Calculation Of Dissociation Constants Research Articles

Thermodynamics of binding of the distal calcium to manganese peroxidase.

We previously demonstrated that manganese peroxidase from Phanerochaete chrysosporiumwas susceptible to thermal inactivation due to release of the distal calcium, which maintained the distal heme environment of the enzyme [Sutherland, G. R. J., Zapanta, L. S., Tien, M., & Aust, S. D. (1997) Biochemistry 36, 3654-3662]. In this investigation the binding of calcium to the distal calcium binding site of manganese peroxidase was studied by optical absorption spectroscopy and isothermal titration calorimetry. The dissociation constant for the distal calcium binding site was 11 +/- 1 microM and the Hill coefficient was 1.1 +/- 0.1. The binding of calcium was accompanied by decreases in enthalpy and entropy that were large compared to those of other calcium binding proteins. The decreases were consistent with the large conformational changes proposed to occur in manganese peroxidase as a result of the binding and release of the distal calcium. Studies involving binding of the hydrophobic fluorescent probe, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt (bis-ANS), to manganese peroxidase indicated that the active, calcium-containing form of the enzyme had less exposed hydrophobic surface area, which would contribute to an increase in enthalpy and entropy upon calcium binding. Therefore, the negative changes in enthalpy and entropy associated with calcium binding were attributed to a large increase in the structural rigidity and compactness of the enzyme. The dissociation constant for calcium decreased and the rate of thermal inactivation decreased with decreasing pH. However, both the ability of calcium to prevent thermal inactivation of manganese peroxidase and the rate of calcium binding decreased as the pH decreased. Therefore it was proposed that, at lower pH, calcium binding to manganese peroxidase was more thermodynamically favorable, but the rate of calcium binding decreased because the flexibility of the calcium binding site, and in turn exposure of the ligands to the incoming ion, decreased.

Structure/calcium affinity relationships of site III of calmodulin: testing the acid pair hypothesis using calmodulin mutants.

Calmodulin mutants in which the calcium binding affinity of site IV was greatly reduced by a D133E mutation were prepared using site-specific, cassette-mediated mutagenesis as a multisite calcium binding protein model to examine structure/calcium affinity relationships in site III of calmodulin. Tryptophan was introduced in position 92 of the calmodulin mutants as a fluorescent label to monitor the calcium-induced structural changes in the C-terminal domain of calmodulin. The five calmodulin mutants, 3xCaM, 3zCaM, 4xCaM, 4zCaM, and 4xzCaM, were designed so that there were three or four acidic amino acid residues in chelating positions of site III with acid pairs on either the X and/or Z coordinating axes. The calcium dissociation constant of site III, KIII, of the five calmodulin mutants changes in a descending order from 3xCaM (237 microM), 3zCaM (140 microM), 4xCaM (5.8 microM), 4zCaM (3 microM), to 4xzCaM (2 microM), and these KIII values are significantly lower than that of F92W/D133E calmodulin (335 microM) in which three acidic residues with no acid pairs were present in site III [Wu, X., & Reid, R. E. (1997) Biochemistry 36, 3608-3616]. These results indicate that the calcium affinity of site III increases when the number of the acidic chelating residues increases from three to four, when the number of acid pairs increases from zero to one and further to two, and when the location of the acid pair is changed from the X axis to the Z axis. This study provides the first evidence that the acid pair hypothesis which correlates the nature of the chelating residues with the calcium affinity of the hlh motif is applicable to a multisite calcium binding protein model. The Hill coefficients indicate that reversal of the sequence of filling of the calcium binding sites in the C-terminal domain from IV --> III to III --> IV also changes the site cooperativity from positive to negative. The cooperativity returns to positive when the proteins are titrated in the presence of a calmodulin-binding peptide. Data from the present study also demonstrate that calmodulin mutants with a decreased calcium affinity have a reduced efficiency in phosphodiesterase regulation at low calcium concentrations (50 microM). However, high calcium concentrations (15 mM) restore the phosphodiesterase regulatory activity of the calmodulin mutants to a level obtained with F92W calmodulin, indicating that the mutations alter calcium regulation of calmodulin-mediated phosphodiesterase activity without affecting the interaction between calmodulin and the enzyme.

