[Kinetics of binding of I-125-labeled heparin and its low-molecular-weight fraction CY 222 to the endothelium in vitro].
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Binding Sites↗
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Ascites tumor mitochondrial hexokinase II. Effect of binding on kinetic properties.
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Adenine Nucleotides↗
Progesterone-binding components of chick oviduct. IX. The kinetics of nuclear binding.
A cell-free system was used to study the kinetics of progesterone-receptor interaction with purified nuclei prepared from estrogen-primed chick oviducts. The binding process was a saturable phenomenon in both target and nontarget tissues. More nuclear acceptor sites were available to target tissue (similar to 9000 sites per oviduct nucleus) than in nontarget tissues (similar to 1000 to 3000 sites per nucleus), but the binding constant was essentially the same (Kd similar to 10-8 M). A second much smaller class of higher affinity sites (Kd similar to 10-11M) may exist. Its presence was detected by Scatchard plot nonlinearity at very low concentrations of added receptor-hormone complex (similar to 10-10 to 10-12M). The current study focused on the prevalent class of acceptor sites which was more readily detectable. Receptor binding to these sites was highly sensitive to salt. More sites were exposed at 25 degrees than at 0 degrees. Binding to these sites was inhibited in a nonselective fashion by the addition of protein. Although receptors may be activated by temperature or conditions of high ionic strength, these conditions could not capacitate more than 30 to 40% of the progesterone-receptor proteins for binding. Rate studies suggested that temperature plays a minimal role in nuclear uptake of activated receptors. Such a finding is consistent with a diffusion-limited uptake process.
Ammonium Sulfate↗
Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles.
Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein-protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K(D)s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the DeltaG(bind) was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the DeltaG(bind) from electrostatic steering was assessed from the dependence of the k(a) on ionic strength. Release of solvent H(2)O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein-protein binding. The mAb binding is enthalpy driven and has a slow k(d). TM binding appears to be entropy driven and has a fast k(a). The favorable entropy of the thrombin-TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed.
Allosteric Regulation↗
Copper(II) complexes with N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine: synthesis, characterization, DNA-binding thermodynamical and kinetic studies.
Copper(II) complexes (Cu-L, L=N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine) were synthesized and characterized by elemental analyses, IR spectra and conductance measurement. The interaction of the copper(II) complex with calf thymus DNA was studied by means of UV melting experiments, fluorescence spectra and circular dichroic spectra. Using ethidium bromide as a fluorescence probe, the binding mode of the complexes Cu-L with calf-thymus DNA was studied spectroscopically. The results indicate that the complexes Cu-L perhaps interact with calf-thymus DNA by both intercalative and covalent binding. Kinetics of binding of the cupric complexes to DNA was studied for the first time using ethidium bromide as a fluorescence probe with stopped-flow spectrophotometer under pseudo-first-order condition. The stronger binding of two steps in the process of the complexes Cu-L interacting with DNA was observed, and the probable interaction process was discussed in detail. The corresponding k(obs) and E(a) of binding to DNA (where k(obs) is the observed pseudo-first-order rate constant, E(a) is the observed energy of activation) were obtained.
Amines↗
Identification of two affinity states of low affinity receptors for Escherichia coli heat-stable enterotoxin: correlation of occupation of lower affinity state with guanylate cyclase activation.
Two distinct affinity states of low affinity Escherichia coli heat-stable enterotoxin (ST) receptors in rat intestinal membranes, with dissociation constants of 0.12 and 2.5 nM, were identified. Kinetic binding studies demonstrated biphasic association kinetics, whereas dissociation was unimodal. These studies also confirmed that ligand bound to each receptor state in an independent bimolecular reaction. In contrast, equilibrium binding studies yielded linear Scatchard plots, indicative of a single class of noninteractive binding sites, with a Kd = 2.3 nM. Close agreement of the dissociation constants determined by kinetic and equilibrium methods suggested that receptors were in the lower affinity state at equilibrium. Several models, including binding site heterogeneity, cooperativity, and ligand-induced alterations in receptor conformation were inconsistent with these observations. Indeed, these data were most consistent with a two-step binding process involving a third component. Comparison of the ligand dependence of enzyme activation (EC50 = 124 nM) and the calculated fractional receptor occupancy of the lower affinity component at 5 min (EC50 = 40 nM) demonstrated that occupation of the lower affinity state of low affinity ST receptors correlated with guanylate cyclase activation. The close correlation between receptor occupation and enzyme activation suggests that there are no spare receptors for ST in intestinal membranes. These data resolve the previously observed discrepancy between the affinity of receptors for ST and the potency of this ligand for activating guanylate cyclase. Receptor affinity state alterations may represent a common mechanism for receptor-effector coupling of particulate guanylate cyclases.
