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At least 253 records · Page 14Linked to original sources
PubMed1995

Irreversible binding kinetics of Bacillus thuringiensis CryIA delta-endotoxins to gypsy moth brush border membrane vesicles is directly correlated to toxicity.

To examine the binding of Bacillus thuringiensis delta-endotoxins, CryIAa, CryIAb, and CryIAc, to Lymantria dispar (gypsy moth) brush border membrane vesicles (BBMV), saturation kinetic analyses were conducted according to a two-step interaction scheme [formula: see text] for delta-endotoxin binding to BBMV, rather than the one-step reversible binding presented in prior reports. The order of toxicity of the delta-endotoxins, as measured by the dose required for a 50% inhibition of weight gain (ID50), was CryIAa (77.3 ng) > CryIAb (157 ng) > CryIAc (187 ng). While both the maximum extent of binding, Bmax, and the half-maximum insertion rate concentration, K1/2, was observed to be indirectly related to toxicity, the rate constant of irreversible binding, k2, was found to be directly correlated to toxicity.

Animals↗
PubMed2006

Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells.

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds, reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles, indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5 to 8 microm2/s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, and U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes.

Binding Sites↗
PubMed1980

Binding kinetics of vinyl chloride and vinyl bromide at very low doses.

Vinyl chloride and vinyl bromide are metabolically activated by liver microsomal enzymes to intermediates that covalently bind to proteins and nucleic acids. Several lines of evidence suggest the involvement of the epoxides, i.e., chloroethylene oxide or bromoethylene oxide. Proven targets for alkylation are adenine, cytosine and guanine moieties in nucleic acids, and sulfhydryl groups of proteins. For all the halogenated ethylenes studied so far, including vinyl chloride and vinyl bromide, metabolism in vivo is a dose-dependent, saturable process. The metabolic capacity of rats is saturated at atmospheric concentrations of 250 ppm vinyl chloride and 55 ppm vinyl bromide. As recent reports describe a diminishment of hepatocellular glutathione in rats after exposure to vinyl chloride concentrations of 50 ppm and more, we carried out a series of experiments measuring covalent binding of vinyl chloride metabolites after exposure to different concentrations of 14C-vinyl chloride. In all of these experiments, including one of an exposure to only 2 ppm vinyl chloride, hepatic covalent protein binding was related to the dose of vinyl chloride which was actually metabolized, and the ratio between bound and metabolized material was constant. This strongly suggests that hepatic glutathione levels must have only a very limited impact on covalent protein binding of vinyl chloride metabolites, an assumption which is supported by a lacking effect of a pretreatment with diethylmaleate. A scheme of hepatocellular compartimentation of metabolic steps is proposed which serves to explain these findings.

Alkylation↗
PubMed1977

Mechanisms of lymphocyte activation. Binding kinetics of phytohemagglutinin to human lymphocytes.

The interactions of phytohemagglutinin (PHA) with normal human lymphocytes were studied utilizing radioiodinated leukoagglutinin (125I-LPHA) over a concentration spectrum encompassing the entire range of lymphocyte metabolic responses. 125I-LPHA binding was temperature-, pH-, and time-dependent. Ligand association was rapid with a t1/2 of 3 to 5 min, reaching steady state in 30 min at 22 degrees. Receptor specificity was demonstrated by the high receptor affinity for 125I-LPHA and by quantitative inhibition of 125I-LPHA binding with LPHA and 127I-LPHA but not with concanavalin A or bovine serum albumin. Under our experimental conditions there was no measurable degradation of 125I-LPHA and no detectable shedding of 125I-LPHA receptors or receptor-125I-LPHA complexes. Equilibrium studies of 125I-LPHA interactions with specific lymphocyte membrane receptors generated a complex curvilinear Scatchard plot. This, added to progressive deceleration of the dissociation reaction inversely proportional to receptor occupancy by 125I-LPHA, reflects changing receptor affinity for the ligand and suggests site-site interactions of the negative cooperativity type. These interactions which appear to be common to all lymphocyte subpopulations, preclude accurate calculation of lymphocyte binding capacity for 125I-LPHA and of physically meaningful affinity constants. Although the fate and role of a small fraction of apparently nondissociable 125I-LPHA remains to be elucidated, occupancy-dependent receptor affinity for 125I-LPHA, dissociation of receptor-125I-LPHA complexes, retention of binding properties by cell-exposed 125I-LPHA, and the large numbers of spare surface receptors for 125I-LPHA might represent important mechanisms for modulating cell activation by 125I-LPHA.

Binding Sites↗
PubMed1996

Interaction of phosphorylated FcepsilonRIgamma immunoglobulin receptor tyrosine activation motif-based peptides with dual and single SH2 domains of p72syk. Assessment of binding parameters and real time binding kinetics.

