Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “binding kinetics”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22Linked to original sources
PubMed2005

Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis.

Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.

Binding Sites↗
PubMed1975

Kinetic and binding studies of Mn (II) and fructose 1,6-bisphosphate with rabbit liver hexosebisphosphatase.

The separate interaction of the substrate fructose 1,6-bisphosphate and a metal ion cofactor Mn2+ with neutral hexosebisphosphatase has been studied under equilibrium conditions at pH 7.5 with gel filtration and electron paramagnetic resonance measurements, respectively. Binding data for both ligands to the enzyme yielded nonlinear Scatchard plots that analyze in terms of four negatively cooperative binding sites per enzyme tetramer. Graphical estimates of the binding constants were refined by a computer searching procedure and nonlinear least squares analysis. These results are qualitatively similar to those obtained from binding studies involving teh alkaline enzyme, a modified form of hexosebisphosphatase whose pH optimum is in the alkaline pH region. Both forms of the enzyme enhance the proton relaxation rate of water protons by a factor of approximately 7 to 8 at 24 MHz, demonstrating similar metal ion environments. Teh activator Co(III)-EDTA did not affect Mn2+ binding to the neutral enzyme. In the presence of (alpha + beta)methyl-D-fructofuranoside 1,6-bisphosphate, however, two sets--each containing four Mn2+ binding sites--were observed per enzyme tetramer with loss of the negatively cooperative interaction. These results are viewed in terms of four noncatalytic and four catalytic Mn2+ binding sites. Parallel kinetic investigations were conducted on the neutral enzyme to determine specific activity as a function of Mn2+ and fructose 1,6-bisphosphate concentration. A pro-equilibrium sequential pathway model involving Mn2+-enzyme and the Mn2+-fructose 1,6-bisphosphate complex both as substrate and as an allosteric inhibitor satisfactorily fit the kinetic observations. All possible enzyme species were computed from the determined binding constants and grouped according to the number of moles of Mn2+-fructose 1,6-bisphosphate complex bound to the Mn2+-enzyme, and individual rate constants were calculated. The testing of other models and their failure to describe the kinetic observations are discussed.

Animals↗
PubMed1997

Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells.

The Ca2+ binding kinetics of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer, which determine the time course of Ca2+ changes after photolysis of DM-nitrophen, were studied in bovine chromaffin cells. The in vivo Ca2+ association rate constants of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer were measured to be 5.17 x 10(8) M-1 s-1, 3.5 x 10(7) M-1 s-1, and 1.07 x 10(8) M-1 s-1, respectively. The endogenous Ca2+ buffer appeared to have a low affinity for Ca2+ with a dissociation constant around 100 microM. A fast Ca2+ uptake mechanism was also found to play a dominant role in the clearance of Ca2+ after flashes at high intracellular free Ca2+ concentrations ([Ca2+]), causing a fast [Ca2+]i decay within seconds. This Ca2+ clearance was identified as mitochondrial Ca2+ uptake. Its uptake kinetics were studied by analyzing the Ca2+ decay at high [Ca2+]i after flash photolysis of DM-nitrophen. The capacity of the mitochondrial uptake corresponds to a total cytosolic Ca2+ load of approximately 1 mM.

Acetates↗
PubMed1995

Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated with a novel large-scale purification method.

A novel, large-scale method for the purification of cytochrome-c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme a/protein ratio of 9.1 mmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. N-terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.-W. & Capaldi, R.A. (1992) J. Biol. Chem. 267, 22,481-22,485]. 15 of the N-terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (M(r) 5364). We conclude that subunit XII postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome-c oxidase contains eleven rather than twelve subunits. We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25% of charged amino acids and hydropathy analysis predicts one membrane-spanning helix. The purified enzyme had a turnover number of 1500 s-1 and the ionic-strength dependence of the Km value for cytochrome-c was similar to that described for other preparations of cytochrome-c oxidase. This was also true for the cyanide-binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide-binding kinetics and was resistant to formate incubation, indicating that chloride was bound to the binuclear center. When the enzyme was isolated in the absence of chloride, approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of optical and EPR spectra for cytochrome-c oxidase purified with our method.

Amino Acid Sequence↗
PubMed1986

Analysis of one-component saturable systems such as ligand binding, enzyme kinetic, uptake, and transport data.

