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PubMed1993

Comparative analysis of the kinetics of binding and internalization of IL-5 in murine IL-5 receptors of high and low affinity.

The high affinity IL-5R consists of at least two chains, alpha and beta. IL-5R alpha binds IL-5 with low affinity. IL-5R beta is required to construct the high affinity IL-5R, although IL-5R beta does not bind IL-5 by itself. To characterize the roles of IL-5R alpha and IL-5R beta on the association, dissociation, and internalization of IL-5, we compared the binding kinetics and the internalization of 35S-labeled IL-5 by high affinity IL-5R (dissociation constant approximately 150 pM) bearing T88-M with those by low affinity IL-5R (dissociation constant approximately 30 nM) bearing MOPC104E. We found that association kinetics of IL-5 to either natural receptor were similar. The maximal binding of IL-5 to both high and low affinity IL-5R was rapid (within 10 min). The dissociation of IL-5 from low affinity IL-5R was rapid (t1/2 < 30 min), but that from the high affinity IL-5R was remarkably slower (t1/2 > 120 min). The internalization of IL-5 was observed only in T88-M, but not in MOPC104E, suggesting that IL-5 internalization is mediated via high affinity IL-5R. Association and dissociation kinetics observed in natural cell line were mostly reproduced by the kinetic analysis of reconstituted IL-5R on transfectants with either the IL-5R alpha or the IL-5R alpha and IL-5R beta genes. However, transfectants that expressed IL-5R alpha significantly internalized IL-5, although the level was much lower than observed with IL-5R alpha beta transfectants. These results suggest that IL-5R alpha may be involved in the internalization of IL-5, whereas IL-5R beta is responsible for slowing the dissociation and the efficient internalization of IL-5 by stabilizing the ligand-receptor complex.

Animals↗

PubMed2004

Human receptor kinetics, tissue binding affinity, and stability of mometasone furoate.

Mometasone furoate (MF) is a topically used glucocorticoid with high anti-inflammatory potency. In contrast to the wealth of data derived from clinical studies, information about the molecular pharmacology of the compound is lacking or contradictory. Thus, we elucidated the characteristics of receptor binding kinetics and receptor affinity in a bioassay. Metabolite formation was determined in human plasma and lung tissue as well as binding affinity to human lung tissue. Fast and extensive association of MF to the human glucocorticoid receptor was observed while the dissociation of the MF-receptor complex was faster compared to fluticasone propionate (FP). The relative receptor affinity of MF was calculated as 2200 (dexamethasone = 100, FP = 1800) and confirmed in a bioassay measuring the induction of the glucocorticoid regulated protein CD163 in human monocytes. In plasma and human lung tissue MF formed a 9,11-epoxy degradation product. The binding affinity of MF to human lung tissue was low compared to FP due to fast redistribution from tissue into plasma. These molecular pharmacological properties are in accordance with clinical data.

Antigens, CD↗

PubMed1981

Kinetics of binding of methyl alpha- and beta-D-galactopyranoside to peanut agglutinin: a carbon-13 nuclear magnetic resonance study.

The binding kinetics of methyl alpha- and methyl beta-D-galactopyranoside to the anti-T lectin from peanuts were studied by 13C NMR, employing methyl galactopyranosides specifically enriched in 13C at C-1. Association and dissociation rate constants, as well as their activation parameters, are reported. The association rate constants, 4.6 X 10(4) M-1 s-1 for the alpha-galactopyranoside and 3.6 X 10(4) M-1 s-1 for the beta-galactopyranoside, are several orders of magnitude below those expected for a diffusion-controlled process. For both anomers, the association rate constant was temperature independent, implying that the association process occurs without a significant activation enthalpy. However, a considerable association activation entropy was found for both ligands. The dissociation rate constants were in the range of 9-46 s-1 within a temperature range of 5-35 degrees C for the alpha-galactopyranoside, and in the range of 9-39 s-1 within a temperature range of 5-25 degrees C for the beta-galactopyranoside. A considerable dissociation activation enthalpy of ca. 10 kcal mol-1 was found for both anomers. A two-step binding model, consistent with the present NMR data and with previous UV and CD spectroscopic data, is presented.

Circular Dichroism↗

PubMed1980

Stopped-flow studies on drug-protein binding. 1. Kinetics of warfarin binding to human serum albumin.

