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1.
Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.  相似文献   

2.
M S Silver  A R Fersht 《Biochemistry》1983,22(12):2860-2866
The availability of epsilon DNA, a fluorescent ssDNA derivative, has made it possible to examine quantitatively the interactions between recA protein and single-stranded polynucleotides. Fluorescence titrations of epsilon DNA with recA protein and vice versa establish that each recA protein monomer covers 5.5 epsilon DNA nucleotides and that the dissociation constant of the recA-epsilon DNA complex is 10 nM. Fluorescence titrations of recA protein-epsilon DNA mixtures with poly(dT) establish that each recA protein monomer covers 5.1 poly(dT) nucleotides and that the dissociation constant of the recA-poly(dT) complex is 0.03 nM. Observations on how the addition of ssDNA affects the fluorescence of recA protein-epsilon DNA mixtures establish that the dissociation constant of the recA-ssDNA complex exceeds 20 microM. Stopped-flow kinetics in which excess recA protein binds to epsilon DNA indicate that k2 = 6 X 10(6) M-1 s-1 for the process. A more approximate kinetic technique indicates that recA protein binds to epsilon DNA at least one-tenth as fast as to poly(dT); the rate constant for dissociation of recA-epsilon DNA exceeds that for recA-poly(dT) by at least 30-fold. epsilon DNA is proven to be a versatile reagent for studying single-stranded polynucleotide-protein interactions. Not only can its own complexes with protein be investigated but also, under suitable circumstances, it can be used as a fluorescent probe to explore complexes incorporating nonfluorescent polynucleotides.  相似文献   

3.
4.
P A Whitson  K S Matthews 《Biochemistry》1986,25(13):3845-3852
The dissociation kinetics for repressor-32P-labeled operator DNA have been examined by adding unlabeled operator DNA to trap released repressor or by adding a small volume of concentrated salt solution to shift the Kd of repressor-operator interaction. The dissociation rate constant for pLA 322-8, an operator-containing derivative of pBR 322, was 2.4 X 10(-3) s-1 in 0.15 M KCl. The dissociation rate constant at 0.15 M KCl for both lambda plac and pIQ, each of which contain two pseudooperator sequences, was approximately 6 X 10(-4) s-1. Elimination of flanking nonspecific DNA sequences by use of a 40 base pair operator-containing DNA fragment yielded a dissociation rate constant of 9.3 X 10(-3) s-1. The size and salt dependences of the rate constants suggest that dissociation occurs as a multistep process. The data for all the DNAs examined are consistent with a sliding mechanism of facilitated diffusion to/from the operator site. The ability to form a ternary complex of two operators per repressor, determined by stoichiometry measurements, and the diminished dissociation rates in the presence of intramolecular nonspecific and pseudooperator DNA sites suggest the formation of an intramolecular ternary complex. The salt dependence of the dissociation rate constant for pLA 322-8 at high salt concentrations converges with that for a 40 base pair operator. The similarity in dissociation rate constants for pLA 322-8 and a 40 base pair operator fragment under these conditions indicates a common dissociation mechanism from a primary operator site on the repressor.  相似文献   

5.
Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.  相似文献   

6.
S P Jordan  L Waxman  D E Smith  G P Vlasuk 《Biochemistry》1990,29(50):11095-11100
Tick anticoagulant peptide (TAP) is a 60 amino acid protein which is a highly specific inhibitor of human blood coagulation factor Xa (fXa) isolated from the tick Ornithodoros moubata [Waxman, L., Smith, D. E., Arcuri, K. E., & Vlasuk, G. P. (1990) Science 248, 593-596]. Due to the limited quantities of native TAP, a recombinant version of TAP produced in Saccharomyces cerevisiae was used for a detailed kinetic analysis of the inhibition interaction with human fXa. rTAP was determined to be a reversible, slow, tight-binding inhibitor of fXa, displaying a competitive type of inhibition. The binding of rTAP to fXa is stoichiometric with a dissociation constant of (1.8 +/- 0.02) x 10(-10) M, a calculated association rate constant of (2.85 +/- 0.07) x 10(6) M-1 s-1, and a dissociation rate constant of (0.554 +/- 0.178) x 10(-3) s-1. Binding studies show that 35S-rTAP binds only to fXa and not to DFP-treated fXa or zymogen factor X, which suggests the active site of fXa is required for rTAP inhibition. That rTAP is a unique serine proteinase inhibitor is suggested both by its high specificity for its target enzyme, fXa, and also by its unique structure.  相似文献   

7.
The interaction of nucleic acid with the Escherichia coli DNA-binding protein has been studied by fluorescence emission spectroscopy and sedimentation velocity analysis. The protein binds to single-strand DNA with an apparent equilibrium dissociation constant of 2 X 10(-9). It binds to the homopolymers poly (dA) and poly (dT) slightly more tightly, but has a larger apparent equilibrium dissociation constant to poly (dC). The protein also binds tightly to ribohomopolymers and to tRNA, but not to duplex DNA. By the use of defined-length oligonucleotides, it has been shown that the protein binds to DNA in a highly cooperative manner. The extent of cooperativity is seen as the difference in binding between an isolated monomeric protein molecule bound to DNA and two or more molecules binding to contiguous sites.  相似文献   

8.
9.
Arc repressor is tetrameric when bound to operator DNA   总被引:10,自引:0,他引:10  
B M Brown  J U Bowie  R T Sauer 《Biochemistry》1990,29(51):11189-11195
The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.  相似文献   