To examine the characteristics of the interaction of the FcepsilonRIgamma ITAM with the SH2 domains of p72(syk), the binding of an 125I-labeled dual phosphorylated FcepsilonRIgamma ITAM-based peptide to the p72(syk) SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72(syk) SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcepsilonRIgamma ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

Amino Acid Sequence↗
PubMed2001

Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca2+-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca2+ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca2+-occupied domain with vesicles. Ca2+ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k(off)) and increasing the association rate constant (k(on)) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 microM Ca2+, k(off) and k(on) are 8.9 s(-1) and 1.2 x 10(10) M(-1) x s(-1), respectively. The k(off) value increases to 150 s(-1) when free Ca2+ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (tau = k(off)(-1)) from 110 ms in the presence of Ca2+ to 6.7 ms when Ca2+ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain-membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl2 in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k(on) and increasing k(off) measured in the presence of 200 microM Ca2+. These species do not affect the k(off) value when Ca2+ is rapidly removed. Studies with PKC betaII reveal that Ca2+-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k(off) for full-length PKC betaII increases when Ca2+ is rapidly removed, reducing tau from 11 s in the presence of Ca2+ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC betaII-membrane ternary complex in the presence of Ca2+, with rapid membrane release triggered by removal of Ca2+. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.

Binding Sites↗
PubMed1983

Monoiodoinsulin labelled in tyrosine residue 16 or 26 of the B-chain or 19 of the A-chain. II. Characterization of the kinetic binding constants and determination of the biological potency.

The binding affinity to insulin receptors in isolated rat adipocytes at 37 degrees C of the four isomers of [125I]monoiodoinsulin was ranked as B26 greater than B16 = A14 greater than A19. It was demonstrated that the difference in affinity was mainly due to a change in the association rate constant, rather than in the dissociation rate constant. At steady state in the binding process the fraction of cell-associated 125I-activity eluting from a Sephadex G-50 Fine column at a position identical to that of iodoinsulin was greater than 90% and independent of the position of the iodine. It was also shown that the formation of [125I]-monoiodotyrosine as a consequence of receptor-mediated degradation was proportional to the respective binding affinities of the four isomers. The two isomers with binding affinities different from that of [A14-Tyr-125I]monoiodoinsulin (i.e. the B26 and the A19 isomers, respectively) were shown to have biological potencies which corresponded within +/- 8% to the observed changed binding affinities. In cultured human lymphocytes of the IM-9 line the hierarchy of binding affinities at 37 degrees C was B26 greater than B16 greater than A14 greater than A19, and in cultured human colon adenocarcinoma cells of the HT-29 line the binding affinities were ranked in the order B26 greater than B16 greater than A14 greater than or equal to A19 indicating that the functional properties of the insulin receptor vary within cell types and/or species.

Adipose Tissue↗
PubMed1984

Different forms of the oxysterol-binding protein. Binding kinetics and stability.

Based upon measurements of the sedimentation coefficient and the Stokes radii, three forms of the oxysterol-binding protein were identified. The unliganded binding protein was the largest (7.7 S, Stokes radius = 71.6 A, Mr = 236,000) was relatively asymmetric (f/f0 = 1.7), and was composed of at least three subunits. Binding of 25-hydroxycholesterol was associated with a reduction in the size of the protein (7.5 S, Stokes radius = 50 A, Mr approximately 169,000) and an increase in symmetry (f/f0 = 1.4), due to the loss of a subunit of Mr approximately 67,000. At pH 6 or lower, the Mr = 169,000 sterol-protein complex was altered so that reversible dissociation to give a smaller (4.2 S, Stokes radius = 53 A, Mr = 97,000) more asymmetric (f/f0 = 1.8) sterol-protein complex occurred when it was sedimented in a sucrose gradient buffered at pH 7.4 containing 0.3 M KCl and 2.5 M urea. Irreversible dissociation of the 7.5 S, Mr = 169,000 form to a 4.2 S form occurred spontaneously when the complex in whole cytosol buffered at pH 7.8 was allowed to stand overnight at 0 degree C, or when the partially purified complex was incubated at pH 5.5 at 0 degree C for several days. The partially purified, unliganded binding protein was unstable at 0 degree C (approximately 75% loss of binding activity in 24 h) whereas the liganded protein was stable for 7 days at 0 degree C although irreversible conversion to a 4.2 S form occurred under some conditions. Rates of sterol binding and dissociation were increased in the presence of 2.5 M urea at pH 7.4 or when the pH was lowered to 5.5 Kd values were not greatly altered under the various incubation conditions.

Animals↗
PubMed1996

Analysis of mass transport-limited binding kinetics in evanescent wave biosensors.

