amnion and chorion only when excess unlabeled EGF addition was withheld. The quantification of grains revealed the presence of a significantly (P less than 0.01) greater number of grains in chorion than in amnion, supporting the conclusion of the binding data. The physiological significance of EGF binding to fetal membranes and decidua is not known, but the considerable amount of EGF binding reported here suggests that they are target tissues for EGF.

66 citations


Journal ArticleDOI
01 Sep 1984-Planta
TL;DR: In-vitro data indicated that the relative ability of allosteric activation to dominate over allosterIC inhibition increases markedly with both pH and temperature.
Abstract: The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K a =1.5 μM), while feedback inhibition was effected by l-tyrosine (K i =15 μM) or by l-phenylalanine (Ki=15 μM). At pH 6.1, all three effectors acted competitively, influencing the apparent K m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K i for l-phenylalanine was elevated over 30-fold to 500 μM, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n′=1.8.

61 citations


Journal ArticleDOI
01 May 1984-Diabetes
TL;DR: It is indicated that prolonged exposure of Fao hepatoma cells to insulin produces a type of downregulation characterized by a decrease in insulin receptor number and a concomitant increase in receptor affinity.
Abstract: We have studied the effects of chronic exposure to insulin on the binding and the biologic activity of the hormone using a well-differentiated cell line (Fao) derived from the Reuber H35 rat hepatoma. Prolonged incubation (24 h) with 10(-6) M insulin produced a 20-25% decrease in binding of tracer concentrations (2 X 10(-11) M) of 125I-insulin, and a leftward shift of the curve for inhibition by unlabeled insulin. Scatchard analysis of the binding data revealed that a 75-80% decrease in the number of binding sites had occurred in the insulin-treated cells, but was accompanied by an increase in apparent receptor affinity. Kinetic studies suggested negative cooperativity in insulin binding and indicated that the change in affinity was accounted for by a decrease in the rate of dissociation. Both the decrease in receptor number and the increase in affinity were dependent on time, temperature, and the insulin concentration during the treatment period. Both effects were also blocked by cycloheximide, suggesting that they required new protein synthesis. Plasma membranes isolated from downregulated cells retained both the change in receptor number and affinity. Anti-receptor antibodies present in two human sera (B-2 and B-9) inhibited 125I-insulin binding in downregulated cells with equal or slightly greater sensitivity than in control cells. The changes in insulin binding were accompanied by changes in insulin's biologic effects in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

54 citations


Journal ArticleDOI
TL;DR: Data taken together indicate that (+/-)-[3H]nicotine binds with specificity to multiple sites in the rat brain P2 preparation with a complexity not addressed heretofore.
Abstract: (−)-Nicotine may bind to as many as 5 sites in the rat brain P2 preparation: A very high affinity site (KD∼2.2×10−11 M); a positive cooperativity site; a high affinity site (KD∼5.2×10−9 M); a low affinity site (KD∼4.5×10−5 M) and a very low affinity site. The curvilinear nature of both Scatchard plots and kinetic curves indicates the presence of multiple binding sites. Evidence for a positive cooperativity site includes: (1) The configuration of Scatchard plots (at low concentrations) of saturation as well as inhibition curves for (−)- and (+)-nicotine. (2) The Hill number of 1.37 for the binding of low concentrations of (±)-[3H]nicotine. (3) Selectivity among cholinergic drugs for producing positive cooperativity. (4) Markedly different specificities of drugs for the positive cooperativity site. Thus while only (+)- and (−)-nicotine interacted with the very high affinity site, acetylcholine, atropine, mecamylamine, lobeline, carbachol, (+)-nicotine and (−)-nicotine enhanced the binding of (±)-[3H]nicotine and cytisine, anabasine, cotinine and choline selectively inhibited binding at the high affinity site. Several lines of evidence indicate that there is stereospecificity. (+)-Nicotine was more potent than (−)-nicotine in inducing positive cooperativity whereas (−)-nicotine was 80 times more potent than (+)-nicotine in inhibiting binding at the high affinity site. Further, the specificity of the binding sites can be altered by changing the concentration of the buffer which gives additional evidence for the lability of the nicotine binding site. Although the pharmacologic significance of the different binding sites has not been determined, these data taken together indicate that (±)-[3H]nicotine binds with specificity to multiple sites in the rat brain P2 preparation with a complexity not addressed heretofore.