Statistical methods for analyzing enzyme kinetic or drug binding assays involving simple first-order kinetics have been reviewed. The Eadie-Hofstee format combines many attractive features. Specifically, this plot is as efficient as any of the alternatives, shows no tendency to produce grossly aberrant values, does not require explicit weighting, does not require an iterative fitting process, gives the parameters of interest and their errors of estimate directly, and is unbiased (after correction). A correction for the bias inherent in the Eadie-Hofstee format has been developed. A numerical example and computer program are given in appendices.

Biological Transport↗
PubMed1997

Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding.

The association of the TATA binding protein (TBP) to eukaryotic promoters is a possible rate-limiting step in gene expression. Slow promoter binding might be related to TBP's ability to occlude its DNA binding domain through dimerization. Using a "pull-down" based assay, we find that TBP dimers dissociate slowly (t1/2 = 6-10 min), and thus present a formidable kinetic barrier to TATA binding. At 10 nM, TBP appears to exist as a mixed population of monomers and dimers. In this state, TATA binding displays burst kinetics that appears to reflect rapid binding of monomers and slow dissociation of dimers. The kinetics of the slow phase is in excellent agreement with direct measurements of the kinetics of dimer dissociation.

DNA↗
PubMed1989

Kinetics of binding of chicken cystatin to papain.

The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions, i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 microM. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 X 10(6) M-1 s-1 at 25 degrees C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25 degrees C of 31.5 kJ mol-1 and an entropy of activation at 25 degrees C of -7 J K-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of 9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with an excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystatins↗
PubMed1975

Prothrombin fragments. Ca2+ binding and activation kinetics.

The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots of these data indicate that prothrombin (Mr = 70,000) has 10 to 11 Ca2+ binding sites which can be differentiated in terms of their binding affinity. Six of these Ca2+ binding sites have log Kassoc = 3.5 and all are found intact in the NH2-terminal segment (activation intermediate 3, Mr = 23,000) of the prothrombin molecule. Four or five additional weaker binding sites for Cz2+ with log Kassoc = 2.7 present in prothrombin are found intact in the remaining COOH-segment (activation intermediate 1, Mr = 51,000) of the prothrombin molecule. Upon further activation the Ca2+ binding sites residing in intermediate 1 are found intact in activation intermediate 4 (which constitutes the NH2-terminal segment of the intermediate 1 molecule). The remaining COOH-terminal portion (activation intermediate 2, Mr = 41,000) of the intermediate 1 molecule has no affinity for Ca2+. The activation of prothrombin and activation intermediates 1 and 2 was studied using these activators: Factor Xa alone, Factor Xa-Ca+, AND Factor Xa-Ca2+-phospholipid. The rate of thrombin production from prothrombin was progressively increased as Ca2+ and phospholipid were added to the system, whereas no significant increase in the rates of activation of intermediate 1 and 2 was observed. When Factor V was added to the Factor Xa-Ca2+-phospholipid system, the rate of activation of intermediate 1 was greatly enhanced. In the absence of Ca2+, Factor V had no effect on the rate of thrombin formation from intermediate 1. Factor V had no stimulatory effects on the rate of intermediate 2 activation. However, in the presence of an equimolar amount of intermediate 4, Factor V accelerated the conversion of intermediate 2 to thrombin. These studies indicate that the Ca2+ binding sites of the prothrombin molecule are contained in the "pro" fragment (intermediates 3 and 4) of the prothrombin molecule. Intermediate 1 and intermediate 2, both of which lack the strong Ca2+ binding sites of prothrombin, are poor substrates for the Factor Xa-Ca2+-phospholipid complex activation when compared to prothrombin. The addition of Factor V to the catalyst results in acceleration of the activation rate of intermediate 1 and an equimolar mixture if intermediates 2 and 4. These results lead us to conclude that the strong Ca2+ binding sites are the sites of phospholipid binding (intermediate 3), whereas the seak binding sites are the sites of Factor V binding (intermediate 4).

Binding Sites↗
PubMed2006

Electrostatic steering at acetylcholine binding sites.

The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.

Acetylcholine↗
PubMed2000

Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor.

The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy. These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC. The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR. Analytical ultracentrifugation further indicated that the complex comprised 2EGF:2sEGFR, consistent with the model proposed recently by Lemmon et al. (1997). Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF:sEGFR interaction and that more complex interaction mechanisms are operative. Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity. Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2-20 nM and the other with a KD of 400-550 nM.