We have studied the binding of warfarin to human serum albumin (HSA) with the stopped-flow method. At 37 degrees C the rate constant for the velocity of dissociation of the stable warfarin-HSA complex is 10 S-1 (t50% = 0.07 s). Concentration and temperature dependent association constants for warfarin binding to HSA have been measured (2.5 x 10(5) M-1 S-1 at 6 degrees C, 9.8 x 10(5) M-1 S-1 at 22 degrees C and 15.3 x 10(5) M-1 S-1 at 37 degrees C). Our experimentally obtained relaxation constants are best explained by the existence of 5 equivalent low affinity binding sites for warfarin on the HSA molecule, each capable of conversion into a high affinity site. The measured energy of activation for this conversion is 57.5 kJ M-1.

Humans↗

PubMed1986

Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388D1 macrophage-like cells.

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5 X 10(-3) dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1-10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.

Animals↗

PubMed1984

Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding.

The extended X-ray absorption fine structure (EXAFS) data show differences between the active site structures of different cytochrome oxidase preparations. In the resting (as isolated) state of the Yonetani preparation, the bridging atom between Fe3+a3 and Cu2+a3 is present [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465], whereas in another preparation (e.g., Hartzell-Beinert), this atom seems to be bound only to Fe3+a3 in a significant fraction of the molecules. Both preparations bind cyanide in a multiphasic fashion, suggesting that the resting cytochrome oxidase is not homogeneous but rather is a mixture of several forms. The proportion of these forms as detected by cyanide binding kinetics differs for different preparations. However, upon reduction and reoxidation (conversion to the "oxygenated" form) the cyanide binding kinetics become monophasic and all preparations of the oxygenated form bind cyanide at the same rate. Thus, a combination of structural and kinetic approaches seems necessary for evaluation of the nature of the active site of cytochrome oxidase in its various forms.

Animals↗

PubMed2004

Real-time measurement of in vitro peptide binding to soluble HLA-A*0201 by fluorescence polarization.

Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 degrees C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three beta-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate k(on) values (1 x 10(4) to 8.7 x 10(4) M(-1) s(-1)) and slow k(off) values (1.9 x 10(-4) to 4.3 x 10(-4) s(-1)), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies.

Cross Reactions↗

PubMed1987

Striatal D1- and D2-dopamine receptor sites are separately detectable in vivo.

We have characterized in particulate fractions of normal rat striatum the in vivo binding kinetics, binding affinity, and pharmacological profiles of [3H]SCH 23390, a ligand selective for the D1-subtype of dopamine (DA) receptor, and compared these to [3H]spiperone, a ligand classically associated with the D2 DA receptor subtype. The pharmacological specificity of each ligand's in vivo binding is very similar to binding to striatal homogenates in vitro. While similar maximum numbers (Bmax) of striatal binding sites exist in vivo compared to in vitro for both ligands, binding affinities in vivo for both ligands are reduced 125- to 200-fold compared to in vitro. In vivo binding of [3H]SCH 23390 to striatum is not increased by dopamine denervation produced by 6-hydroxydopamine lesions of the nigrostriatal pathway. In vivo binding of [3H]SCH 23390 and [3H]spiperone to striatum is not significantly reduced by increased synaptic concentration of dopamine following D-amphetamine administration. 125I-SCH 23982, the iodinated analogue of SCH 23390, localizes very highly to dopaminergic forebrain areas following i.v. administration. External imaging of mammalian and human brain D1-receptors is potentially feasible with this ligand.

Animals↗

PubMed1998

Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships.

The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol(-1). Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nM and Bmax of 94 fmol mg(-1) of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs = 0.104 min(-1); K1 =0.034 min(-1): T1/2=20 min; K(+1)=0.07 min nM(-1). Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(delta), OP2(kappa) and OP3(mu). Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)-NH2([F/G]NC(1-13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH = NC-NH2-NC(1-13)-NH2 > > NC(1-12)-NH2 > NC(1-13)-OH > > NC(1-11)-NH2, and for NC and NC(1-13)-NH2 analogues: [Tyr1]NC-NH2 > or = [Leu1]NC(1-13)-NH2 > or = [Tyr1]NC(1-13)-NH2 > or = [F/G]NC(1-13)-NH2 > > [Phe3]NC(1-13)-NH2 > [DF/G]NC(1-13)-NH2. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 microM indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor.

Animals↗

PubMed1996

Interbacterial binding among strains of pathogenic and commensal oral bacterial species.