It is shown that currently used methods for analyzing surface plasmon resonance or resonant mirror biosensor data do not adequately take into account the effects of mass transport on the kinetics of ligand association and dissociation. Conventional analyses may yield arbitrary apparent reaction rate constants lying between the mass transport rate constant and the true intrinsic chemical binding rate constants, depending on the choice of ligand concentrations used in the experiments. A new kinetic analysis of biosensor data, based upon a phenomenological two-compartment approximate description of transport, is presented and tested on experimental data and on simulated data generated with a computer model for combined mass transport and reversible binding to a single class of immobilized sites. Results of the analysis indicate the extent to which the experimental binding progress curve is transport controlled and whether or not values of chemical rate constants may be validly extracted from the data. The new analysis is independent of the details of the transport process, simple in its application, and in favorable cases permits determination of the correct values of chemical rate constants that are 10- to 100-fold greater than those that can be correctly evaluated by previous analyses.

Biological Transport↗
PubMed2005

Scanning electrochemical microscopy as a probe of Ag+ binding kinetics at Langmuir phospholipid monolayers.

A new method has been developed for measuring local adsorption rates of metal ions at interfaces based on scanning electrochemical microscopy (SECM). The technique is illustrated with the example of Ag+ binding at Langmuir phospholipid monolayers formed at the water/air interface. Specifically, an inverted 25 microm diameter silver disc ultramicroelectrode (UME) was positioned in the subphase of a Langmuir trough, close to a dipalmitoyl phosphatidic acid (DPPA) monolayer, and used to generate Ag+ via Ag electro-oxidation. The method involved measuring the transient current-time response at the UME when the electrode was switched to a potential to electrogenerate Ag+. Since the Ag+/Ag couple is reversible, the response is highly sensitive to local mass transfer of Ag+ away from the electrode, which, in turn, is governed by the interaction of Ag+ with the monolayer. The methodology has been used to determine the influence of surface pressure on the adsorption of Ag+ ions at a phospholipid (dipalmitoyl phosphatidic acid) Langmuir monolayer. It is shown that the capacity for metal ion adsorption at the monolayer increased as the density of surface adsorption sites increased (by increasing the surface pressure). A model for mass transport and adsorption in this geometry has been developed to explain and characterise the adsorption process.

Adsorption↗
PubMed1992

Binding kinetics of an anti-dinitrophenyl monoclonal Fab on supported phospholipid monolayers measured by total internal reflection with fluorescence photobleaching recovery.

Fluorescence photobleaching recovery with total internal reflection illumination (TIR-FPR) has been used to measure the dissociation kinetics of a fluorescein-labeled anti-dinitrophenyl monoclonal Fab specifically bound to supported monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine. The fluorescence recovery curves were not monoexponential; when analyzed as a sum of two exponentials, the rates and fractional recoveries were approximately 1 s-1 (approximately 50%) and approximately 0.1 s-1 (approximately 30%). The data did not change as a function of the Fab solution concentration, indicating that the fluorescence recovery curves were not influenced by the rate of diffusion in bulk solution. Also, the recovery curves were independent of the size of the illuminated area, indicating that surface diffusion did not significantly contribute to the rate and shape of the fluorescence recovery. The measured off rates and apparent association constant (1.6 x 10(5) M-1) were analyzed with the theoretical formalism for a proposed mechanism that accounts for the nonmonoexponential kinetics.

Animals↗
PubMed1981

Explanation of anomalous binding kinetics with a high yield immobilized enzyme system.

The activities of glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) from commercial preparations do not give typical adsorption curves upon immobilization on non-porous polyethylenimine-coated glass microbeads. The cause of this effect with glucose oxidase was investigated. Protein binding exhibited a rectangular hyperbolic adsorption isotherm, approaching saturation at high concentrations, however, enzyme activities did not. The isotherm for activities exhibited a maxima which corresponded to less than 50% saturation with regard to total protein adsorption. The enzyme preparation was found to contain small quantities of several low molecular weight impurities as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These impurities apparently compete with glucose oxidase for binding. When large excesses of protein are added to beads, the binding of impurities becomes significant and the amount of enzyme activity per unit of bead is reduced.

Catalase↗
PubMed1991

Binding kinetics of quinuclidinyl benzilate and methyl-quinuclidinyl benzilate enantiomers at neuronal (M1), cardiac (M2), and pancreatic (M3) muscarinic receptors.