Amino Acid Sequence↗
PubMed1982

Role of actinomycin pentapeptides in actinomycin-deoxyribonucleic acid binding and kinetics.

Results are reported on equilibrium and kinetic experiments probing the DNA binding properties of a series of actinomycin analogues differing at the 3'-amino acid position. While the parent compound, actinomycin D, contains proline at this position on both pentapeptide lactone rings, the analogues under consideration here contain either azetidine-2-carboxylic acid, pipecolic acid, or 4-ketoproline on one or both pentapeptide rings. This study extends our earlier results on doubly substituted analogues [Shafer, R.H., Burnett, R. R., & Mirau, P.A. (1980) Nucleic Acids Res. 8, 1121]. DNA binding constants were determined from Scatchard plots constructed from visible absorption data and covered the range of (0.3-9) X 10(6) M-1 for the whole series of analogues. The thermal denaturation temperature of calf-thymus DNA was increased by 3-17 degrees C. DNA dissociation kinetics, along with enthalpies and entropies of activation, were also determined. The time constant for the slowest dissociation process ranged from 278 to 10 900 s. The strongest DNA binding analogue, in terms of the largest binding constant, the largest increase in DNA thermal denaturation temperature, and the slowest DNA dissociation rate, was actinomycin V, which has 4-ketoproline in the beta peptide ring, while the weakest DNA binding analogue has pipecolic acid on both peptide rings. Evidence is presented for one peptide ring exerting a greater influence than the other in the interaction with DNA. Also, the possible role of cis-trans isomerization about one or two peptide bonds in determining the slow DNA binding kinetics is discussed.

Animals↗
PubMed2003

Kinetic analysis of human topoisomerase IIalpha and beta DNA binding by surface plasmon resonance.

Topoisomerase IIbeta binding to DNA has been analysed by surface plasmon resonance for the first time. Three DNA substrates with different secondary structures were studied, a 40 bp oligonucleotide, a four way junction and a 189 bp bent DNA fragment. We also compared the DNA binding kinetics of both human topoisomerase isoforms under identical conditions. Both alpha and beta isoforms exhibited similar binding kinetics, with average equilibrium dissociation constants ranging between 1.4 and 2.9 nM. We therefore conclude that neither isoform has any preference for a specific DNA substrate under the conditions used in these experiments.

Antigens, Neoplasm↗
PubMed2001

Polyoxometalate HIV-1 protease inhibitors. A new mode of protease inhibition.

Nb-containing polyoxometalates (POMs) of the Wells-Dawson class inhibit HIV-1 protease (HIV-1P) by a new mode based on kinetics, binding, and molecular modeling studies. Reaction of alpha(1)-K(9)Li[P(2)W(17)O(61)] or alpha(2)-K(10)[P(2)W(17)O(61)] with aqueous H(2)O(2) solutions of K(7)H[Nb(6)O(19)] followed by treatment with HCl and KCl and then crystallization affords the complexes alpha(1)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(1)()1) and alpha(2)-K(7)[P(2)W(17)(NbO(2))O(61)] (alpha(2)()1) in 63 and 86% isolated yields, respectively. Thermolysis of the crude peroxoniobium compounds (72-96 h in refluxing H(2)O) prior to treatment with KCl converts the peroxoniobium compounds to the corresponding polyoxometalates (POMs), alpha(1)-K(7)[P(2)W(17)NbO(62)] (alpha(1)()2) and alpha(2)-K(7)[P(2)W(17)NbO(62)] (alpha(2)()2), in moderate yields (66 and 52%, respectively). The identity and high purity of all four compounds were confirmed by (31)P NMR and (183)W NMR. The acid-induced dimerization of the oxo complexes differentiates sterically between the cap (alpha(2)) site and the belt (alpha(1)) site in the Wells-Dawson structure (alpha(2)()2 dimerizes in high yield; alpha(1)()2 does not). All four POMs exhibit high activity in cell culture against HIV-1 (EC(50) values of 0.17-0.83 microM), are minimally toxic (IC(50) values of 50 to >100 microM), and selectively inhibit purified HIV-1 protease (HIV-1P) (IC(50) values for alpha(1)()1, alpha(2)()1, alpha(1)()2, and alpha(2)()2 of 2.0, 1.2, 1.5, and 1.8 microM, respectively). Thus, theoretical, binding, and kinetics studies of the POM/HIV-1P interaction(s) were conducted. Parameters for [P(2)W(17)NbO(62)](7)(-) were determined for the Kollman all-atom (KAA) force field in Sybyl 6.2. Charges for the POM were obtained from natural population analysis (NPA) at the HF/LANL2DZ level of theory. AutoDock 2.2 was used to explore possible binding locations for the POM with HIV-1P. These computational studies strongly suggest that the POMs function not by binding to the active site of HIV-1P, the mode of inhibition of all other HIV-1P protease inhibitors, but by binding to a cationic pocket on the "hinge" region of the flaps covering the active site (2 POMs and cationic pockets per active homodimer of HIV-1P). The kinetics and binding studies, conducted after the molecular modeling, are both in remarkable agreement with the modeling results: 2 POMs bind per HIV-1P homodimer with high affinities (K(i) = 1.1 +/- 0.5 and 4.1 +/- 1.8 nM in 0.1 and 1.0 M NaCl, respectively) and inhibition is noncompetitive (k(cat) but not K(m) is affected by the POM concentration).

