New capabilities in determining the binding parameters for ligand-receptor interaction

A new method for determining the binding parameters of ligand-receptor interaction is suggested. The method is based on the application of the so-called coordinate of dilution, suggested by us earlier. We demonstrated that it is possible to determine the binding characteristics of ligand-receptor interaction using either the measurement of the concentration of the ligand-receptor complex at a state of equilibrium or the concentration of free receptors at different dilutions of the studying ligand-receptor mixture. The method also allows the determination of the concentration of the ligand in a pre-existing ligand-receptor mixture without preliminary separation of the interacting counterparts. For this reason the suggested method could be especially useful when the studying very labile receptors for which purification from the corresponding ligand is very difficult or impossible.     

Analysis of ligand-receptor binding by the difference method

A new method has been proposed for analysis of experimental data on ligand-receptor binding at equilibrium. This method makes it possible to detect heterogeneity of a receptor system in cases where the contribution of the high-affinity site to total binding is rather small and the problem of graphic discrimination of a model cannot be solved unambiguously by other methods. The difference method permits us to exclude experiments on measuring nonspecific binding. A computer program for analysis of ligand-receptor binding has been worked out in which the difference method and traditional methods of binding isotherm analysis are realized. Numerical modeling has shown that the best strategy in experimental data processing is the treatment of total binding isotherms by both the difference method and regression analysis, including the nonspecific binding constant as one of the regression parameters.     

非饱和土壤水力参数的模型及确定方法

土壤水力参数的选取和确定是土壤中水分运动和污染物迁移预测的基础,本文在国内外研究成果的基础上,综述了土壤水力参数的模型(土壤水分特征曲线模型、土壤导水率模型)及其直接测定法和间接推求法,并比较了土壤水力参数的模型及确定方法的适用性与局限性,以便为生态环境建设和农业可持续发展研究中土壤水力参数的选取提供依据。     

A computerized approach towards the estimation of binding parameters to characterize cooperative and non-cooperative multicomponent ligand-receptor interactions

A mathematical model has been developed to analyse multicomponent ligand-receptor interaction data directly from binding experiments without resorting to approximations such as linearization. This approach may be applied to analyze binding data for radioreceptor systems involving thyroid and steroid hormones, and drugs on neurotransmitters. Affinity constants, maximal binding capacity, cooperativity and nonspecific binding may be calculated for multiple binding systems.     

Probabilistic description of the system "ligand-receptor"

The theory of probabilities was used to describe the ligand-receptor interaction. Mean and variance of number ligand-receptor complexes are calculated. It is shown that the mean number of ligand-receptor complexes coincides with that obtained from the law of conservation masses. Proceeding from a ratio of mean and expectation it is shown that the variance of the number of ligand-receptor complexes should be taken into account with concentration of ligand-receptor complexes component less than 1 fmol.     

Dynamics of the Adair model for ligand-receptor binding

The dynamics of the Adair model for ligand-receptor binding involving the case with interacting receptors is investigated. Using the methods of formal reaction kinetics, the existence, uniqueness, and asymptotic stability of equilibria within the model are demonstrated. The approximate solutions to the Adair model are found.     

A comparison of methods for determining compartmental analysis parameters

The traditional method for determining compartmental analysis parameters relies on a visual selection of data points to be used for regression of data from each cellular compartment. This method is appropriate when the compartments are kinetically discrete and are easily discernible. However, where treatment effects on compartment parameters are being evaluated, a more objective method for determining initial parameters is desirable.

Three methods were examined for determining initial isotopic contents and half-times of ⁸⁶Rb elution from cellular compartments using theoretical data with known parameters. Experimental data from roots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) and barley (Hordeum vulgare L.) intact seedlings were also used. The three methods were a visually assisted, linear regression on data of semilog plot of isotope elution versus time, a microcomputer-assisted, linear regression on semilog plot where maximization of the square of the correlation coefficient (r²) was the criterion to determine data points needed for each regression and a mainframe computer-assisted, direct nonlinear regression on elution data using a model of the sum of three exponential decay functions. The visual method resulted in the least accurate estimates of compartmental analysis parameters. The microcomputer-assisted and nonlinear regression methods calculated the parameters equally well.

     

Ligand-receptor interactions: a new method for determining the binding parameters

A new experimental procedure and new plot coordinates that allow determination of the binding parameters of ligand-acceptor interaction have been proposed. Instead of titration of a constant concentration of receptors with changing concentrations of ligand, as requested by the well-known methods of Klotz and Scatchard, a series of sequential dilutions of the reacting ligand-receptor mixture is suggested. This allows the application of a new coordinate system that transforms the binding isotherms into straight lines. The case of one acceptor with two classes of receptors with different binding constants is also considered briefly, where the correspondent graphs are nonlinear. It is suggested that in some cases this approach can be a simple and convenient substitute of the broadly used methods of Klotz and Scatchard.     