We analyzed the competition kinetics of quinuclidinyl benzilate (QNB) and QNB methiodide enantiomers on human NB-OK1 neuroblastoma (M1), rat cardiac (M2), and rat pancreas (M3) muscarinic binding sites. The association rate constants of the four drugs depended on the receptor subtype studied and were lower with pancreas (M3) (1-9 x 10(5) M-1 sec-1) than with cardiac (M2) (1-5 x 10(6) M-1 sec-1) and NB-OK1 (M1) (1-5 x 10(6) M-1 sec-1) binding sites. At each receptor subtype, we observed no significant difference between the association rate constants of the R- and S-enantiomers of either QNB or QNB methiodide. Receptor stereoselectivity, when present, was associated with differences in unlabeled drug dissociation rate constants. The dissociation rate constant varied much more than the association rate constant, when either (R)-QNB dissociation from the three subtypes (half-life, 77 min to greater than 340 min; best fit, 40 days) or dissociation of the four drugs from each receptor subtype (half-lives varying from 1.4 min to 4 hr at M1 receptors, 1.1 to 77 min at M2 receptors, and 3.5 min to greater than 340 min at M3 receptors were obtained by competition kinetics analysis) was compared.

Animals↗
PubMed1987

Tight binding of divalent cations to monomeric actin. Binding kinetics support a simplified model.

Using the fluorescent Ca2+ selective chelator Quin2 to induce and measure the dissociation of Ca2+ from actin, we have recently found that actin binds Ca2+ and Mg2+ much more tightly than previously thought (Gershman, L.C., Selden, L.A., and Estes, J.E. (1986) Biochem. Biophys. Res. Commun. 135, 607-614). In this report, we show that the kinetics of dissociation of Ca2+ from Ca-actin and Mg2+ from Mg-actin closely parallel the fluorescence changes in 1,5-I-N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)-actin, suggesting that the 1,5-I-AEDANS-actin fluorescence directly reflects slow first-order cation exchange rather than a slow Mg2+-induced isomerization as originally proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Measuring divalent cation exchange directly, we have determined the dissociation rate constants for Ca2+ (k-Ca) and Mg2+ (k-Mg), the equilibrium dissociation constants for Ca2+ (KCa), and the ratio of cation binding affinities, KMg/Kca, to actin over the pH range 7-8. We have found that k-Ca is 5-10 times greater than k-Mg and KMg is about 4 times greater than KCa. From the data we calculate the association rate constants for Ca2+ (kCa) and Mg2+ (kMg) to be about 7 X 10(6) M-1 s-1 and 2 X 10(5) M-1 s-1, respectively. kCa appears to be diffusion-limited, but kMg is significantly smaller due to the characteristics of the Mg2+ aquo ion. These findings are consistent with a simple first-order binding model for the tight binding of divalent cations to actin.

Actins↗
PubMed2004

Proximal and distal influences on ligand binding kinetics in microperoxidase and heme model compounds.

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound heme complexes to study proximal and distal influences on ligand rebinding kinetics. We report kinetics of CO rebinding to microperoxidase (MP) and 2-methylimidazole ligated Fe protoporphyrin IX in the 10 ns to 10 ms time window. We also report CO rebinding kinetics of MP in the 150 fs to 140 ps time window. For dilute, micelle-encapsulated (monodisperse) samples of MP, we do not observe the large amplitude geminate decay at approximately 100 ps previously reported in time-resolved IR measurements on highly concentrated samples [Lim, M., Jackson, T. A., and Anfinrud, P. A. (1997) J. Biol. Inorg. Chem. 2, 531-536]. However, for high concentration aggregated samples, we do observe the large amplitude picosecond CO geminate rebinding and find that it is correlated with the absence of the iron-histidine vibrational mode in the time-resolved Raman spectrum. On the basis of these results, the energetic significance of a putative distal pocket CO docking site proposed by Lim et al. may need to be reconsidered. Finally, when high concentration samples of native myoglobin (Mb) were studied as a control, an analogous increase in the geminate rebinding kinetics was not observed. This verifies that studies of Mb under dilute conditions are applicable to the more concentrated regime found in the cellular milieu.

Animals↗
PubMed1995

The influence of a metastable structure in plasmid primer RNA on antisense RNA binding kinetics.

Replication of the ColE1 group plasmids is kinetically regulated by the interaction between plasmid-encoded primer RNA II and antisense RNA I. The binding is dependent on alternative RNA II conformations, formed during the transcription, and effectively inhibits the primer function within some time interval. In this paper, the folding pathways for the wild type and copy number mutants of ColE1 RNA II are studied using simulations by a genetic algorithm. The simulated pathways reveal a transient formation of a metastable structure, which is stabilized by copy number mutations. The folding kinetics of the proposed conformational transitions is calculated using a model of a multistep refolding process with elementary steps of double-helical stem formation or disruption. The approximation shows that the lifetime of the metastable structure is relatively long and is considerably increased in the mutants, resulting in a delay of the formation of the stable RNA II structure, which is the most sensitive to the inhibition by the antisense RNA I. Thus the effect of copy number mutations can be interpreted as a compression of the time window of effective inhibition due to an increased time spent by the RNA II in the metastable state. The implications of metastable foldings in RNA functioning are discussed.

Algorithms↗