Strong interspecies adherence was demonstrated among the periodontal pathogens Treponema denticola, Bacteroides forsythus and Porphyromonas gingivalis, and between these pathogens and the commensal plaque organism Streptococcus crista. Adherence showed specificity and demonstrated saturation binding kinetics. Binding between B. forsythus and P. gingivalis appeared to be a unimodal protein-protein interaction. Binding between the other organisms was at least bimodal involving interactions between combinations of proteins and carbohydrates with a variety of sugar specificities. Salivary molecules prevented adherence between T. denticola and S. crista, and serum eliminated B. forsythus binding to P. gingivalis. All other interactions occurred to some degree in the presence of serum and saliva. Such interbacterial binding interactions may be important in the establishment of periodontopathic plaque.

Adhesins, Bacterial↗

PubMed1996

Proton-linked subunit kinetic heterogeneity for carbon monoxide binding to hemoglobin from Chelidonichthys kumu.

The pH dependence of CO binding kinetics to Chelidonichthys kumu hemoglobin (Hb) and human adult Hb has been investigated between pH 2.0 and 9.0 at 20 degrees C. For both Hbs, CO binding kinetics is characterized by two proton-linked transitions, with different pKa values for alpha- and beta-chains in C. kumu Hb, leading to a relevant functional kinetic heterogeneity at most pH values. On the other hand, in human adult Hb the CO binding does not display a functional heterogeneity. Lowering the pH from 9 to 6 brings about a decrease of the CO binding rate constants, to a different extent for human adult Hb and the two chains of C. kumu Hb. Further lowering the pH from 6 to 2 induces an enhancement of CO binding rate constants, probably related to the protonation of proximal HisF8 Nepsilon atom and the cleavage (or severe weakening) of the HisF8-Fe bond. The presence of physiological concentrations of ATP (approximately 3 mM) affects the pH dependence of CO binding kinetics to C. kumu. Moreover, the effect of temperature (between 8 degrees C and 38 degrees C) on CO binding kinetics has been investigated in the absence of ATP at different pH values. These results allow to interpret the functional kinetic heterogeneity of C. kumu Hb on the basis of different regulatory aspects in the alpha- and beta-subunits, as suggested by structural considerations.

Adult↗

PubMed1988

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly.

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.

Algorithms↗

PubMed1991

Kinetics of the binding of immunoglobulins, antibodies and virus haemagglutination inhibitors to kaolin.

Kaolin is widely used in diagnostic virology, mainly to remove serum lipoproteins that may interfere with antibody assaying. The binding kinetics of antibody to kaolin at different pH values and with varying amounts of kaolin indicated a uniform and characteristic binding pattern for IgG with maximum adsorption at pH 5 and no adsorption at pH above 9. To avoid loss of IgG antibody adsorption with kaolin should therefore be performed at pH greater than or equal to 9. The amount adsorbed increased with the amount of kaolin used. The IgM pattern was less uniform with maximum adsorption of total IgM at about 7.0, the amount adsorbed being highly dependent on kaolin concentration. Serum lipoproteins were rapidly and strongly adsorbed independent of pH from 7 to 11 and independent of the lipoprotein content of the serum. The amount of kaolin used was, however, critical.

Adsorption↗

PubMed2006

Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface.

It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology.

Antigen-Antibody Complex↗

PubMed2000

On the kinetics of distamycin binding to its target sites on duplex DNA.

Distamycin A is a well known polyamide antibiotic that can bind in the minor groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side antiparallel dimer. The association phase of the distamycin binding reaction has not been studied in either of its binding modes, because of the lack of an adequate UV or CD signal at the low concentrations needed to monitor the fast bimolecular reaction. We report a significant increase in fluorescence amplitude, accompanied by a small red shift, on binding distamycin to its specific target sites. This signal can be used to monitor drug binding in steady-state and time-resolved processes. Distamycin shows extremely fast association with the 1:1 binding site, with a bimolecular rate of 7 x 10(7) M(-1) small middle dots(-1) and also fairly rapid dissociation ( approximately 3 s(-1)). When DNA is in excess, there is a slow component in the association reaction whose rate decreases strongly with increasing DNA concentration. Binding of the drug to the 2:1 site occurs in two distinct steps: fast, sequential binding of each drug molecule to the DNA with a bimolecular rate comparable to that at the 1:1 site, followed by a slow ( approximately 4 s(-1)) equilibration to the final population. Dissociation from the 2:1 site is approximately 40-fold slower than from the 1:1 site. This study provides the groundwork for analysis of the binding kinetics of longer polyamides and covalently linked polyamides that have recently been shown to inhibit transcription in vivo.

Anti-Bacterial Agents↗

PubMed1996

Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport.