Anti-HIV Agents↗
PubMed1996

Kinetics of association and dissociation of two enantiomers, NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI 980), to tubulin.

The kinetics of binding of R- and S-enantiomers were studied by the fluorescence stopped-flow technique. For the R-enantiomer, the time course of the increase in fluorescence is best fitted by a sum of two exponentials. In pseudo-first-order conditions, the first observed rate constant showed a linear concentration dependence whereas the second showed a hyperbolic one. The dissociation rate constants were determined independently by displacement experiments with 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). The two exponential phases were assumed to be due to a two-step binding mechanism: an initial binding followed by a conformational change. This is different from colchicine and MTC binding, where the two phases show a hyperbolic concentration dependence and are attributed to the parallel binding to different isoforms of tubulin [Banerjee, A., & Luduena, R. F. (1992) J. Biol. Chem. 267, 13335-13339]. R-isomer binding did not discriminate between the tubulin isoforms. The temperature dependence of all the rate constants were measured, and the entire thermodynamic reaction path was constructed. For the S-isomer, the direct fluorescence stopped-flow study showed that the signals were largely imputable to the fluorescence of the binding at low-affinity sites [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10674-10682]. Therefore, we exploited the competition between R- and S-isomers to determine the binding kinetics of the S-isomer to the R-site. The observed rate constants for competitive binding showed a linear concentration dependence, thus allowing us to calculate the association rate constant of the S-isomer to the R-site. The kinetics of displacement of the S-isomer by MTC allowed the dissociation rate constant for the S-isomer to be determined. The binding of both enantiomers to tubulin in presence of tropolone methyl ether (analog of the colchicine C ring) was decreased, indicating the involvement of the C subsite.

Animals↗
PubMed1988

Kinetics of binding of phospholipase A2 to lipid/water interfaces and its relationship to interfacial activation.

The time-course of binding of phospholipase A2 and prophospholipase A2 to vesicles and micelles of a variety of substrate and nonhydrolyzable phospholipid analogs is obtained by monitoring the change in the fluorescence intensity of Trp-3 on the protein or of the 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chromophore on the surface of the vesicles. The time-dependent increase in the fluorescence intensity of phospholipase A2 is observed only under conditions where catalysis and equilibrium binding are also observed. The overall kinetics of binding is described by two rate constants. A rapid second-order rate constant (ka) for binding of both the proteins is 2.10(7) per s per mol expressed in terms of phospholipids as monomers, and 10(10) per s per mol expressed in terms of vesicles. This is probably a diffusion-limited encounter of the protein with vesicles as the first step in binding. An additional first-order rate constant (kb = 4 per s) was also discerned for the binding of phospholipase A2 but not for prophospholipase A2. The rate of desorption of the bound iphospholipase A2 in the presence of EGTA is very slow (less than 0.0002 per s), whereas the rate of desorption of the bound prophospholipase A2 is much more rapid (2.9 per s). The mechanistic significance of these rate constants is elaborated in terms of the differences in the rates of interfacial catalytic turnover of phospholipase A2 and prophospholipase A2. As shown elsewhere (Jain et al. Biochim. Biophys. Acta 860, 435-447) the hydrolysis of anionic vesicles by phospholipase A2 occurs in the scooting mode such that the bound enzyme remains on the target vesicles for several thousand catalytic turnover cycles. On the other hand, as shown in this paper, the kinetics of hydrolysis by prophospholipase A2 is dominated by its intervesicle exchange. Therefore, interfacial catalysis by prophospholipase A2 in the hopping mode would involve an on- and an off-step in each cycle, resulting in a catalytic turnover number of about 1.2 per s. A change from the hopping to the scooting mode of catalysis thus provides the kinetic basis for activation of interfacial catalysis by phospholipase A2 compared to that for prophospholipase A2.