A simple and convenient approach for evaluation of the parameters of ligand-receptor interaction. Receptor blocking index and its application

A new approach for determination of the parameters for ligand-receptor interaction, which is based on so-called dilution coordinates, was developed earlier. Equations that allow evaluation of not only the affinity of ligand-receptor interaction but also of the amount of free (or occupied by corresponding ligand) receptors were suggested. The most important advantage of this approach as compared with well-known methods is the ability to determine the binding parameters for ligand-receptor interaction even for the cases in which ligand and receptor are already present in a mixture and separation of counterparts from each other is technically difficult or even impossible. Due to this reason, the proposed approach can be especially useful for studying interactions between highly-labile biological receptors and corresponding ligands as found in vivo. In the present paper I continue to consider how to determine the binding parameters for a given ligand-receptor interaction if the value of receptor blocking index is determined experimentally.     

Optimization of methods for immobilizing antibodies against the carcino-embryonic antigen on insoluble matrices. Equilibrium parameters of the interaction of the immobilized antibodies with the antigen

To obtain highly efficient immunosorbents for solid-phase immunoassay and affinity chromatography, methods for immobilization of antibodies against the carcino-embryonic antigen (CEA) on insoluble matrices were optimized. The immunosorbents obtained were characterized by equilibrium parameters of the reaction between immobilized anti-CEA and CEA calculated from rather a simple kinetic model. This model describes the interaction of the monovalent antigen with two independent types of binding sites. The role of some amino acid residues of anti-CEA in the interaction with CEA was investigated. The effects of immobilization density and the spacer arm length on the functional properties of the immobilized antibodies were studied. The optimal immunosorbent was used to purify 125I-CEA by immunoaffinity chromatography.     

New systems of coordinates in the study of ligand-receptor interaction

Two new coordinate systems that allow to determine the parameters of ligand-receptor interaction are suggested. These coordinate systems principally differ from the well-known coordinates of Klotz and Scatchard. It was shown that suggested coordinates were simpler and more convenient then coordinates of Klotz and Scatchard and in some cases was more informative. The case when a ligand interacts with two classes of non-identical independent receptors was also considered.     

The binding of the B-chain of ricin to Burkitt lymphoma cells. A new approach to ligand-receptor interaction studies

It is shown that conformational changes of receptor proteins brought about by binding of a ligand induce changes in the lipid environment of the receptor that can be monitored by fluorescent lipid probes. On this basis a new approach to studies of ligand-receptor binding is proposed. Using the interaction of the ricin B-chain with Burkitt lymphoma cells as an example and fluorescent labelled sphingomyelin as a probe, the ligand-induced changes of fluorescence anisotropy were shown to be concentration-dependent and to permit determination of the binding constant and the number of receptor-binding sites. The method was found to be specific and highly sensitive, allowing detection of the action of one RB molecule per cell. Scatchard analysis of the binding of 125I-RB demonstrated the presence on the cell surface of two binding sites with Kd approximately 10(-10) and approximately 10(-8) M, respectively. Only the high-affinity sites were detected by the fluorescence technique. Saturation of these sites resulted in maximum inhibition of protein synthesis.     

Analysis of structural and physico-chemical parameters involved in the specificity of binding between alpha-amylases and their inhibitors

Enzyme-inhibitor specificity was studied for alpha-amylases and their inhibitors. We purified and cloned the cDNAs of two different alpha-amylase inhibitors from the common bean (Phaseolus vulgaris) and have recently cloned the cDNA of an alpha-amylase of the Mexican bean weevil (Zabrotes subfasciatus), which is inhibited by alpha-amylase inhibitor 2 but not by alpha-amylase inhibitor 1. The crystal structure of AI-1 complexed with pancreatic porcine alpha-amylase allowed us to model the structure of AI-2. The structure of Zabrotes subfasciatus alpha-amylase was modeled based on the crystal structure of Tenebrio molitor alpha-amylase. Pairwise AI-1 and AI-2 with PPA and ZSA complexes were modeled. For these complexes we first identified the interface forming residues. In addition, we identified the hydrogen bonds, ionic interactions and loss of hydrophobic surface area resulting from complex formation. The parameters we studied provide insight into the general scheme of binding, but fall short of explaining the specificity of the inhibition. We also introduce three new tools-software packages STING, HORNET and STINGPaint-which efficiently determine the interface forming residues and the ionic interaction data, the hydrogen bond net as well as aid in interpretation of multiple sequence alignment, respectively.     

Biospecific chromatography of lipolytic enzymes and methods for determining their activity

The review concerns application of affinity chromatography for isolation of phospholipases and lipases, as well as the methods for determining their activities. Main emphasis is laid on the preparation of biospecific supports with lipid ligands as well as on development of new methods for assaying lipolytic activity.     