51 citations


Journal ArticleDOI
TL;DR: The binding parameters of a number of ADP or ATP analogs to the adenine nucleotide carrier in mitochondria and inside-out submitochondrial particles have been explored by means of two specific inhibitors, carboxyatractyloside and bongkrekic acid.

37 citations


Journal ArticleDOI
TL;DR: Results are extended to include ligands of any shape and cooperative interactions, and data on the cooperative binding of polymyxin to charged lipid bilayers are reevaluated.

33 citations


Journal ArticleDOI
TL;DR: The experimental results are shown to be consistent with two alternative binding mechanisms, and the first model, incorporating the notion of base selectivity, is in quantitative agreement with published work on ethidium binding to DNAs of different base compositions and with nmr measurements of bound ligand lifetimes.
Abstract: The relaxation kinetics of binding of ethidium to calf-thymus DNA as studied previously by the temperature-jump method with absorption detection [Bresloff, J. & Crothers, D. M. (1975) J. Mol. Biol.95, 103] is reanalyzed in terms of a series of models for DNA-ligand interactions that include cases with and without internal and bimolecular direct transfer of ligands between different binding modes or different binding sites. The experimental results are shown to be consistent with two alternative binding mechanisms. Both models include bimolecular “direct transfer” steps and a site size of two base pairs per site. In the first model, there exist two distinct modes of binding to the DNA double helix at each binding site. In the second model, sites containing at least one GC base pair constitute a different and stronger class of binding sites than those containing only AT base pairs. The existence of a direct transfer pathway between two classes of bound ligands is supported by the linear increase of reciprocal relaxation times far beyond the concentration regions, where off-rates should have become rate limiting. The second model, incorporating the notion of base selectivity, is in quantitative agreement with published work on ethidium binding to DNAs of different base compositions and with nmr measurements of bound ligand lifetimes.

33 citations


Journal ArticleDOI
TL;DR: The effect of higher ionic strength on the binding of CTAB with lysozyme at pH 9.0 is evidenced by lower binding ratios and decreased intensities of the UV difference bands, thus indicating the involvement of electrostatic interactions.
Abstract: Binding of lysozyme with cetyltrimethylammonium bromide (CTAB) and dodecyl-trimethylammonium bromide (DTAB) at various detergent concentrations and pH was studied at 25°C by equilibrium dialysis technique. In the case of CTAB, binding isotherms at pH 5.0, 7.0, and 9.0 show cooperative binding at all the concentrations of the detergent and the binding ratios increase with pH. Cooperative binding is also shown by DTAB at all the concentrations and pH, but the binding ratios are lower compared to CTAB. The Gibb's free energy change calculated on the basis of Wyman's binding potential concept increases with pH, indicating increased binding strength of CTAB at higher pH.The UV difference spectra of CTAB and DTAB with lysozyme and its model compounds such as L-Trp, L-Tyr.HCl and L-Phe show two peaks at 297 nm and 250 nm at pH 9.0 indicating the possible involvement of tryptophans as the binding sites along with the carboxylate anion or the phenolic group of a tyrosine on lysozyme. The effect of higher ionic strength on the binding of CTAB with lysozyme at pH 9.0 is evidenced by lower binding ratios and decreased intensities of the UV difference bands, thus indicating the involvement of electrostatic interactions. However, the hydrophobic interactions between the detergents and the aromatic amino acid residues in lysozyme contribute more to the binding strength.The binding of these cationic detergents by lysozyme induces conformational changes in the enzyme. They are followed by the circular dichroism (CD) technique which shows a decrease in the aromatic bands in the 320-250 nm region. In the 250-200 nm region, the I¸ 222 values obtained at various concentrations of CTAB in the complex indicate an increase in the I±-helical content indicating a more ordered structure. The CD spectra of lysozyme-DTAB complex were found to be very similar to those of CTAB complex, but the effect was less pronounced probably due to the decrease in the hydrocarbon chain length.Therefore, it is concluded that hydrophobic interactions play an important role in the detergent binding, whereas electrostatic interactions play only a minor role. © 1984 BY THE JOURNAL OF BIOCHEMISTRY.

