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1.
Guy Goertz Jacques Longchampt Odette Crepy Odette Judas Max-Fernand Jayle 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,451(1):287-304
The present work deals with the determination of association constants at equilibrium by a non-graphical method in binding systems containing one specific receptor. Equations have been derived from that originally described by Lea (Biochim. Biophys. Acta, 322, 68–74), the terms of which are obtained from the data of simple displacement curves of a bound radioactive ligand by unlabelled competitors identical or different in nature. By knowing the function relating the variations of the bound ligand (B) to the affinity constant (Ki) and the quantity (Mi) of competitor for a given system, it is possible to calculate any of these parameters when the two others are measured. Thus, it becomes easy to compare the relative affinities of differents receptors for the same ligand or that of one receptor for various labelled or unlabelled ligands. Furthermore, theoretical displacement curves can be drawn and compared to experimental data, when only knowing the affinity constant of a specific binding system in given conditions. These modes of calculation have been tested in a study of interactions between various steroids and a fraction of human serum proteins precipitated by ammonium sulfate (30–45%) and containing the sex hormone-binding globulin. Association constants thus obtained agree well with those reported in the literature and determined by graphical procedures. 相似文献
2.
Cytochrome P-450-dependent 14 alpha-demethylation of lanosterol in Candida albicans. 总被引:2,自引:0,他引:2
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Analysis of receptor-ligand binding characteristics can be greatly hampered by the presence of non-specific binding, defined as low-affinity binding to non-receptor domains which is not saturable within the range of ligand concentrations used. Conventional binding analyses, e.g. according to the methods described by Scatchard or Klotz, relate the amount of specific receptor-ligand binding to the concentration of free ligand, and therefore require assumptions on the amount of non-specific binding. In this paper a method is described for determining the parameters of specific receptor-ligand interaction which does not require any assumption or separate determination of the amount of non-specific binding. If the concentration of labelled free ligand is constant, a plot of Fu/(B0*-B*) versus Fu yields a linear relationship, in the case of a single receptor class, in which Fu is the concentration of unlabelled free ligand, B0* is the total amount of labelled bound ligand in the absence of unlabelled ligand and B* is the total amount of labelled bound ligand in the presence of an unlabelled ligand concentration Fu; all of these data are readily obtained from binding studies. This linear relationship holds irrespective of the amount of non-specific binding, and the values for receptor density, ligand dissociation constant and a constant for non-specific binding can be readily obtained from it. If the concentration of labelled free ligand is not a constant for all data points, data are first converted according to a straightforward normalization procedure to permit the use of this relationship. The presence of multiple receptor classes with dissociation constants in the range of the ligand concentrations used results in a negative deviation from this linearity, and therefore the presence of multiple receptor classes can be discriminated unequivocally from non-specific binding. Both theoretical and practical advantages of the present method are described. The method, which will be referred to as the linear subtraction method, is illustrated using the binding of tumour promoters and polypeptide growth factors to their specific cellular receptors. 相似文献
3.
An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods. 相似文献
4.
We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E. coli. It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs. At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1. The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques. This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application. 相似文献
5.
P M Silverman 《Archives of biochemistry and biophysics》1973,157(1):234-242
The number of RNA sites bound by the Qβ RNA polymerase and the affinity of the enzyme for different RNA sites was measured by equilibrium partition of enzyme and enzyme-nucleic acid complexes between two liquid polymer phases. At 0 °C and in the absence of other components required for RNA synthesis the enzyme bound to many regions of Qβ RNA, f2 RNA, 16S rRNA, double-stranded RNA, and circular DNA. Under conditions of enzyme excess, the maximum numbers of enzyme molecules bound per molecule of Qβ RNA, f2 RNA, and 16S rRNA were 32, 26, and 12, respectively. The enzyme bound to most, if not all, of the Qβ RNA sites with the same affinity. Nevertheless, the association constant for enzyme binding to Qβ RNA was more than 10-fold greater than for binding to f2 RNA over a wide range of salt concentrations. 相似文献
6.