10.
We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.  相似文献   

11.
The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH相似文献   

12.
Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.  相似文献   

13.
The binding of [125I] alpha-latrotoxin to synaptosomes from the rat brain is studied. It is shown that the constant rate of toxin association with the synaptosome receptor at 37 degrees C is equal to 8.2 +/- 1.3 x 10(7) M-1.s-1, while that of synaptosomal membrane -7.6 +/- 2.7 x 10(6) M-1 s-1. Depolarization of the synaptosome membrane induced by 55 mM KCl decreases the binding rate of toxin to the receptor, the rate constant being equal to 3.9 +/- 1.5 x 10(7) m-1 s-1. The pattern of the dissociation process of the toxin-receptor complex of synaptosomes and of synaptosomal membrane is different. In the first case dissociation follows two stages with the rate constants 3.6 x 10(-3) s-1 and 1.2/10(-4) s-1, in the second case it follows one stage with the constant equalled 2.0 x 10(-5) s-1. The quantity of the toxin binding sites on synaptosomes may vary under the action of agents modifying the activity of calcium fluxes which are induced by alpha-latrotoxin. It is supposed that a decrease in the ATP level in synaptosomes as well as deenergy of the surface membrane leads to a change in the state of the alpha-latrotoxin receptor.  相似文献   

14.
Contacts between the factor TUF and RPG sequences   总被引:13,自引:0,他引:13  
The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.  相似文献   

15.
B Zeeberg  M Caplow 《Biochemistry》1979,18(18):3880-3886
The dissociation constant for GDP binding to the E site of tubulin isolated by chromatography on Sepharose 6B is equal to 6.1 X 10(-8) M, as determined by the Hummel-Dryer procedure. This is smaller than any previously reported value, and the discrepancy with earlir results is analyzed.By use of a recently described column centrifugation procedure [Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899], it was established that GDP and GTP bind to the same site. GTP is bound 2.8-fold tighter than GDP, and the dissociation constant is 2.2 X 10(-8) M. A new method for the determination of dissociation constants for a protein-bound ligand, based on a quantitative analysis of the loss of ligand during exclusion chromatography, is presented. This has been used to determine that the dissociation constant for GDP bound to tubulin is equal to 5.5 X 10(-8) M, in excellent agreement with that determined independently from the Hummel-Dryer method. A previous theoretical treatment [Dixon, H. B. F. (1976) Biochem. J. 159, 161-162] of ligand loss during exclusion chromatography is discussed.  相似文献   

16.
Pressure-jump study of the kinetics of ethidium bromide binding to DNA   总被引:4,自引:0,他引:4  
Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
Uptake and destruction of 125I-CSF-1 by peritoneal exudate macrophages   总被引:3,自引:0,他引:3  
The binding and uptake of the colony-stimulating factor CSF-1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2 degrees C, and at 37 degrees C. At 2 degrees C, 125I-CSF-1 was bound irreversibly to the cell surface. At 37 degrees C, 90% of the cell surface associated 125I-CSF-1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact 125I-CSF-1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand-cell interactions at either temperature, and alternative approaches were developed. At 2 degrees C, the equilibrium constant (Kd less than or equal to 10(-13) M) was derived from estimates of the rate constants for the binding (kon congruent to 8 x 10(5) M-1 s-1) and dissociation (koff less than or equal to 2 x 10(-7) s-1) reactions. At 37 degrees C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, koff = 0.7 min-1 (t1/2 = 10 min) and internalization, kin = 0.07 min-1 (t 1/2 = 1 min). Approximately 15 min after internalization, low-molecular weight 125I-labeled degradation products began to appear in the medium. Release of this degraded 125I-CSF-1 was kinetically first order over three half-lives (Kd = 4.3 x 10(-2) min-1, t1/2 = 16 min). Thus CSF-1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.  相似文献   

18.
Using the rapid-mixing/photocross-linking technique developed in our laboratory, we have investigated the kinetics of interaction between Escherichia coli RNA polymerase and pAR1319, a recombinant plasmid DNA containing the bacteriophage T7 A2 early promoter. By monitoring the time-dependent density of bound RNA polymerase along the relaxed circular DNA molecule using this technique, we have been able to demonstrate kinetic evidence for linear diffusion of RNA polymerase along DNA in a different system from that previously described (Park, C. S., Hillel, Z., and Wu, C.-W. (1982) J. Biol. Chem. 251, 6950-6956). The nonspecific association rate constant kon was measured to be 7.7 x 10(4) M-1 s-1 at a DNA chain concentration of 22.4 nM. By taking advantage of the fact that rapid mixing displaces bound protein molecules from DNA, but leaves them within the domain of the DNA, the rate of intradomain binding of RNA polymerase to pAR1319 DNA was determined to be 8.2 s-1. Since the plasmid is described by a radius of gyration of 0.22 microns, the intradomain concentration of base pairs could be calculated. Using this concentration (180 microM), the rate constant for intradomain nonspecific association of RNA polymerase to pAR1319 DNA was estimated to be 4.6 x 10(4) M-1 s-1. In addition, a mathematical model has been used to fit the other two important rate constants to the experimental data: koff, which describes the dissociation of RNA polymerase from nonspecific binding sites, and D1, the one-dimensional diffusion coefficient of the enzyme along the DNA molecule. In this model, the circular DNA molecule is described as a ring of interconnected binding sites which together comprise a DNA "domain." RNA polymerase, which enters the domain via three-dimensional diffusion and binds to each site, is allowed to diffuse linearly between adjacent sites and three-dimensionally on and off the DNA molecule. The rate equations for the time-dependent occupancy of each site by RNA polymerase could be written, based on general principles. By solving the resulting family of differential equations, koff and D1 were determined to be 0.3 s-1 and 1.5 x 10(-9) cm2 s-1, respectively.  相似文献   

19.
Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus-Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus-Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus-Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 x 10(-9) M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of 相似文献   

20.
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