26 citations


Journal ArticleDOI
TL;DR: Competitive binding curves for 125I-insulin on a cultured rat hepatoma indicated that low competitive insulin concentrations increased label binding, and at insulin levels below 10 ng/ml, the binding curves exhibited sigmoidicity consistent with positive cooperativity.

Journal ArticleDOI
TL;DR: A sensitive ESR method which allows a direct quantitative determination of nucleic acid binding affinities of proteins under physiologically relevant conditions has been applied to the gene 5 protein of bacteriophage fd, and an intrinsic binding constant, Kint, and cooperativity factor, omega, of 60 are given.

Journal ArticleDOI
TL;DR: Ionic binding of the surfactant is weakened and hydrophobic binding strengthened by increasing ionic strength, and the stability of the Surfactant-protein complexes is discussed in relation to the Stability of surfactants micelles.

Journal ArticleDOI
TL;DR: The equilibrium positive cooperativity in high-affinity acetylcholine binding previously inferred from the data is deceiving; the curvature in Scatchard representations is a consequence of long-lived nonequilibrium distributions between high-Affinity and lower affinity receptor conformers.
Abstract: Studies of the binding of (3H)acetylcholine to receptor-rich membranes of Torpedo californica electric organ under conditions that normally iead to a state of equilibrium did not give rise to equilibrium binding curves. Instead, the acetylcholine receptor was found to develop very long lived metastable states resulting in hysteresis in binding. Under conditions where the concentration of free (3H)acetylcholine is both less than 0.1 NM and smaller or comparable to the total receptor concentration, the degree of binding of acetylcholine depends on the rate, Le., the mode, of increasing the acetyl- choline concentration (rapid mixing vs. dialysis). The equi- librium positive cooperativity in high-affinity acetylcholine binding previously inferred from the data is deceiving; the curvature in Scatchard representations is a consequence of long-lived nonequilibrium distributions between high-affinity and lower affinity receptor conformers. By manipulation of the experimental conditions, true equilibrium binding, resulting in a linear Scatchard binding curve, was obtained and yielded the apparent equilibrium constant, K = 5 f 1 nM at 4 OC. The stoichiometry of the high-affinity site associated with this K value was found to be one acetylcholine per receptor mo- nomer (M, 250 000) when carefully standardized (3H)- acetylcholine analyzed for both radiopurity and acetylcholine concentration was used. While our fresh membrane fragments prepared in the presence of 4 mM Ca2+ revealed up to twice as many '251-a-bungarotoxin sites in 0.1% nonionic detergent relative to those assayed in the absence of detergent, nonionic %e binding of acetylcholine (AcCh)' (A in equations and schemes) by the acetylcholine receptor (AcChR; R in equa- tions and schemes) is a key reaction in the chemical control of the bioelectric excitation of cholinergic membranes (for monographs, see Nachmansohn (1959) and Katz (1 969)). The nicotinic AcChR protein of fish electric organs, which regulates the rapid Na+-K+ fluxes through the electroplax membranes, exhibits major conformational variability consistent with its functional properties (for recent reviews, see Karlin (1980), Changeux (1 98 l), Adams (198 l), Conti-Tronconi & Raftery (1982) and Taylor et al. (1983)l. Electrophysiological data from frog neuromuscular junctions, which in their electri- cal-chemical behavior are similar to that of fsh electric organs, suggested that the AcChR channel coexists in at least two conformational states prior to any AcCh binding: an acti- vatable resting state (RJ and an inactivated desensitized state (Rh) with a binding affinity for AcCh that is higher than that

Journal ArticleDOI
TL;DR: The binding of 1,25‐dihydroxyvitamin D3 to its chick intestinal receptor does not fit well to the linear regression line of Scatchard's model, but the Hill analysis and K d and n H are strongly correlated and suggest the existence of two ligand binding sites located in subunits for the 1, 25(OH)2D3 receptor.