Porschke D 《European biophysics journal : EBJ》2012,41(5):415-424
The kinetics of cAMP receptor (CAP) binding to promoter DNA has been studied by stopped-flow electric-dichroism at a reduced
salt concentration, where the coupling of non-specific and specific binding can be observed directly. Amplitudes, rise and
decay times of dichroism transients provide detailed information about the reaction and the structure of intermediates over
more than six orders of magnitude on the time scale. CAP binding during the first milliseconds after mixing is indicated by
an increase of both rise- and decay-time constants. A particularly large increase of rise times reflects initial formation
of non-symmetric complexes by protein binding to non-specific sites at DNA ends. The increase of the hydrodynamic dimensions
continues up to ~1 s, before a decrease of time constants reflects transition to compact states with bent DNA up to the time
range of ~103 s. The slow approach to CAP-induced DNA bending is due to non-specific complexes, which are formed initially and are converted
slowly to the specific complex. At the salt concentration of 13.5 mM, conversion to specific complexes with bent DNA is completed
after ~40 s at pH 8 compared to >103 s at pH 7, resulting from a higher affinity of CAP to non-specific sites at pH 7 than 8 by a factor of ~100. Thus, under
the given conditions non-specific sites delay rather than facilitate formation of the specific complex with bent DNA. Experimental
data obtained for a non-specific DNA clearly indicate the impact of pseudo-sites. The different electro-optical parameters
have been combined in global fits. 相似文献
7.
The relationship between effector binding and inhibition of activity in D-3-phosphoglycerate dehydrogenase
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The binding of L-serine to phosphoglycerate dehydrogenase from Escherichia coli displays elements of both positive and negative cooperativity. At pH 7.5, approximately 2 mol of serine are bound per mole of tetrameric enzyme. A substantial degree of positive cooperativity is seen for the binding of the second ligand, but the binding of the third and fourth ligand display substantial negative cooperativity. The data indicate a state of approximately 50% inhibition when only one serine is bound and approximately 80-90% inhibition when two serines are bound. This is consistent with the tethered domain hypothesis that has been presented previously. Comparison of the data derived directly from binding stoichiometry to the binding constants determined from the best fit to the Adair equation, produce a close agreement, and reinforce the general validity of the derived binding constants. The data also support the conclusion that the positive cooperativity between the binding to the first and second site involves binding sites at opposite interfaces over 110 A apart. Thus, an order of binding can be envisioned where the binding of the first ligand initiates a conformational transition that allows the second ligand to bind with much higher affinity at the opposite interface. This is followed by the third ligand, which binds with lesser affinity to one of the two already occupied interfaces, and in so doing, completes a global conformational transition that produces maximum inhibition of activity and an even lower affinity for the fourth ligand, excluding it completely. Thus, maximal inhibition is accomplished with less than maximal occupancy of effector sites through a mechanism that displays strong elements of both positive and negative cooperativity. 相似文献
8.
9.
The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions. 相似文献
10.
Sarkar D Azaro MA Aihara H Papagiannis CV Tirumalai R Nunes-Düby SE Johnson RC Ellenberger T Landy A 《Journal of molecular biology》2002,324(4):775-789
The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination. 相似文献
11.
A method using binding site "neighbor-effect" parameters (NEPs) is introduced to evaluate the effects of interaction between adjacent ligands on their binding to an infinite linear lattice. Binding site overlap is also taken into account. This enables the conditional probability approach of McGhee & von Hippel to be extended to more complex situations. The general equation for the isotherm is v/LF = SFKF, where v is the ratio of bound ligands to lattice residues, LF is the free ligand concentration, SF is the fraction of binding sites that are free, and KF is the average association constant of a free site. Solutions are derived for three cases: symmetric ligands, and asymmetric ligands on isotropic or anisotropic lattices. For symmetric ligands there is one NEP, E, which is the ratio of the average binding affinity of a free site if the status of the lattice residue neighboring one end of the site is unspecified (left to chance) to the affinity when this residue is free (holding the other neighbor constant). Thus KF is KE2, where K is the affinity of an isolated site. If a site is n residues long, SF is f ffn-1, where f = 1 - nv is the fraction of residues that are free and ff is the conditional probability that a free residue is bordered on a given side by another free residue. The expression for ff is 1/(1 + x/E), where x is v/f, E is (1 - x + [(1 - x)2 + 4x omega]1/2)/2, and omega is the co-operativity parameter. The binding of asymmetric ligands to an isotropic lattice is described by two NEPs; the last case involves four NEPs and a bound ligand orientation parameter. For each case, the expected length distribution of clusters of bound ligands can be calculated as a function of v. When Scatchard plots with the same intercepts and initial slope are compared, it is found that ligand asymmetry lowers the isotherm (relative to the corresponding symmetric ligand isotherm), whereas lattice anisotrophy raises it. 相似文献
12.