Ligand-binding enhances the affinity of dimerization of the extracellular domain of the epidermal growth factor receptor.

We studied the dimerization of the recombinant soluble extracellular domain of the epidermal growth factor receptor (sEGFR) in response to EGF-binding using multi-angle laser light scattering with size exclusion chromatography (SEC-MALLS). In the absence of EGF, sEGFR behaved as a monomer. However, upon EGF-binding, sEGFR formed a dimer with the stoichiometry of two EGF molecules bound to two sEGFR molecules [(EGF)2-(sEGFR)2]. We analyzed the chemical equilibrium of the dimer formation by SEC-MALLS using a dissociation constant of 0.25 microM for the binding of EGF to sEGFR. The calculated dissociation constant for EGF-induced sEGFR dimerization was found to be 2.4 +/- 0.9 microM. These experiments demonstrated that EGF induces receptor dimerization and that two EGF molecules are bound to an EGF-receptor dimer.

Function of conserved tryptophans in the Aspergillus niger glucoamylase 1 starch binding domain.

Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.

Basal transcription factors TBP and TFIIB and the viral coactivator E1A 13S bind with distinct affinities and kinetics to the transactivation domain of NF-kappaB p65.

Transactivation domains (TADs) are able to contact several components of the basal transcription apparatus and co-activator molecules. In order to study these interactions in biophysical detail, binding of the well-characterized TAD from the human transcription factor NF-kappaB p65 (RelA) to the basal transcription factors TBP and TFIIB and the viral co-activator protein E1A 13S was chosen as a model system to investigate the kinetics and affinities of such protein-protein interactions by surface plasmon resonance analysis. The TAD of NF-kappaB p65 showed remarkably different affinities and kinetics in binding to the various proteins. The real-time kinetic measurements revealed an association rate constant (kass) of 2.3 x 10(6)/M/s for the interaction between the p65 TAD and TBP. The association rate constants of the p65 TAD were much weaker for TFIIB (6.8 x 10(4)/M/s) and for the E1A 13S protein (4.9 x 10(4)/M/s). The dissociation rate constants (kdiss) were determined to be 7.9 x 10(-4)/s for TBP, 1.6 x 10(-3)/s for TFIIB and 1.3 x 10(-3)/s for the E1A protein. Accordingly, the calculated dissociation constants (Kd) differed between 3.4 x 10(-10)M for the strongly binding TBP protein and 2.3 x 10(-8)M and 2.6 x 10(-8)M for the weaker binding TFIIB and E1A 13S proteins respectively. Non-linear analysis of the appropriate part of the sensorgrams revealed monophasic association and dissociation kinetics for binding between the p65 TAD and all three interaction partners. The remarkable differences in protein affinities add another aspect to a more detailed understanding of formation of the transcription preinitiation complex. The co-transfection of TBP and E1A 13S stimulated NF-kappaB p65-dependent gene expression, showing the biological significance of these interactions.

Conservative D133E Mutation of Calmodulin Site IV Drastically Alters Calcium Binding and Phosphodiesterase Regulation

Two calmodulin mutants, F92W and F92W/D133E, were prepared using site-specific cassette-mediated mutagenesis to examine the structure/calcium affinity relationships of cation chelating residues in calcium binding sites III and IV. The mutant, F92W, was prepared to produce a strong fluorescent label to follow the calcium-induced structural changes in the C-terminal domain of the protein. A second mutant, F92W/D133E, was prepared to destroy the calcium binding to site IV and thereby eliminate cooperativity between sites III and IV. The macroscopic calcium dissociation constants of the two sites in the C-terminal domain were derived from the calcium titration data that had been fitted to a two-site Hill equation. The calcium dissociation constants of site III and site IV in the F92W/D133E mutant were 335 microM and 2.76 mM, respectively. These values were significantly greater than the values of 14 and 1 microM for site III and site IV in F92W calmodulin, respectively. These results suggested that a very conservative D133E mutation in the +Z position of the site IV Ca2+-binding loop drastically decreased the calcium binding affinity of the site (2760-fold) and also significantly reduced that of site III in the same domain (24-fold). The D/E calmodulin mutant also had a 3-fold lower phosphodiesterase activation activity with a 25-fold lower affinity for this enzyme than that of F92W calmodulin in the presence of low calcium concentration (50 microM). However, the maximum phosphodiesterase activation activity of the F92W/D133E mutant and the affinity of this mutant for the enzyme were similar to those of F92W calmodulin in the presence of high calcium concentration (15 mM), suggesting that the D133E mutation altered calcium regulation of calmodulin mediated phosphodiesterase activity without affecting calmodulin interaction with the enzyme.