Journal ArticleDOI
TL;DR: The two-state model of cooperative binding is shown to be incompatible with the results of this analysis, as the Monod-Wyman-Changeux parameters derived from the same experimental data demand free energy couplings of an order higher than the second.
Abstract: The number of protein subunits that must be liganded to effect changes in subunit interaction may be characterized by defining an order for the free energy couplings between these two processes. From available data on the chemical equilibrium of stripped hemoglobin A with oxygen, I show that couplings are unequivocally of first order. The two-state model of cooperative binding is shown to be incompatible with the results of this analysis, as the Monod-Wyman-Changeux parameters derived from the same experimental data demand free energy couplings of an order higher than the second.

Journal ArticleDOI
TL;DR: Different equilibrium binding constants of the three anthracyclcine antibiotics correspond to different mean attachment times of the antibiotics at the polymer chain, which positively correlate with the inhibitory action of these drugs on in vitro DNA synthesis.

Journal ArticleDOI
TL;DR: It is concluded that the low basallevel of the RecA441 protein in a recA453-441 cell is sufficient to cleave the λ repressor, under conditions where a normal basal level of RecA430 protein is also present allowing the formation of mixed multimers on single-stranded DNA regions normally present in the cell.
Abstract: Induction of prophage lambda occurs in recA441 mutant lysogens after a shift to 42 degrees C in the presence of adenine. If the synthesis of RecA441 protein is maintained at a low basal level by the presence of a second mutation in the recA441 gene, recA453, induction of prophage lambda is prevented. The ability to induce prophage lambda is restored by the introduction, on a transducing phage, of a second recA gene carrying the recA430 mutation; by itself, the RecA430 protein is devoid of activity against the lambda repressor (Rebollo et al. 1984). In order to explain how the RecA430 protein might complement the RecA441 protein to provide lambda repressor cleavage in a recA453-441 (recA430) diploid lysogen, we characterized the cleavage reaction catalysed by a mixture of these proteins in vitro. Our results suggest that, in the presence of dATP, the RecA441 and RecA430 proteins form mixed multimers on single-stranded DNA, in which the RecA441 protein molecules enhance the DNA binding affinity of RecA430 protein molecules, but RecA430 protein molecules support no cleavage of the lambda repressor. Although the effects of the RecA430 and single-strand binding (SSB) proteins are similar in vitro, we show that the SSB protein cannot substitute for the RecA430 protein in restoring lambda repressor cleavage in a recA453-441 lysogen. Comparison of the stimulatory effect of long single-stranded DNA with that of (dA)14 oligonucleotides on the RecA441 protein-directed cleavage of the lambda repressor in the presence of various nucleoside triphosphates (NTPs) indicates that the cooperative binding of the RecA441 protein to single-stranded DNA stabilizes the RecA protein-DNA complexes so that they remain intact long enough to support cleavage of the lambda repressor. We conclude that the low basal level of the RecA441 protein in a recA453-441 cell is sufficient to cleave the lambda repressor, under conditions where a normal basal level of RecA430 protein is also present allowing the formation of mixed multimers on single-stranded DNA regions normally present in the cell.

Journal ArticleDOI
TL;DR: An empirical mathematical model employing this rationale provided a satisfactory fit for the binding of fatty acyl-CoA to citrate synthase and suggested that its complex isotherm resulted from binding in two classes of sites, i.e. two cooperative nucleotide sites and other sites.