Animals↗
A novel nanolayer biosensor principle.
A method for eliminating the mass transport limitation on biosensor surfaces is introduced. The measurement of macromolecular binding kinetics on plane surfaces is the key objective of many evanescent wave (e.g. total internal reflection fluorescence (TIRF)), and surface plasmon resonance (SPR) based biosensor systems, allowing the determination of binding constants within minutes or hours. However, these methods are limited in not being rigorously applicable to large macromolecules like proteins or DNA, since the on-rates are transport limited due to a Nernst diffusion layer of 5-10 microm thickness. Thus, for the binding of fibrinogen (340 kDa) to a surface current SPR biosensors will show a mass transport coefficient of ca. 2 x 10(-6) m/s. In a novel approach with an immiscible fluid vesicle (e.g. air bubble), it has been possible to generate nanoscopic fluid films of ca. 200 nm thickness on the sensor surface of an interfacial TIRF rheometer system. The thickness of the liquid film can be can be easily probed and measured by evanescent wave technology. This nanofilm technique increases the mass transport coefficient for fibrinogen to ca. 1 x 10(-4) m/s eliminating the mass transport limitation, making the binding rates reaction-rate limited. From the resulting exponential kinetic functions, lasting only 20-30s, the kinetic constants for the binding reaction can easily be extracted and the binding constants calculated. As a possible mechanism for the air bubble effect it is suggested that the aqueous fluid flow in the rheometer cell is separated by the air bubble below the level of the Nernst boundary layer into two independent laminar fluid flows of differing velocity: (i) a slow to stationary nanostream ca. 200 nm thick strongly adhering to the surface; and (ii) the bulk fluid streaming over it at a much higher rate in the wake of the air bubble. Surprising properties of the nanofluidic film are: (i) its long persistence for at least 30-60s after the air bubble has passed (2.5s); and (ii) the absence of solute depletion. It is suggested that a new liquid-liquid interface (i.e. a "vortex sheet") between the two fluid flows plays a decisive role, lending metastability to the nanofluidic film and replenishing its protein concentration via the vortices-thus upholding exponential binding kinetics. Finally, the system relaxes via turbulent reattachment of the two fluid flows to the original velocity profile. It is concluded that this technique opens a fundamentally novel approach to the construction of macromolecular biosensors.
Biosensing Techniques↗
An experimental and theoretical study of the morphine binding capacity and kinetics of an engineered opioid receptor.
Electrochemical real-time monitoring of ligand binding to an engineered opioid receptor specific for morphine is reported. In the particular systems studied, 90% of the binding was found to be completed after only 85-120 s. Thus, the binding kinetics has proven to be more rapid than previously believed. The observed association rate constant for the morphine binding reaction was calculated to be 215 M(-1)s(-1). A theoretical analysis of the experimental binding data suggested that the binding sites of the engineered opioid receptor could best be described by a model having two populations of binding sites: K(D)=40 microM (13 micromol/g) and K(D)=205 microM (29 micromol/g). Furthermore, a theoretical model was developed in order to explain the observed binding of the engineered opioid receptor. This model suggested that the binding sites on the polymer surface are up to 5.1A deep and they allow 100% of the ligand (morphine) to anchor itself into the site. The predicted theoretical maximum binding capacity for the reported receptor is calculated to be approximately 2 mmol/g polymer (based on an increase of cavity density).
Binding Sites↗
A kinetic study of erythrocyte-DNA/anti-DNA immune complex association and dissociation reactions in systemic lupus erythematosus.