We describe an affinity chromatography method to isolate specific RNAs and RNA-protein complexes formed in vivo or in vitro. It exploits the highly selective binding of the coat protein of bacteriophage R17 to a short hairpin in its genomic RNA. RNA containing that hairpin binds to coat protein that has been covalently bound to a solid support. Bound RNA-protein complexes can be eluted with excess R17 recognition sites. Using purified RNA, we demonstrate that binding to immobilized coat protein is highly specific and enables one to separate an RNA of interest from a large excess of other RNAs in a single step. Surprisingly, binding of an RNA containing non-R17 sequences to the support requires two recognition sites in tandem; a single site is insufficient. We determine optimal conditions for purification of specific RNAs by comparing specific binding (retention of RNAs with recognition sites) to non-specific binding (retention of RNAs without recognition sites) over a range of experimental conditions. These results suggest that binding of immobilized coat protein to RNAs containing two sites is cooperative. We illustrate the potential utility of the approach in purifying RNA-protein complexes by demonstrating that a U1 snRNP formed in vivo on an RNA containing tandem recognition sites is selectively retained by the coat protein support. 相似文献
13.
Interaction of phenylthiolato-(2,2'',2"-terpyridine)platinum(II) cation with DNA. 总被引:2,自引:2,他引:0
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The interaction between a novel aromatic thiolato derivative from the family of DNA-intercalating platinum complexes, phenylthiolato-(2,2',2"-terpyridine)platinum(II)-[PhS(ter py)Pt+], and nucleic acids was studied by using viscosity, equilibrium-dialysis and kinetic measurements. Viscosity measurements with sonicated DNA provide direct evidence for intercalation, and show that at binding ratios below 0.2 molecules per base-pair PhS(terpy)Pt+ causes an increase in contour length of 0.2 nm per bound molecule. However, helix extension diminishes at greater extents of binding, indicating the existence of additional, non-intercalated, externally bound forms of the ligand. The ability of PhS(terpy)Pt+ to aggregate in neutral aqueous buffers at a range of ionic strengths and temperatures was assessed by using optical-absorption methods. Scatchard plots for binding to calf thymus DNA at ionic strength 0.01 (corrected for dimerization) are curvilinear, concave upward, providing further evidence for two modes of binding. The association constant decreases at higher ionic strengths, in accord with the expectations of polyelectrolyte theory, although the number of cations released per bound unipositive ligand molecule is substantially greater than 1. Stopped-flow kinetic measurements confirm the complexity of the binding reaction by revealing multiple bound forms of the ligand whose kinetic processes are both fast and closely coupled. Thermal denaturation of DNA radically alters the shapes of binding isotherms and either has little effect on, or enhances, the affinity of potential binding sites, depending on experimental conditions. Scatchard plots for binding to natural DNA species with differing nucleotide composition show that the ligand has a requirement for a single G X C base-pair at the highest-affinity intercalation sites. 相似文献
14.