Molecular and Functional Characterization of the Urokinase Receptor on Human Mast Cells

The urokinase receptor system is involved in several biological processes including extracellular proteolysis, cell invasion, and chemotaxis. Mast cells are multifunctional perivascular cells that play an important role in the regulation of microenvironmental events. We report that primary human mast cells and the human mast cell line HMC-1 express the receptor for urokinase. As assessed by Northern blotting and reverse transcription polymerase chain reaction technique, purified human lung mast cells and HMC-1 cells expressed urokinase receptor mRNA in a constitutive manner. Using a toluidine blue/immunofluorescence double staining technique and monoclonal antibodies, surface expression of urokinase receptor was demonstrable in lung, skin, uterus, heart, and tonsil mast cells, whereas the low density lipoprotein receptor-related protein was not detectable. Binding of monoclonal antibody VIM5 (recognizing the urokinase binding domain of urokinase receptor) to HMC-1 could be blocked by high molecular weight but not low molecular weight urokinase. Binding analyses performed with 123I-urokinase revealed expression of 271,000 +/- 55,000 high affinity urokinase binding sites per HMC-1 cell, with a calculated dissociation constant of 1. 29 +/- 0.3 nM. Purified urokinase induced dose-dependent migration of primary mast cells and HMC-1 in a chemotaxis assay without inducing release of histamine. The mast cell agonist stem cell factor also induced migration of HMC-1 and caused up-regulation of expression of urokinase receptor mRNA. Together, our data show that human mast cells express functional receptors for urokinase. Expression of urokinase receptors on mast cells may have implications for mast cell-dependent microvascular processes associated with fibrinolysis, migration, or local tissue repair.

Characterization of quaternary structure of interleukin-8 and functional implications

A number of studies have addressed the importance of the dimer interface by mutating residues at the dimer interface and designing monomers that show no propensity to dimerize. All of them consistently indicate that a monomeric unit is sufficient to bind to a neutrophil receptor for functional activation. Similarly, calculation of Kd values and functional studies of monomeric analogs of NAP-2 and melanoma growth stimulating activity (MGSA) that cannot dimerize indicate that the monomer is the functionally relevant species. The chapter discusses the association state of IL-8 and related chemokines as probed by different techniques. The various studies that directly probe the importance of the dimer interface at the amino acid level are also discussed. Of the different techniques available to study the significance of the dimer interface, calculating dissociation constants by sedimentation equilibrium and designing nonassociating monomers address the issue in an unequivocal manner.

Epitope contiguous chains and antibody recognition in HIV-1 synthetic peptide antigens.