Analysis of selective, high protein-protein binding interaction of cohesin-dockerin complex using biosensing methods

Optical biosensors that use fluorescence are promising tools for the analysis of target materials such as protein, DNA and other biomaterial. To analyze the binding properties of a protein-protein interaction, we constructed fluorescent biomarkers based on the cohesin-dockerin interaction, which coordinates the assembly of cellulolytic enzymes and scaffolding proteins to produce a cell surface multiprotein complex known as the "cellulosome" in some anaerobic bacteria. Our 2D-PAGE results displayed diverse binding profiles to the dockerin containing cellulosomal proteins produced by Clostridium cellulovorans grown on different carbon sources, such as Avicel, xylan and AXP (Avicel:xylan:pectin (3:1:1)). Fluorescence intensity analysis indicated that EngE and EngH bound more efficiently to Coh6 than to Coh2 or Coh9 (2-fold to 6-fold and 1.5-fold to 5-fold, respectively), while others cellulosomal proteins displayed similar results. In addition, both an enzyme-linked interaction assay (ELIA) and surface plasmon resonance (SPR) analyses demonstrated that both EngE and EngH preferentially bound cohesin6 versus the other two cohesin molecules. This work demonstrated the analysis of the binding patterns between interacting proteins using fluorescent biomarkers. We also illustrated the potential of this sensitive approach to quantify specific target analytical materials via the example of the cohesin-dockerin interaction.     

Cytometric method of determining the parameters of the interaction of synthetic immunostimulants with the lymphocytic surface

The cytofluorimetric method was suggested for the determination of parameters of polymer adsorption on the cellular surface. Multiple equilibrium in the homogeneous cellular population with N independent binding sites and in the heterogeneous population of cells with the Poisson distribution of adsorption sites was analysed. A possibility was shown of cytofluorimetric drawing of the adsorption isotherm using mean fluorescence intensity of cells in the equilibrium suspension. The equilibrium association constant and the mean number of binding sites were estimated for adsorption of poly-L-lysin on the surface of human myelogenous leukemic cells (cellular line K-252).     

CD36 peptides enhance or inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction.

CD36 (glycoprotein IV or IIIB) is an integral plasma membrane protein of wide cellular distribution and functions as a receptor site for thrombospondin (TSP), an adhesive protein important in cell-cell and cell-matrix interactions. OKM5, a monoclonal anti-CD36 antibody, has been reported to block CD36 cell adhesive functions suggesting that the OKM5 epitope on CD36 is functionally important. A panel of 10 synthetic CD36 peptides was made. One peptide, P139-155, specifically inhibited the immunoadsorption of CD36 by OKM5, and P139-155 was directly immunoadsorbed by OKM5, indicating that CD36 sequence 139-155 represents part of the OKM5 epitope. TSP bound to immobilized P139-155 in a dose-dependent and saturable manner. Surprisingly, P139-155 significantly augmented, instead of inhibited, binding of CD36 to TSP. This peptide did not induce platelet aggregation but augmented ADP- and collagen-induced aggregation in platelet-rich plasma. Another CD36 peptide, P93-110, which had no effect on OKM5 immunoadsorption, blocked binding of CD36 to immobilized TSP and partially inhibited collagen-induced platelet aggregation. P93-110 by itself did not bind to TSP; however, in the presence of P139-155, there was a marked enhancement of P93-110 binding to TSP, with a stoichiometry consistent with the trimeric nature of TSP. The data suggest that CD36-TSP interaction is a two-step process; the sequence 139-155 region of CD36 binds first to TSP, triggering a change in TSP to reveal a second site, which binds the 93-110 region of CD36 with high affinity. CD36 peptides can be used as stimulators or inhibitors in cellular adhesive events involving TSP-CD36 interaction. Conformational changes leading to the exposure or activation of high affinity binding sites may occur in both the receptor and the ligand upon cell-cell and cell-matrix adhesion.     

Structural features of pregna-D'-pentaranes determining their interaction with three rat proteins

Competition of a number of progesterone 16alpha,17alpha-cycloalkane derivatives with 3H-labeled ligands for the binding sites of the rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16alpha,17alpha-cyclopropanoprogesterone, and 16alpha,17alpha-cyclopent-3'-enoprogesterone and the selective ligands for serum pentaranophilin are 6alpha-methyl-16alpha,17alpha-cyclohexanopregna-1,4-diene-3,20-dione and 3beta-hydroxy-16alpha,17alpha-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D' we studied decreased the affinity of ligands for all the three proteins. The substitution of the delta5-3beta-hydroxy grouping for the delta4-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3',4'-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.