Analysis of glucagon-receptor interactions on isolated canine hepatocytes. Formation of reversibly and irreversibly cell-associated hormone 总被引:1,自引:0,他引:1
We have investigated the interactions of ligand with the canine hepatic glucagon receptor. Whereas time courses for radiolabeled glucagon binding to receptor and dissociation from receptor revealed fast and slow components at both 30 and 4 degrees C, time courses of ligand dissociation revealed a third component of irreversibly cell-associated (nondissociable) ligand only at the higher temperature. Related experiments identified that (a) the initial rate of formation of nondissociable ligand was slower than that of dissociably bound hormone; (b) the fraction of ligand bound to nondissociable sites achieved a plateau during extended incubations, whereas that bound to dissociable sites was seen to rise and then slowly to fall; (c) the kinetics of formation of a nondissociable ligand was consistent with linked, sequential reactions; (d) dissociable ligand-receptor complexes formed at 4 degrees C were converted to nondissociable complexes during subsequent incubation at 30 degrees C, and (e) nondissociable sites were filled by prior incubation of cells with unlabeled ligand. Analysis of receptor-bound hormone resulting from the incubation of cells with 125I-labeled glucagon and selected concentrations of either glucagon or [[127I]iodo-Tyr10]glucagon at steady state revealed in each case four components of receptor-bound ligand: those corresponding to high and low affinity components of dissociably bound ligand and to high and low affinity components of nondissociably bound ligand. Implications of these findings are considered in terms of mechanisms for the formation of irreversibly bound hormone and for the distribution of hormone among the various components of hepatic glucagon-binding sites. 相似文献
15.
Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase.
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The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of "used" columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand. 相似文献
16.
We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex. 相似文献
17.
18.
Functional heterogeneity in O2 or CO binding of sites of dissociated molluscan hemocyanin polypeptide chains (Helix pomatia and Octopus vulgaris) has been estimated by an analysis of accurate noncooperative binding curves. Three types of experiments were performed: pure O2 or CO binding, competitive displacement of one ligand by the other, and simultaneous removal of both gases from protein partially saturated with O2 and CO. The data were analyzed in terms of a model which has two fractions of sites with different properties for O2 and CO. The relative proportion of the different binding sites and their affinity constant values were found by the combined use of the three different procedures. All species show a marked functional heterogeneity of sites for O2 binding, while for CO binding it has been observed only in the case of H. pomatia beta-hemocyanin. Moreover, in all three molluscan hemocyanins examined, the two classes of O2-binding sites, although present in different proportions within the polypeptide chains, display similar affinity constant values. The data reported show a good consistency with results obtained using digested and isolated domains, providing confidence in the analytical procedure used. From comparison of the O2/CO affinity ratios (KO2, KCO) of each class it may be suggested that the difference in O2 affinity of two kinds of binding sites is related to a different local structure of the active sites. The results, moreover, unequivocally confirm that binding and displacement of two gaseous ligands to hemocyanin occur by a simple competitive mechanism, although the binding site is structurally complex and the two ligands are bound with different geometries. 相似文献
19.
20.
This paper describes a generally applicable method for quantitative investigation of ligand-dependent binding of a regulatory protein to its target DNA at equilibrium. It is used here to analyse the coupled binding equilibria of cAMP receptor protein from Escherichia coli K12 (CRP) with DNA and the physiological effector cAMP. In principle, the DNA binding parameters of CRP dimers with either one or two ligands bound are determinable in such an approach. The change of protein fluorescence was used to measure CRP binding to its recognition sequence in the lac control region and to non-specific DNA. Furthermore, the binding of cAMP to preformed CRP-DNA complexes was independently studied by equilibrium dialysis. The data were analysed using a simple interactive model for two intrinsically identical sites and site-site interactions. The intrinsic binding constant K and the co-operativity factor alpha for binding of cAMP to free CRP depend only slightly on salt concentration between 0.01 M and 0.2 M. In contrast, the affinity of cAMP for CRP pre-bound to non-specific DNA increases with the salt concentration and the co-operativity changes from positive to negative. This results from cation rebinding to the DNA lattice upon forming the cAMP-CRP-DNA complex from cAMP and the pre-formed CRP-DNA complex. The CRP-cAMP1 complex shows almost the same affinity for specific and non-specific DNA as the CRP-cAMP2 complex, and both displace the same number of cations. It is concluded that the allosteric activation of CRP is induced upon binding of the first cAMP. These results are used to estimate the occupation of the CRP site in the lac control region in relation to the cAMP concentration in vivo. Under physiological conditions the lac promoter is activated by the CRP dimer complexed with only one cAMP. Furthermore, a model for the differential activation of various genes expressed under catabolite repression is presented and discussed. 相似文献