Five peptides derived from human immuno deficiency virus (HIV-1) gp41 transmembrane protein have been synthesized: M9 (610-618), M12 (598-609), M15 (600-614), M21 (584-604) and M23 (587-609). These sequences partially overlap in the region vicinal to the immunodominant epitope CSGKLIC, between two cysteine residues 603-609 and three of them (M12, M15 and M23) include this complete heptapeptide. M23, the longer peptide, includes an hydrophilic chain in addition to the heptapeptide loop. The purpose of this work was to determine the influence of contiguous chains to the heptapeptide loop on antibody recognition in fluid and solid phases, and dissociation constants (KD) of each sequence with human anti-HIV-1 antibodies. Two peptides, M13 and M23, overlapped on this loop, were found to be more reactive. Antigen-antibody dissociation constants were determined for both peptides by competition enzyme-linked immunosorbent assay, using each peptide alternatively as the solid phase-immobilized antigen. In addition to the influence of solid-phase antigen on calculated dissociation constants (a phenomenon described by Seligman, 1994), the inhibitory effect of M15 in liquid phase on antibody binding to solid phase M23 was higher than exerted by M23 in solution over antibody binding to M15 on solid phase. On the basis of peptide sequence and predicted antigenicity, this behavior appeared to be contradictory. It is assured that the possible origin of this phenomenon is due to unfavorable conformation of the longer peptide. Even though synthetic peptides mimic mainly sequential epitopes, conformational preferences in fluid or solid phase play an important role in epitope functionality. In particular, addition of residues to known immunodominant sequences may not always amplify antibody recognition if conformation provokes steric hindrance in the native epitope.

The Developmentally Regulated Avian Ch21 Lipocalin Is an Extracellular Fatty Acid-binding Protein

Ch21, a developmentally regulated extracellular protein expressed in chick embryos and in cultured chondrocytes, was expressed in the baculovirus system, and the recombinant protein was purified to homogeneity by gel-filtration chromatography. Separation of two isoforms was achieved on an ion-exchange column. Previous work had shown that Ch21 belongs to the superfamily of lipocalins, which are transport proteins for small hydrophobic molecules. Studies were performed to identify the Ch21 ligand. By analysis of recombinant Ch21 on native polyacrylamide gel electrophoresis and by Lipidex assay, the binding of fatty acid to the protein was shown and a preferential binding of long-chain unsaturated fatty acids was observed. Both isoforms had the same behavior. The binding was saturable. Stoichiometry was about 0.7 mol of ligand/mol of protein. The protein binds the ligand in its monomeric form. Calculated dissociation constants were 2 X 10(-7) M for unsaturated fatty acids and 5 X 10(-7) M for stearic acid. The binding was specific; other hydrophobic molecules, as retinoic acid, progesterone, prostaglandins, and long-chain alcohols and aldehydes did not bind to the protein. Short-chain fatty acids did not bind to the protein. Ch21, also present in chicken serum, represents the first extracellular protein able to selectively bind and transport fatty acid in extracellular fluids and serum. We propose to rename the Ch21 protein as extracellular fatty acid-binding protein (Ex-FABP).

Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing

We characterized the fluorescence probe Fura-2 for calcium measurements using frequency-domain phase-modulation fluorometry. By the use of different excitation wavelengths from 345 to 380 nm, the apparent calcium dissociation constants can be altered from 41 nM to 1.92 μM Ca 2+. This change in apparent Kd results from changes in the relative extent of excitation of the calcium-bound and calcium-free forms, and the excitation wavelength-dependent contribution of each form to the intensity decay. These results indicate that lifetime-based measurements with Fura-2 can be used for imaging of calcium over a wide range of concentrations. An additional favorable feature of Fura-2 is that the calcium-free form can be almost exclusively excited at wavelength of 390 nm or longer, and can thus be used as a reference providing the lifetime in the absence of calcium, without removing the calcium. Additionally, exposure of Fura-2 to intense illumination shifts but does not distort the frequency response. For cellular imaging, these favorable properties of Fura-2 may allow calibration of the calcium concentrations without the use of ionophores.

Mitochondrial selenium-75 uptake and regulation revealed by kinetic analysis.