The influence of mass transport on ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor, was investigated. A one-dimensional computer model for the mass transport of ligand between the bulk solution and the polymer gel and within the gel was employed, and the influence of the diffusion coefficient, the partition coefficient, the thickness of the matrix, and the distribution of immobilized receptor were studied for a variety of conditions. Under conditions that may apply to many published experimental studies, diffusion within the matrix was found to decrease the overall ligand transport significantly. For relatively slow reactions, small spatial gradients of free and bound ligand in the gel are found, whereas for relatively rapid reactions strong inhomogeneities of ligand within the gel occur before establishment of equilibrium. Several types of deviations from ideal pseudo-first-order binding progress curves are described that resemble those of published experimental data. Extremely transport limited reactions can in some cases be fitted with apparently ideal binding progress curves, although with apparent reaction rates that are much lower than the true reaction rates. Nevertheless, the ratio of the apparent rate constants can be semiquantitatively consistent with the true equilibrium constant. Apparently "cooperative" binding can result from high chemical on rates at high receptor saturation. Dissociation in the presence of transport limitation was found to be well described empirically by a single or a double exponential, with both apparent rate constants considerably lower than the intrinsic chemical rate constant. Transport limitations in the gel can introduce many generally unknown factors into the binding progress curve. The simulations suggest that unexpected deviations from ideal binding progress curves may be due to highly transport influenced binding kinetics. The use of a thinner polymer matrix could significantly increase the range of detectable rate constants.

Binding Sites↗

PubMedDate not supplied

Kinetics of drug binding to human serum albumin: allosteric and competitive inhibition at the benzodiazepine binding site by free fatty acids of various chain lengths.

The inhibition of dansylsarcosine (DS) binding at the benzodiazepine binding site of human serum albumin has been studied in the presence of saturated and unsaturated free fatty acids (FFA) of various chain lengths (C6-C20, C18:1, C18:2). In order to determine the mechanism of displacement, velocity constants for association (k2) and dissociation (k-2) and binding constants (KA and KA') have been measured using the stopped-flow method. The inhibitory effect of FFA on DS binding kinetics at site II is dependent of their structure. With increasing amounts of FFA the association velocity constant of DS binding decreases from 520 s-1 (fatty acid free albumin) by a factor of 3-10 and affinity decreases according to FFA chain length. Inhibition is strongest in the presence of caprylic, capric and lauric acid (C8-C12) i.e. with more than one mole FFA per mole albumin, DS association could no longer be measured. Short chain caproic and the long chain FFA C14-C20 showed only a less inhibitory effect since in the presence of a twofold excess k2 ranged between 100 and 200 s-1. Dissociation velocity of DS from the benzodiazepine binding site could be measured in relationship to FFA chain length using ibuprofene, another drug binding at site II. Dissociation velocity constants k-2 remained constant up to 2 moles FFA per mole albumin (k-2 = 16-18 s-1). A rise in k-2 to 70 s(-1) was seen, however, when 2-4 moles capric, lauric, myristic and palmitic (C10-C16) acid were bound, whereas no change was observed when increasing concentrations of caproic, caprylic, stearic and arachic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗

PubMed1990

Distribution and kinetics of GABAB binding sites in rat central nervous system: a quantitative autoradiographic study.

[3H]GABA quantitative autoradiography was used to examine the binding kinetics and regional distribution of GABAB receptors in rat brain. The regional distribution was compared to that of GABAA receptors. At 4 degrees C, [3H]GABA binding to GABAB receptors reached equilibrium within 45 min. The association and dissociation rate constants for GABAB binding to outer neocortical layers were 2.87 +/- 0.17 X 10(5) min-1 M-1 and 0.0966 +/- 0.0118 min-1, respectively, indicating a dissociation constant of 336 +/- 40 nM. Saturation binding studies in the same region yielded a dissociation constant for GABAB receptors of 341 +/- 41 nM while that of GABAA receptors was 92 +/- 10 nM. While the affinities of each type of GABA receptor were uniform across brain regions, the maximal number of binding sites for both types of GABA receptor varied across regions. The distributions of the two receptors in rat brain were different in the olfactory bulb, cerebellum, thalamus, neocortex, medial habenula and interpeduncular nucleus. Areas high in GABAB binding included the medial and lateral geniculates, the superior colliculus and certain amygdaloid nuclei. Binding to white matter tracts and ventricles was negligible. The distribution of GABAB receptors was in agreement with previously postulated sites of action of baclofen.

Animals↗