Journal ArticleDOI
TL;DR: Calorimetric titrations of the apo beta 2 subunit with PLP at 35 degrees C have shown that also at this temperature positive cooperativity between the two binding sites occurs and a complete set of site-specific thermodynamic parameters has been established.
Abstract: The energetics of binding of the coenzyme pyridoxal 5'-phosphate (PLP) to both the apo beta 2 subunit and the apo alpha 2 beta 2 complex of tryptophan synthase from Escherichia coli has been investigated as a function of pH and temperature by direct microcalorimetric methods. At 25 degrees C, pH 7.5, the binding process proceeds in the time range of minutes and shows a biphasic heat output which permits resolution of the overall reaction into different reaction steps. Binding studies on the coenzyme analogues pyridoxal (PAL), pyridoxine 5'-phosphate (PNP), and pyridoxine (POL) to the protein as well as a comparison of these results with data from studies on PLP binding to epsilon-aminocaproic acid have led to a deconvolution of the complex heat vs. time curves into fast endothermic contributions from electrostatic interaction and Schiff base formation and slow exothermic contributions from the interactions between PLP and the binding domain. The pH-independent, large negative change in heat capacity of about -9.1 kJ/(mol of beta 2 X K) when binding PLP to beta 2 is indicative of major structural changes resulting from complex formation. The much smaller value of delta Cp = -1.7 kJ/(mol of beta 2 X K) for binding of PLP to alpha 2 beta 2 clearly demonstrates the energetic linkage of protein-protein and protein-ligand interactions. Calorimetric titrations of the apo beta 2 subunit with PLP at 35 degrees C have shown that also at this temperature positive cooperativity between the two binding sites occurs. On the basis of these measurements a complete set of site-specific thermodynamic parameters has been established.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal ArticleDOI
TL;DR: It is concluded that the site-site interactions of a negatively cooperative type, which are negligible at lower temperatures, exist among rat glomerular angiotensin II receptors at the physiological temperature, and that the binding study may as well be performed at 37 degrees C for the purpose of investigating quantitative correlation between the hormone binding and its biological effect.

Journal ArticleDOI
TL;DR: The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques as discussed by the authors.
Abstract: The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal Article
TL;DR: It is shown that cooperative effects arising upon binding of proteins and other ligands to DNA can be divided into two groups depending on the symmetry of interactions between the bound ligand molecules.
Abstract: Cooperative effects arising upon binding of biologically active ligands to DNA are considered. Equations are derived which enable one to describe the binding of two different ligands to DNA. We also consider the case when ligand can form two type of DNA complexes. The cooperative binding of the ligand in the vicinity of saturation level of binding can be described with a good accuracy by equation derived for the non-cooperative adsorption of the same ligand with some effective binding constant Keff. It is shown that cooperative effects arising upon binding of proteins and other ligands to DNA can be divided into two groups depending on the symmetry of interactions between the bound ligand molecules. In particular, if such interactions favor the formation of dimeric ligand species on the DNA, Keff approximately a1/2, where a is the ligand-ligand interaction constant. If cooperative interactions favor the formation of aggregates of unrestricted size, then Keff approximately aL+Y, where L is the size of the binding site for the ligand on DNA.

Journal ArticleDOI
TL;DR: The bilirubin-binding abilities of human serum albumin, α-fetoprotein and ligandin were investigated by employing absorbance spectral measurement, peroxidation and fluorescence measurement techniques, and no significant difference was found between adult serum and cord serum albumins.
Abstract: The bilirubin-binding abilities of human serum albumin, α-fetoprotein and ligandin were investigated by employing absorbance spectral measurement, peroxidation and fluorescence measurement techniques. The binding constants of the proteins from adult serum and cord serum were very similar. However, those of α-fetoprotein were slightly smaller than those of albumin. Ligandin had two cooperative binding sites for bilirubin, and the binding constants were of the same order as those of the weaker binding sites of albumin. A study of the effect of linolic acid revealed that the bilirubin bound to α-fetoprotein was more easily liberated in the presence of linolic acid than that bound to albumin binding. The drug-binding abilities of these proteins were also examined, and no significant difference was found between adult serum and cord serum albumins. However, α-fetoprotein appeared not to exhibit drug-binding ability when the peroxidation method was employed. The biological role of α-fetoprotein in fetal plasma may be similar to that of albumin.

