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PubMed1989

Hepatic 123I-insulin binding kinetics in non-insulin-dependent (type 2) diabetic patients after i.v. bolus administration.

Insulin binding kinetics in the liver were studied in non insulin dependent (Type 2) diabetic patients, by i.v. bolus administration of 123I-insulin. Eight Type 2 diabetic patients were compared with six male volunteers. Uptake of 123I-insulin by liver and kidneys was measured by dynamic scintigraphy with a gamma camera during 30 min. Images of liver and kidneys appeared within 2-3 min after administration of 123I-insulin at a dose of 1 mCi (37 MBq). Peak radioactivity for the liver was found 7.5 +/- 0.2 and 6.9 +/- 0.3 min after injection for the healthy and the diabetic subjects, respectively (N.S.). The percentage 123I-insulin hepatic uptake was not significantly different for the diabetic and the healthy subjects. Although a large variation exists for maximal uptake of radioactivity within both groups, the data suggest that binding differences in the liver in Type 2 diabetic patients, as compared to healthy subjects, may not account for hepatic insulin resistance.

Adult↗
PubMed2000

An Evaluation of Cellular Analyte-Receptor Binding Kinetics Utilizing Biosensors: A Fractal Analysis.

A fractal analysis is presented for cellular analyte-receptor binding kinetics utilizing biosensors. Data taken from the literature can be modeled by using (a) a single-fractal analysis and (b) a single- and a dual-fractal analysis. Case (b) represents a change in the binding mechanism as the reaction progresses on the biosensor surface. Relationships are presented for the binding rate coefficient(s) as a function of the fractal dimension for the single-fractal analysis examples. In general, the binding rate coefficient is rather sensitive to the degree of heterogeneity that exists on the biosensor surface. For example, for the binding of mutagenized and back-mutagenized forms of peptide E1037 in solution to salivary agglutinin immobilized on a sensor chip, the order of dependence of the binding rate coefficient, k, on the fractal dimension, D(f), is 13.2. It is of interest to note that examples are presented where the binding coefficient (k) exhibits an increase as the fractal dimension (D(f)) or the degree of heterogeneity increases on the surface. The predictive relationships presented provide further physical insights into the binding reactions occurring on the surface. These should assist us in understanding the cellular binding reaction occurring on surfaces, even though the analysis presented is for the cases where the cellular "receptor" is actually immobilized on a biosensor or other surface. The analysis suggests possible modulations of cell surfaces in desired directions to help manipulate the binding rate coefficients (or affinities). In general, the technique presented is applicable for the most part to other reactions occurring on different types of biosensors or other surfaces. Copyright 2000 Academic Press.

Journal Article↗
PubMed2003

Scopolamine-induced convulsions in fasted mice after food intake: determination of blood glucose levels, [3H]glutamate binding kinetics and antidopaminergic drug effects.

The present study was performed to evaluate the role(s) of hypoglycemia, changes in [(3)H]glutamate binding kinetics and dopaminergic activity in the occurrence of scopolamine-induced convulsions in fasted mice after food intake. Plasma glucose levels and density (B(max)) and affinity (K(d)) of [(3)H]glutamate binding sites in whole brain synaptic membranes were determined in animals fed ad lib or fasted for 48 h and treated intraperitoneally (i.p.) with 3 mg/kg scopolamine or saline and allowed to eat for 5 min. Fasting for 48 h decreased plasma glucose levels. After refeeding, plasma glucose concentrations increased in saline treated animals, but remained unchanged in scopolamine treated animals which consumed less food. Fasting for 48 h also produced significant changes in the kinetics of [(3)H]glutamate binding. The B(max) and K(d) of the binding sites decreased in fasted animals. These changes were partially antagonized by scopolamine treatment and food intake. For the evaluation of the contribution of dopaminergic activity, another group of mice fasted for 48 h and pretreated (i.p.) with saline or dopamine antagonists, 2 mg/kg chlorpromazine or 2 or 4 mg/kg haloperidol, were treated 10 min later with either saline or 3 mg/kg scopolamine. Then 20 min later, they were allowed to eat ad lib and were observed for 30 min for the incidence and onset of clonic convulsions. Pretreatment of both 2 mg/kg chlorpromazine and 4 mg/kg haloperidol markedly suppressed the convulsions. These results indicate that the decrease in the [(3)H]glutamate binding induced by fasting, its antagonism by scopolamine treatment and food intake, and the dopaminergic hyperactivity may be possible factors contributing to the occurrence of convulsions.