Decreased binding capacity of the erythrocyte complement receptor (RBC CR1) in systemic lupus erythematosus (SLE) may contribute to abnormal handling of circulating immune complexes in these patients. Decreased numbers of RBC CR1 have been reported in SLE, but, since binding is a function of both receptor number and receptor binding kinetics, we measured kinetic parameters for the interaction of complement (C) containing [3H]DNA:anti-DNA immune complexes (IC) with normal control (NC) and SLE RBC. Experiments were performed at five temperatures ranging from 7-37 degrees C. The parameters measured included: (1) the maximum quantity of DNA:anti-DNA:C which could bind per RBC, S; (2) the association rate constant, ka, for the binding of DNA:anti-DNA:C to RBC; (3) the dissociation rate constant, kd, for the dissociation of bound DNA:anti-DNA:C IC from RBC; (4) the steady-state constant, Kss (ka/kd); and (5) the energies of activation for association, Eaa, and dissociation, Ead. Although the relative amount of bound DNA:anti-DNA:C per RBC was significantly decreased in SLE patients compared to NC (P less than 0.001), the mean values for Kss, ka, kd, Eaa and Ead did not differ significantly between the two groups. These data suggest the following: (1) RBC CR1 binding and dissociation of DNA:anti-DNA:C are consecutive reactions resulting in steady-state concentrations of free and RBC-bound IC; (2) at steady-state times, the ratio of RBC bound to unbound DNA:anti-DNA:C are governed by kinetic factors; (3) since the binding kinetics of SLE and NC RBC are not significantly different, the decreased binding activity described by other investigators can only be due to a decreased number of CR1 per RBC; and (4) values for Eaa and Ead suggest that the rate-determining steps in IC association with and dissociation from RBC involves making and breaking of hydrogen bonds.
Antibodies, Antinuclear↗
Structural and functional characterization of oversulfated chondroitin sulfate/dermatan sulfate hybrid chains from the notochord of hagfish. Neuritogenic and binding activities for growth factors and neurotrophic factors.
Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of D-glucuronic acid (GlcUA)- and L-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765-9776), suggest physiological roles of the hybrid chains in the development of the brain.
Animals↗
The kinetics of glucocorticoid binding to the soluble specific binding protein of mouse fibroblasts.
The kinetics of binding of glucocorticoids to the soluble, specific binding protein of mouse fibroblasts has been examined. The rate at which both potent and weak glucocorticoids achieve binding equilibrium is very slow. Second order rate constants of association range from 3 times 10-5 M- minus 1 min- minus 1 for cortisol to 6.7 times 10-5 M- minus 1 min- minus 1 for triamcinolone acetonide. Studies of the rates of binding at high steroid concentrations suggest that the slow rate of binding may be explained by a two-step mechanism. Active glucocorticoids, regardless of their potency, bind initially in a rapid manner to form a weak complex with the binding protein. The dissociation constant for the weak binding reaction is 0.87 times 10- minus 7 M for triamcinolone acetonide and 2.4 times 10- minus 7 M for cortisol. The weak binding complex becomes converted slowly to a tight complex. The first order rate constants for this conversion and the rate constants of dissociation from the tight complex have been determined for cortisol, dexamethasone and triamcinolone acetonide. The binding affinity of steroids of different biological potency is correlated with their rate of dissociation from this second tight binding state.
Animals↗
Characterization of the molecular mode of action of the sulfonylurea, glimepiride, at beta-cells.