Uptake of Na2(75)SeO3 by mitochondria of the larvae of the insect C. cephalonica reared at different dietary selenium (Se) levels revealed: 1. A proportional increase in the uptake with externally added Na2(75)SeO3 in concentrations upto 25.32 microM; and 2. At each added selenite concentration, an increase up to 60 min, with linearity up to 15-30 min. A differential affinity for Na2(75)SeO3 was elicited in the mitochondrial protein fractions of different dietary Se groups and correlated well with the pattern and the ratio of distribution of incorporated 75Se in protein to nonprotein fractions. Kinetic studies on 75Se uptake by whole mitochondria negated passive diffusion of selenite and revealed a trend of negative cooperativity, confirmed by Hill and Scatchard plots. Half saturation value was estimated to be approx 13 nmole Se/mg mitochondrial protein. Scatchard plot for 75Se uptake was biphasic and the high affinity binding sites were estimated to be around 5 nmole/mg mitochondrial protein. Calculated dissociation constants revealed maximal affinity for 75Se in the 1.5 ppm group (KSe 0.0034 nM) and minimal in the basal group (KSe 0.007 nM). In the mitochondria of all the three dietary Se groups, the estimated low affinity sites amounted to be 15-19 nmole/mg mitochondrial protein. Inherent Se in the mitochondria of the high Se group positively enhanced the incorporation of 75Se in the mitochondrial protein fraction. About 20-30% of the total uptake was indicated to be energy linked as revealed by studies with respiratory inhibitors. Addition of sulfite and sulfate (5-25 microM) in the medium, inhibited 75Se uptake by 35-55%, suggestive of the involvement of the dicarboxylate port. Thiol interactive 75Se uptake was confirmed by the inhibition mediated by mersalyl and NEM up to 50-70%. The study revealed thiol-selenite interactions of metabolic significance during selenite uptake.

Kinetics and Thermodynamics of Virus Binding to Receptor.: STUDIES WITH RHINOVIRUS, INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1), AND SURFACE PLASMON RESONANCE

We have studied the kinetics and thermodynamics of a virus interacting with its receptor using human rhinovirus serotype 3 (HRV3), soluble intercellular adhesion molecule-1 (ICAM-1, CD54) containing Ig superfamily domains 1-5 (sICAM-1), and surface plasmon resonance. There were two classes of binding sites for sICAM-1 on HRV3, each comprising about 50% of the total sites, with association rate constants of 2450 +/- 300 and 134 +/- 11 M-1 s-1. These rates are low, consistent with binding to a relatively inaccessible site in the rhinovirus canyon. By contrast, three monoclonal antibodies bound to sICAM-1 with a single rate constant of 17,000-48,000 M-1 s-1. The dissociation rate constant for HRV3 was 1.7 +/- 0.1 x 10(-3) s-1, giving calculated dissociation constants of 0.7 +/- 0.1 and 12.5 +/- 1.2 microM. Agreement was good with saturation binding in solution, which showed two sites of similar abundance with KD of 0.55 +/- 0.2 and 5.7 +/- 2.0 microM. A bivalent chimera of ICAM-1 with the IgA1 Fc region bound with KD = 50 and 410 nM, showing 17-fold enhanced affinity. Lowering pH from 8.0 to 6.0 reduced affinity by approximately 50-fold, primarily by reducing the on rate. Thermodynamic measurements showed that binding of ICAM-1 to HRV3 is endothermic, by contrast to binding to monoclonal antibody. The heat that is absorbed of 3.5 and 6.3 kcal/mol for the two classes of ICAM-1 binding sites may contribute to receptor-mediated disruption of virions, which has an activation energy of about 42 kcal/mol.

Ion binding constants for gramicidin a obtained from water permeability measurements

Gramicidin A pores are permeable to water and small monovalent cations. For K, Rb, and Cs there is good evidence from conductances and permeability ratios that a second ion can enter a pore already occupied by another, but for Na this evidence is inconclusive and comparison of tracer fluxes and single channel conductances suggests that second ion entries are prohibited. Partly as a result of the complications of second ion entry there have been widely differing estimates for the dissociation constants for the first ion in the channel. Dani and Levitt (1981, Biophys. J. 35: 485-499) introduced a method for calculating ion binding constants from simultaneous measurements of water fluxes and membrane conductance. They found no evidence for second ion binding and calculated dissociation constants of 115 mM for Li, 69 mM for K, and 2 mM for Tl. It is shown here that the two-ion, four-state model predicts a dependence of water permeability on ion concentration that is difficult to distinguish from the predictions of block by a single ion. Using a modified technique that allows measurement of higher conductances, the first ion dissociation constants have been determined as 80 mM for Na, 40 mM for Rb and 15 mM for Cs. These values and those of Dani and Levitt fall in a smooth sequence. The dissociation constant for Cs is consistent with single channel conductances and flux ratios. There is a discrepancy between this constant for Na and the value, 370 mM, calculated from the single channel conductances and the assumption that a second ion cannot enter or affect an occupied pore. The dissociation constant for Rb is intermediate between those for K and Cs whereas tracer flux measurements (Schagina, Grinfeldt & Lev, 1983. J. Membrane Biol. 73: 203-216) have suggested that Rb interacts much more strongly with the channel than Cs.

Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator indo-1

The fluorescent indicator indo-1 is widely used to monitor intracellular calcium concentration. However, quantitation is limited by uncertain effects of the intracellular environment on indicator properties. The goal of this study was to determine the effects of protein and acidosis on the fluorescence spectra and calcium dissociation constant (Kd) of indo-1. With 350 nm excitation light, the ratio of indo-1 fluorescence in the absence versus the presence of saturating Ca2+ at wavelength lambda (S lambda) and Kd increased with [protein]. At pH 7.3, Kd, S400, and S470, which were 210 nM, 0.033, and 1.433 in the absence of protein, increased to 808 nM, 0.161, and 2.641, respectively, by adding proteins from frog muscle and to 638 nM, 0.304, and 3.039, respectively, by adding proteins from rat heart. Effects of protein on indo-1 fluorescence were reduced at higher [indo-1]. Acidosis (pH 6.3) had separate effects, which were additive to those of protein: in the absence of protein, acidosis increased Kd to 640 nM; frog muscle proteins further increased Kd to 1700 nM. Acidosis also changed S lambda slightly. In summary, interaction with protein or protons alters indo-1 calcium-binding and fluorescence. These findings are consistent with several previous studies and suggest that indo-1 calibration constants need to be derived in the presence of appropriate types of protein, ratio of [indo-1]/[protein], and pH.

Transfer of retinol-binding protein from HepG2 human hepatoma cells to cocultured rat stellate cells.

Rat liver stellate cells were cocultured with HepG2 human hepatoma cells, which are known to synthesize and secrete retinol-binding protein (RBP). Transfer of human RBP from HepG2 cells to stellate cells was studied by cryoimmunoelectron microscopy. In stellate cells, human RBP was found on the cell surface and within endosomes. The transfer of human RBP from HepG2 cells to stellate cells was blocked by addition of RBP antibodies to the culture medium. Very little uptake of RBP was observed when fibroblasts were cocultured with HepG2 cells. In a series of experiments, RBP was bound to its putative cell surface receptor at 4 degrees C, and the stellate cells were washed and then incubated at 37 degrees C in order to allow them to internalize a pulse of RBP. About 50% of the RBP was internalized after 6 min of incubation. The RBP-positive vesicles were initially (after 1-2 min) located close to the cell surface and later were found deeper in the cytoplasm. During the first 10 min, RBP was mainly observed in close association with membranes. After 2 hr, however, most RBP was localized in intracellular vesicles at a distance from the vesicular membranes, suggesting that RBP had been released from its receptor. Saturable binding of RBP to liver cells was demonstrated when cells were incubated with 125I-RBP at 4 degrees C and cell-associated radioactivity was determined. The calculated dissociation constant for the specific binding was 12.7 +/- 3.2 nM. A binding assay was also developed for determination of solubilized RBP receptor. Solubilized proteins from the nonparenchymal liver cells bound about 30 times more 125I-labeled RBP than did parenchymal cells (based on mass of cell protein). These data suggest that RBP mediates the paracrine transfer of retinol from hepatocytes to perisinusoidal stellate cells in liver and that stellate cells bind and internalize RBP by receptor-mediated endocytosis.

A new structure-activity model for Ah receptor binding. Polychlorinated dibenzo-p-dioxins and dibenzofurans.