Animals↗
PubMed2005

Irreversible binding kinetics of neuropeptide Y ligands to Y2 but not to Y1 and Y5 receptors.

Neuropeptide Y (NPY) receptors type 1 (Y1), type 2 Y2) and type 5 (Y5) were tested for their kinetic properties to bind radiolabeled NPY or PYY. Rapid association and dissociation was observed with recombinant (HEK293 cells) and endogenous (SK-N-MC cells) human Y1 and recombinant mouse Y5 receptors. Recombinant (HEK293) and endogenous (SMS-KAN) human Y2 receptors bound both radiolabels comparable to the Y1 receptors, but only minimal ( approximately 20%) dissociation of both radiolabels was observed after long incubation time (>8 h). Furthermore, neither peptide nor small molecule Y2 ligands efficiently competed for binding to Y2 receptors once association binding had been initiated. The Y2-selective antagonist BIIE0246 behaved as an insurmountable antagonist in functional assays when pre-incubated for 30 min before agonist addition, but was a competitive antagonist when co-applied with the agonist. These data show that Y2 receptors in contrast to Y1 and Y5 receptors bind their ligands in an irreversible manner.

Animals↗
PubMed1988

Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements.

The Ca2+-binding kinetics of fura-2 and azo-1 were studied using temperature-jump relaxation methods. In 140 mM KCl at 20 degrees C, the association and dissociation rate constants for fura-2 were 6.02 x 10(8) M-1s-1 and 96.7 s-1, respectively. The fura-2 kinetics were insensitive to pH over the range 7.4 to 8.4. Azo-1 was studied in 140 mM KCl, at pH 7.4, at 10 degrees and 20 degrees C. At 10 degrees C, azo-1 exhibited association and dissociation rate constants of 1.43 x 10(8) M-1s-1 and 777.9 s-1, respectively; while at 20 degrees C, the corresponding values were 3.99 x 10(8) M-1s-1 and 1,177 s-1. The kinetic results demonstrate that fura-2 and azo-1 are well suited to monitoring rapid changes in intracellular [Ca2+].

Azo Compounds↗
PubMed2000

Structure determination and binding kinetics of a DNA aptamer-argininamide complex.

The structure of a DNA aptamer, which was selected for specific binding to arginine, was determined using NMR spectroscopy. The sequence forms a hairpin loop, with residues important for binding occurring in the loop region. Binding of argininamide induces formation of one Watson-Crick and two non-Watson-Crick base pairs, which facilitate generation of a binding pocket. The specificity for arginine seems to arise from contacts between the guanidino end of the arginine and phosphates, with atoms positioned by the shape of the pocket. Complex binding kinetics are observed suggesting that there is a slow interconversion of two forms of the DNA, which have different binding affinities. These data provide information on the process of adaptive recognition of a ligand by an aptamer.

Arginine↗
PubMed1990

Evidence of the receptor nature of the binding sites induced in Tetrahymena by insulin treatment. A quantitative cytofluorimetric technique for the study of binding kinetics.

Tetrahymena pyriformis GL cells pretreated (imprinted) and not pretreated with insulin showed dissimilar quantitative relations of FITC-insulin binding. Displacement of FITC-insulin by unlabelled insulin was considerably less in the control than in the imprinted series. The curve for saturation of the binding sites with FITC-insulin resembled a true saturation curve. The imprinted cells bound considerably more hormone in a shorter time than the control cells at identical levels of exposure. The dissociation of bound hormone from the imprinted cells increased over the control at 23 degrees C, and to a still greater degree at 4 degrees C. The effect of the pH of the medium on the dissociation of bound FITC-insulin also differed between the imprinted and not imprinted cells. Thus the proposed cytofluorimetric assay of binding kinetics demonstrated the actual conditions of receptor activity, and indicated that the induced insulin binding sites of Tetrahymena behaved similarly to 'classical' receptors.