Animals↗
PubMed2000

Stopped-flow and Brownian dynamics studies of electrostatic effects in the kinetics of binding of 7-methyl-GpppG to the protein eIF4E.

The kinetics of binding 7-methyl-GpppG, an analogue of the 5'-mRNA cap, to the cap-binding protein eIF4E, at 20 degrees C, in 50 mM Hepes-KOH buffer, pH 7.2, and 50, 150 and 350 mM KCl, was measured using a stopped-flow spectrofluorometer, and was simulated by means of a Brownian dynamics method. For most of the stopped-flow measurements a single bimolecular step is an inadequate description of the binding mechanism and an additional step is required to accommodate the kinetic data. The rate constants derived from assumed one-step and two-step binding models were determined. The forward rate constants towards the complex formation decrease, and the reverse rate constants increase, with increasing ionic strength. The association rate constants derived from the stopped-flow measurements and the computed diffusional encounter rate constants agree, indicating that the first observed step can be viewed as a diffusionally controlled encounter of the protein and the ligand. Moreover, comparison of experimental and computed bimolecular association rate constants indicate that the experimentally observed decrease of the rate constants with the increasing ionic strength is caused by two factors. The first is less effective steering of the ligand towards the binding site at higher ionic strengths, and the second is that for higher ionic strengths the ligand must be closer to the binding site to induce the fluorescence quenching.

Animals↗
PubMed2005

Fibrillin-1 interactions with heparin. Implications for microfibril and elastic fiber assembly.

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.

Animals↗
PubMed2000

Differential antiplatelet efficacy for various GPIIb/IIIa antagonists: role of plasma calcium levels.

OBJECTIVES: The present study was undertaken to determine the effects of free ionized calcium influenced by either the anticoagulant used (citrate vs. heparin) or directly varying the calcium levels after treatment of blood with citrate on the antiplatelet efficacy of two classes of GPIIb/IIIa antagonists. METHODS: The platelet effects of changes in plasma [Ca(++)] with the different GPIIb/IIIa antagonists were determined using light transmittance aggregometry, direct binding kinetics, and (125)I-fibrinogen binding to activated human platelets. RESULTS: A significantly higher IC50s was shown with heparin (free ionized calcium=1.1 mM) as compared to that with citrate (free ionized calcium=0.12 mM) with class II GPIIb/IIIa antagonists (P<0.01) such as Orbofiban, and Integrilin. In contrast, class I GPIIb/IIIa antagonists such as Roxifiban and Abciximab showed no significant changes in their IC50s in either citrate or heparin. Similar data were shown with other non-calcium chelating anticoagulant such as PPACK as compared to that with heparin. Additionally, similar data were shown with regard to the [Ca(++)] sensitivity for GPIIb/IIIa antagonists from Class II but not Class I in the changes in IC50 values required for the inhibition of (125)I-fibrinogen binding to activated human gel filtered platelets. Additionally, examples from Class I GPIIb/IIIa antagonists such as (3)H-active form of Roxifiban showed no significant changes in its platelet binding affinity in response to change in [Ca(++)]. In contrast, GPIIb/IIIa antagonists from class II such as (3)H-active form of Orbofiban demonstrated significant changes (P<0.01) in its platelet binding kinetics and antiplatelet efficacy in response to changes in Ca(++) concentrations. CONCLUSIONS: These data suggest the impact of the method of blood collection or changes in plasma calcium levels on the antiplatelet efficacy for class II but not class I GPIIb/IIIa antagonists depending on their platelet binding kinetics.

Abciximab↗