A new structure-affinity model for the aromatic hydrocarbon (Ah) receptor is reported. The proposed mathematical model completely eliminates multiple regression analysis in its formulation and overcomes the cross-class comparison inherent to classical quantitative structure-activity relationships. Taking the polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) as model xenobiotics, the binding affinity of a PCDD relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is shown to be analytically related to the electron affinities, entropies, and lipophilicities of PCDD and TCDD. From the calculated dissociation constants of PCDD-Ah receptor complexes, the corresponding equilibrium constants of PCDF-Ah complexes could be computed, in agreement with the experimental observation that the trend in the binding affinities of PCDDs and PCDFs to the Ah receptor are similar. The reported model is capable of quantitatively explaining the quantitatively estimating the in vitro binding affinities of PCDDs, PCDFs, and related xenobiotics to the Ah receptor. Therefore, a halogenated aromatic compound is expected to have a higher affinity for the cytosolic protein than TCDD if it is less lipophilic and has a higher electron affinity and lower entropy. Furthermore, the affinities of structurally related polychlorinated aromatic xenobiotics for the Ah receptor could be computed from their entropies and electron affinities.

Characterization of the novel 5-HT 3 antagonists MDL 73147EF (dolasetron mesilate) and MDL 74156 in NG108-15 neuroblastoma × glioma cells

In radioligand binding experiments, MDL 73147EF and MDL 74156 inhibited the binding of [ 3H]GR65630 to 5-hydroxytryptamine 3 (5-HT 3) binding sites on membranes prepared from NG108-15 neuroblastoma × glioma cells. The calculated dissociation constants (K I) were 20.03 ± 6.58 and 0.44 ± 0.18 nM, respectively (means ± S.E.M., n = 6 and 9, respectively). Application of 5-HT (10–50 μM) to voltage-clamped NG108-15 cells elicited a rapidly desensitizing inward membrane current, characteristic for the activation of 5-HT 3 receptors. The 5-HT-induced membrane current was suppressed in a reversible, concentration-dependent manner by MDL 73147EF and MDL 74156EF. The concentrations required to block half the 5-HT response (IC 50) were 3.8 and 0.1 nM, respectively. It is concluded that both compounds are potent and reversible antagonists at 5-HT 3 receptors in this neuroblastoma cell line.

Binding properties of Pyrularia thionin and Naja naja kaouthia cardiotoxin to human and animal erythrocytes and to murine P388 cells

Pyrularia thionin and snake venom cardiotoxin are strongly basic peptides which induce hemolysis, depolarization of muscle cells and activation of endogenous phospholipase A 2. An earlier study of the hemolysis reaction indicated that the two peptides bind to and compete for the same site on human erythrocytes. A recent study examined the hemolysis induced by both peptides as the phosphate and Ca 2+ content of the reaction mixture was varied. The results of the recent study ( Vernon, L. P. and Rogers, A., Toxicon 30, 701–709) agree with this companion study on the binding of 125I-labeled pyrularia thionin and cardiotoxin to erythrocytes under the same conditions. Added phosphate ion at 5 mM and removal of membrane-bound Ca 2+ by treatment with 10 mM EGTA make more binding sites of the same affinity available to both peptides, which are shown to bind in a competitive fashion to the same site. Addition of 10 mM Ca 2+ to the medium decreases peptide binding due to competitive binding of Ca 2+ to the same site on the membrane. For human erythrocytes the number of binding sites/cell for the thionin ranged from 0.7 to 1.7 × 10 5 and for cardiotoxin from 0.82 to 1.6 × 10 5. The calculated dissociation constants ( K d) from the Scatchard plots ranged from 0.43 to 1.1 μM for the thionin and from 0.40 to 0.98 μM for the cardiotoxin. The binding sites for thionin and cardiotoxin with sheep erythrocytes were 1.7 and 2.0 × 10 4 sites/cell, respectively, and both cow and horse erythrocytes demonstrated 2.7 × 10 4 sites/cell for the thionin. Binding studies with murine P388 cells showed 7.0 and 9.5 × 10 6 sites per cell for Pyrularia thionin and cardiotoxin, respectively.

Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.