Animals↗
PubMed1999

General numerical treatment of competitive binding kinetics: application to thrombin-dehydrothrombin-hirudin.

This paper describes a general numerical method for the determination of rate constants that characterize the binding of a ligand L simultaneously and competitively to two different receptor molecules, R1 and R2. The experimental data consist of changes in the concentration of one receptor (e.g., R1) monitored over time. An example problem is represented by hirudin (L) binding to thrombin (R1) and to a chemical mutant of thrombin (R2). The published experimental data [Wedemeyer et al. (1997) Anal. Biochem. 248, 130-140], previously analyzed by using an appropriate algebraic method, were reanalyzed here by numerical integration [Kuzmic (1996) Anal. Biochem. 237, 260-273]. This general numerical method offers the following advantages. (1) It provides an estimate for the lower limit on feasible values of association rate constants. (2) It is many orders of magnitude more accurate. (3) It is easily extensible to more complicated reaction mechanisms. (4) It uses a simpler formalism and it is thus more accessible to nonmathematicians. (5) A suitable computer program for the analysis of competitive binding kinetics can be obtained via the Internet (http://www.biokin.com).

Animals↗
PubMed2001

A single-fractal analysis of cellular analyte-receptor binding kinetics utilizing biosensors.

A fractal analysis of a confirmative nature only is presented for cellular analyte-receptor binding kinetics utilizing biosensors. Data taken from the literature can be modeled by using a single-fractal analysis. Relationships are presented for the binding rate coefficient as a function of the fractal dimension and for the analyte concentration in solution. In general, the binding rate coefficient is rather sensitive to the degree of heterogeneity that exists on the biosensor surface. It is of interest to note that examples are presented where the binding coefficient, k exhibits an increase as the fractal dimension (D(f)) or the degree of heterogeneity increases on the surface. The predictive relationships presented provide further physical insights into the binding reactions occurring on the surface. These should assist in understanding the cellular binding reaction occurring on surfaces, even though the analysis presented is for the cases where the cellular "receptor" is actually immobilized on a biosensor or other surface. The analysis suggests possible modulations of cell surfaces in desired directions to help manipulate the binding rate coefficient (or affinity). In general, the technique presented is applicable for the most part to other reactions occurring on different types of biosensor or other surfaces.

Animals↗
PubMed1997

Influence of diffusion to fractal surfaces on the binding kinetics for antibody-antigen, analyte-receptor, and analyte-receptorless (protein) systems.

The diffusion-limited binding kinetics of antigen-antibody, ligand-receptor, analyte-receptorless systems for biosensor applications is analyzed within a fractal framework. The analysis presented applies equally well to these types of systems. For example, for the binding of 2-(p-toluidiny)-naphthalene-6-sulfonic acid (2,6-TNS) to beta-cyclodextrin (ligand-receptor system) immobilized on a fiber-optic base inclusate biosensor, an increase in temperature from 4 to 30 degrees C leads to an increase in the fractal dimension, D1 and to a decrease in the binding rate coefficient, k1. For the binding of TRITC-labeled low-density proteins (LDL) directly to an optical fiber-based sensor (analyte-receptorless system), an increase in the LDL concentration from 5 to 50 micrograms ml-1 in solution leads to a decrease in the fractal dimension, D1 and to an increase in the binding rate coefficient, k1. Also, during the binding of human chorionic gonadotropin (hCG) to anti-hCG antibody immobilized on a HPLC column (antigen-antibody system), an increase in temperature from 4 to 50 degrees C leads to an increase in the fractal dimension, D1 and in the binding rate coefficient. k1. The different examples analyzed and presented together for the three different types of systems provide one means of a 'unified analysis,' and a method by which the forward binding rate coefficient, k1 may be controlled, that is, by changing the fractal dimension or 'disorder' on the surface. The analysis should assist in improving the stability, sensitivity, and response time of biosensors wherein different types of binding systems are utilized in the analysis method. More-or-less all of the treatment presented should be applicable to the above types of binding systems occurring in non-biosensor applications also. However, the single-fractal analysis is unable to describe the data over the full time course of some of the experiments.