Glimepiride was compared with glibenclamide for its insulin secretion stimulating and beta-cell membrane depolarizing activity as well as for its binding kinetics to beta-cell membranes and for its beta-cell membrane binding proteins. Steady state, kinetic and competitive binding studies revealed a 3- to 4-fold lower binding affinity of glimepiride to isolated beta-cell membranes and intact beta-cells compared to glibenclamide. Direct photoaffinity labeling of beta-cell membrane proteins with the radiolabeled sulfonylureas identified a 65-kDa binding protein for glimepiride and a 140-kDa binding protein for glibenclamide which may be the basis for the different binding characteristics of the two sulfonylureas. The inhibition of binding and photoaffinity labeling of glimepiride and glibenclamide to the 65- and 140-kDa protein, respectively, by glibenclamide and glimepiride can be explained with allosteric interactions between two distinct sulfonylurea binding proteins, subunits of a KATP protein complex, each regulating the open/closed-state of a common separate pore-forming subunit by allosteric interactions. Whole-cell patch clamp experiments with RINm5F cells demonstrated a 3- to 4-fold lower depolarization activity of glimepiride compared to glibenclamide which correlates well with the lower binding affinity of glimepiride. The lower binding affinity and depolarization activity of glimepiride were not reflected in vitro in perifused islets and the isolated perfused pancreas of the rat. This discrepancy remains to be elucidated. The binding, depolarisation and insulin releasing characteristics of glimepiride and glibenclamide suggest that different sulfonylureas can interact with different components of KATP.
ATP-Binding Cassette Transporters↗
Renal brush border membrane vesicle aminoglycoside binding and nephrotoxicity.
The in vitro binding affinity (KD) of aminoglycoside (AG) antibiotics to renal brush border membranes (BBM) has previously been correlated with several variables (sex, age and toxic potential of specific antibiotics) influencing in vivo AG nephrotoxicity in rats. The initial intent of our study was to extend this correlation to the in vivo difference in sensitivity to tobramycin recently observed between two strains of rats. However, the tobramycin binding affinity (KD) failed to correlate with the in vivo strain difference in AG nephrotoxicity. In addition, a comparison of BBM vesicle tobramycin binding affinity between sexes failed to correlate with an earlier report of in vivo gender differences in tobramycin nephrotoxicity. Comparison of binding capacities for male Sprague-Dawley rats and male Fischer rats yielded the only instance where binding parameters correlated with the reports of in vivo nephrotoxicity comparisons. Preliminary examinations of tobramycin binding using renal BBM vesicles derived from a single human kidney revealed binding kinetics and characteristics similar to rat BBM binding. Tobramycin binding was rapid and saturable for each type of BBM vesicle preparation. Scatchard analyses indicated low affinity, high capacity binding characteristics, as well as a single binding site in each case. Polyaspartic acid, which blocks in vitro BBM tobramycin binding as well as in vivo nephrotoxicity in rats, also blocked binding of tobramycin to human BBM vesicles. These data indicate that AG binding is qualitatively similar for rat and human BBM vesicle preparations. However, examination of the quantitative kinetic aspects of AG binding to BBM vesicles (KD and binding capacities) suggests that these parameters may not be critical determinants in the pathogenesis of Ag nephrotoxicity.
Aminoglycosides↗
In vitro binding of tritiated glucocorticoids directly on unfixed rat brain sections.
We describe a new technique for measuring specific in vitro binding of tritiated adrenal steroids on unfixed cryostat brain sections. The specific binding of [3H]corticosterone represents about 70% of the initial binding. Kinetic studies show that specific binding for [3H]corticosterone reaches equilibrium after 15 min incubation at room temperature. Scatchard analysis of [3H]corticosterone in vitro binding gives a linear plot with an apparent dissociation constant (Kd) and a number of binding sites (Bmax) in the range of 10(-8) M and 100 fmol/mg protein, respectively. [3H]Dexamethasone binding under the same conditions gives a similar Kd and a Bmax of 55 fmol/mg protein. The order of potency for the relative binding affinity for [3H]corticosterone labeled sites is as follows: corticosterone greater than progesterone, dexamethasone, RU 38486 (a "pure" antiglucocorticoid), RU 26988 (a "pure" glucocorticoid), aldosterone greater than estradiol, testosterone. Anatomical studies reveal that sections at the level of the hippocampus bind more [3H]corticosterone and [3H]dexamethasone in vitro than more rostral sections taken at the level of the septum. Adrenalectomy increases the capacity of [3H]corticosterone to bind to these sites and perfusion of the brain to remove transcortin and other blood proteins does not modify [3H]corticosterone binding. We conclude that it is possible to measure in unfixed frozen brain sections glucocorticoid binding sites.
Adrenalectomy↗
On the competition for available zinc.
Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.