Journal ArticleDOI
TL;DR: It is suggested that, in general, charge effects are more likely to enhance the binding of anionic than cationic amphibiles by proteins.
Abstract: A family of fluorescent probes, consisting of 2-p-toluidinylnapththalene-6-sulfonate (TNS) and neutral and cationic sulfonamido derivatives has been utilized to study the influence of electrostatic forces in protein-amphiphile interactions. 2-p-Toluidinylnaphthalene-6-[N-β-ethylammonium chloride] sulfonamide (III) binds to a lower number of discrete sites in bovine serum albumin and sperm whale apomyoglobin than does TNS, and is also bound less efficiently by β-lactoglobulin. The fluorescence characteristics of the bound probes indicate that their environments are hydrophobic, but thatpH and ionic strength influence the binding. The initial binding of III to discrete sites on both apomyoglobin and bovine serum albumin induces the cooperative binding of additional probe molecules. TNS, but not III, fluoresces in α-chymotrypsin solutions. An [N-β-ethyltrimethyl ammonium] sulfonamido derivative (IV), but not TNS, fluoresces in bovine trypsin solutions. Fluorescence-enhancing interactions were detected between TNS, III, and polyvinylpyrrolidone, but not between these probes and ribonuclease A, α-chymotrypsinogen, lysozyme, or γ-globulins. The wheat prolamin A-gliadin binds more TNS than III. The accessibility of all binding sites of gliadin is lower atpH 5.0 than atpH 3.1. It is suggested that, in general, charge effects are more likely to enhance the binding of anionic than cationic amphibiles by proteins.

Journal Article
TL;DR: It is reported that membrane preparations of human myometrium possess binding sites for [3H] RO 5-4864, a specific ligand for the peripheral type of benzodiazepine receptors, which might exert their relaxant activity on uterine muscle directly at peripheral level through specific binding sites.
Abstract: We report that membrane preparations of human myometrium possess binding sites for [3H] RO 5-4864, a specific ligand for the peripheral type of benzodiazepine receptors. Scatchard analysis shows a high-affinity binding site (Kd = 3.1 +/- 1.1 nM) and a class of low-affinity binding sites (Kd greater than 30nM) with positive cooperativity. Displacement and tryptic digestion experiments indicate that both these benzodiazepine binding sites are specific and proteic in nature. So benzodiazepines might exert their relaxant activity on uterine muscle directly at peripheral level through specific binding sites.

Journal ArticleDOI
TL;DR: Evidence is presented justifying the conclusion that, in contrast to the dextran-coated charcoal technique, the widely used technique of separating bound and free TSH with polyethylene glycol is inadequate and yields inaccurate results.

Journal ArticleDOI
TL;DR: It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal.
Abstract: Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.

Journal ArticleDOI
TL;DR: Compared insulin binding, plasma membrane fluidity, and phospholipid composition of three different Friend erythroleukemia clones, a wild type (FLC) a mutant (R3) and the revertant to wild type F+.

Journal ArticleDOI
TL;DR: GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles in the anterior pituitary cells.
Abstract: Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 ± 0.07, 0.78 ± 0.04, 0.31 ± 0.03 and 0.27 ± 0.03 fmol μg−1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10−9M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p > 0.5) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p < 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p < 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells. A single, low-capacity binding site may, or may not suggest the presence of a structurally incomplete form of the receptor protein in microsomes and nuclei. Binding to the secretory granules fraction exhibited only a relatively minor temporal difference compared to the plasma membrane, which may have resulted from an inappropriate conformational state of the receptor protein. Only the binding to the plasma membrane exhibited appropriately both the affinity and temporal requirements of the intact GnRH receptor protein in vitro and in vivo.