Antibodies↗
PubMed1981

Influence of plasma protein binding kinetics on hepatic clearance assessed from a "tube" model and a "well-stirred" model.

The potential influence of protein binding kinetics on elimination from liver sinusoids was evaluated by means of a "well-stirred" model (I) and a "tube" model (II). When the dissociation rate constant (k-1) is at the estimated maximum, equilibrium is maintained during the passage of drug through the eliminating organ, and hence dissociation as such has no limiting effect on elimination. When, however, k-1 is at the estimated minimum, equilibrium is not maintained, the unbound fraction is reduced during the passage, and a significant decrease in the extraction ratio occurs when the unbound fraction is 0.01 or less. The models were furthermore used to investigate the effect of saturation, of both the binding protein and the elimination process, on elimination.

Blood Proteins↗
PubMed1983

Pseudomonas cytochrome C-551 peroxidase. A purification procedure and study of CO-binding kinetics.

A procedure is described for the purification of cytochrome c peroxidase from Pseudomonas aeruginosa involving extraction by sonication, followed by acid precipitation and chromatography on only two types of gel. The final preparation had a purity ratio A407/A280 of 4.2, and was found to be essentially pure by isoelectric focusing. The enzyme was shown to be unstable during degassing under vacuum except in the presence of detergent. The kinetics of CO binding to dithionite-reduced peroxidase were studied with stopped-flow and flash-photolysis techniques, and the results obtained between pH 5 and 7 suggest the existence of two forms of dithionite-reduced enzyme in slow equilibrium.

Carbon Monoxide↗
PubMed1984

Insulin receptors: binding kinetics and structure-function relationship of insulin.

During the last decade, earlier suggestions that insulin acts at the plasma membrane level via combination with receptors have been amply confirmed in studies of 125I-labeled insulin binding kinetics. Efforts have been devoted to the development of homogeneous, stable, and bioactive tracers, and a preparation of monoiodo[TyrA14]insulin showed 100-125% biological activity. The initially simple model of reversible, bimolecular, and noncooperative interaction between receptor and insulin has been revised to include the existence of at least three affinity states that may be linked to modulation of the biological response induced by the insulin-receptor complex. Thus negative cooperativity seems important in reducing oscillations of insulin action with variations in plasma insulin concentration, and formation of a high-affinity state or positive cooperativity may lead to desensitization of receptors. The kinetic phenomena suggest that receptor-binding affinity and function are actively regulated by insulin itself. At present the receptor model is purely functional and does not imply molecular mechanisms. However, recent advances in the analysis of receptor structure and biochemistry promise that the molecular equivalents of the kinetic phenomena may be elucidated in the near future. Furthermore the reaction between receptor and insulin is irreversible because of degradation of receptor-bound insulin, which may result in termination of the metabolic activation. Morphological and biochemical work suggests that internalization of the receptor-insulin complex from the plasma membrane transfers insulin to intracellular organelles like the lysosomes, the Golgi apparatus, or nucleus, where degradation by insulin protease takes place, whereas the receptor is recycled back to the membrane. Recent advances in the studies of biosynthesis and cellular dynamics of receptors indicate that intracellular processing and redistribution of binding sites may play a role in the mechanism of insulin action. Insulin receptors are widely distributed in all cell types, but evidence has accumulated that receptors show tissue and species variations in their functional properties regarding binding affinity, insulin specificity, cooperativity, and insulin degradation and in structural properties such as antigenic determinants and glycosidic composition. Perhaps these differences reflect cellular adaptations and variations in the physiological role of insulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1994

Ouabain binding kinetics of the rat alpha two and alpha three isoforms of the sodium-potassium adenosine triphosphate.