Amino Acid Sequence↗
Copper(II).bleomycin, iron(III).bleomycin, and copper(II).phleomycin: comparative study of deoxyribonucleic acid binding.
The kinetics and mechanism of binding of Cu-(II).bleomycin, Fe(III).bleomycin, and Cu(II).phleomycin to DNA were studied by using fluorometry, equilibrium dialysis, electric dichroism, and temperature-jump and stopped-flow spectrophotometry. The affinity of Cu(II).bleomycin for DNA was greater than that of metal-free bleomycin but less than that of Fe(III).bleomycin. Cu(II).bleomycin exhibited a two-step binding process, with the slow step indicating a lifetime of 0.1 s for the Cu(II).bleomycin.DNA complex. Fe(III).bleomycin binding kinetics indicated the presence of complexes having lifetimes of up to 22 s. DNA was lengthened by 4.6 A/molecule of bound Cu(II).bleomycin and by 3.2 A/bound Fe(III).bleomycin but not at all by Cu(II).phleomycin, suggesting that both bleomycin complexes intercalate while the phleomycin complex does not. However, phleomycin exhibited nearly the same specificity of DNA base release as bleomycin. These results suggest that the coordinated metal ion plays a major role in the binding of metal-bleomycin complexes to DNA but that intercalation is neither essential for DNA binding and degradation nor primarily responsible for the specificity of DNA base release by these drugs.
Animals↗
Differential dissociation kinetics explain the binding preference of insulin-like growth factor binding protein-6 for insulin-like growth factor-II over insulin-like growth factor-I.
Insulin-like growth factor binding protein-6 binds insulin-like growth factor-II with a marked preferential affinity over insulin-like growth factor-I. The kinetic basis of this binding preference was studied using surface plasmon resonance. Binding of insulin-like growth factor-I and insulin-like growth factor-II to immobilized insulin-like growth factor binding protein-6 fitted a two-site binding kinetic model. Insulin-like growth factor-I and insulin-like growth factor-II association rates were similar whereas the dissociation rate was approximately 60-fold lower for insulin-like growth factor-II, resulting in a higher equilibrium binding affinity for insulin-like growth factor-II. The equilibrium binding affinities of a series of insulin-like growth factor-II mutants were also explained by differential dissociation kinetics. O-glycosylation had a small effect on the association kinetics of insulin-like growth factor binding protein-6. The insulin-like growth factor binding properties of insulin-like growth factor binding protein-6 are explained by differential dissociation kinetics.
Binding, Competitive↗
Distinct characteristics of initiation of the classical and alternative complement pathways by Candida albicans.
Candida albicans is a potent activator of the complement system. The objective of this study was to characterize factors that influence the kinetics for activation of C3 and binding of C3 fragments to C. albicans. Factors that were examined included the surface properties of the yeast and contributions of the classical and alternative complement pathways. The results showed that incubation of hydrophobic, hydrophilic, or germinating yeast cells in normal human serum (NHS) containing radiolabeled C3 led to immediate accumulation of C3 on all three cell types, although the rate of accumulation of C3 on germinating cells was lower. An examination of the sites for early C3 binding showed that classical pathway initiation led to immediate, synchronous binding over the entire cell surface. A blockade of the classical pathway by absorption of putative classical pathway initiators or by chelation of calcium limited activation to the alternative pathway. Binding of C3 solely via the alternative pathway was characterized by a significant lag in the initial binding kinetics. In the absence of classical pathway initiation, the early cellular sites for C3 binding appeared as random, asynchronous foci of C3 that appeared to expand with time. The factor(s) mediating rapid deposition of C3 that was characteristic of the classical pathway initiation was reciprocally cross-absorbed by hydrophilic and hydrophobic C. albicans but was not removed by absorption of NHS with Saccharomyces cerevisiae, encapsulated Cryptococcus neoformans, or nonencapsulated C. neoformans. Delayed binding of C3 produced by absorption of serum was largely reversed by addition to the absorbed serum of immunoglobulin G isolated from NHS, indicating a significant role for a naturally occurring anti-C. albicans immunoglobulin C. in classical pathway initiation.
Antibodies, Fungal↗