The Na,K-ATPase has three alpha isoforms which differ in cardiac glycoside sensitivity and tissue distribution. The rodent alpha 1 isoform is relatively resistant to cardiac glycosides, while the alpha 2 and alpha 3 isoforms are quite sensitive. Because both the alpha 2 and alpha 3 isoforms are generally expressed in the same tissue, it has been difficult to differentiate and accurately determine the kinetics of ouabain binding to these isoforms. To more fully understand the interactions of the alpha 2 and alpha 3 isoforms with cardiac glycosides, the association and dissociation rates of ouabain binding were measured in transfected cell lines. cDNA's coding for the rat alpha 2 and alpha 3 isoforms were transfected into NIH 3T3 cells and characterized by Na,K-ATPase activity and [3H]ouabain binding. By individually expressing the alpha 2 and alpha 3 isoforms in ouabain-insensitive NIH 3T3 cells, the ouabain-binding characteristics of each isoform could be accurately determined. The association rate constants of the alpha 2 and alpha 3 isoforms were similar while the dissociation rate constant was 33 times slower for the alpha 3 isoform than the alpha 2 isoform. Calculation of the dissociation constant (Kd) from these rate constants yielded values of 115 and 1.6 nM for rat alpha 2 and alpha 3 isoforms, respectively. Scatchard analysis of the rat alpha 2 isoform produced a similar value for Kd of 37 +/- 9 nM. Inhibition of Na,K-ATPase activity indicates the rodent alpha 1 isoform has an IC50 1000-fold higher than the alpha 2 or alpha 3 isoform at 4.8 x 10(-5) M. The results are consistent with the hypothesis that the order of ouabain affinity between the rat alpha isoforms of the Na,K-ATPase is alpha 3 > alpha 2 >> alpha 1.

3T3 Cells↗
PubMed1998

Fractal Analysis of Antigen-Antibody Binding Kinetics: Membrane Chromatography Applications.

Fractal analysis provides novel physical insights into diffusion-limited reactions occurring at interfaces. In this paper, we analyze the fractal dimension Df values for the kinetics of the binding of gamma-globulin molecules to immobilized protein A on various supports (Unarska, M., et al., J. Chromatgr. 519, 53 (1990)). Fractal analysis data seem to indicate that Sepharose beads (Df = 2.22) increase the "ruggedness" or inhomogeneity of surfaces compared to microporous membranes (Df = 1.29) for chromatography applications. Copyright 1998 Academic Press.

Journal Article↗
PubMed1987

Differential binding kinetics of cholera toxin to intestinal microvillus membrane during development.

A complete randomized block design was used to compare the binding kinetics of cholera toxin to developing rat enterocyte microvillus membranes prepared from newborn, 2-week-old, 4-week-old, and adult animals. Saturation-binding isotherms were generated on 16 independent samples (four blocks) under steady-state and reversible conditions. Scatchard analyses suggested positive cooperative binding to a single class of receptors, and the isotherms were analyzed by both the Hill-Waud and Michaelis-Menten functions. Receptor density varied significantly with age (P = 0.013). An abrupt rise in receptor density occurred after the neonatal period and normalized in the adult animal. The half-dissociation constant also varied significantly with age (P = 0.019). Microvillus membranes from suckling animals had a slightly higher apparent affinity than those from weaned animals. Neither receptor concentration nor membrane purity confounded these observations. Whereas age-related changes in apparent affinity correlated with cellular responses, changes in receptor density did not. This study suggests that developmental changes in membrane structure which influence binding affinity but not receptor density may, in part, contribute to the increased sensitivity of suckling rats to cholera toxin exposure.

Age Factors↗
PubMed1995

Binding kinetics of monoclonal antibody using antigen-beta-galactosidase hybrid protein: application to measurement of peptide antigenicity.

A simple method for determination of binding kinetics of a solid-phase antibody using antigen-beta-galactosidase hybrid protein was evaluated. To minimize conformational change of the antigen binding site of the antibody when directly binding to a microtiter plate, the microtiter plate was precoated with protein A. The binding and free antigen concentrations were directly obtained from the beta-galactosidase activity. This method can be used for analyses of the equilibrium dissociation constant (KD), and the association (Kass) and dissociation (Kdiss) rate constants. Peptide antigenicity was also analyzed by competitive ELISA using this method. Since both antigen-beta-galactosidase and the peptide used are localized in the fluid-phase, the proper affinity constant (KA) of the peptide can be estimated from the KD value of the antigen-beta-galactosidase-antibody interaction, and from the IC50 value of the peptide.

Amino Acid Sequence↗
PubMed1990

Species differences in the binding kinetics of 25-hydroxyvitamin D3 to vitamin D binding protein.

The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay.

Analysis of Variance↗