The possible mechanism of binding interaction of insulin molecule with its receptor.

Detailed structural comparisons and investigation of DPI, 2 Zn insulin and some other derivatives of insulin were performed by the least-squares superimposition technique and the graphics technique. It is pointed out in this paper that the binding interaction with the receptor molecule should take place mainly on an amphipathic surface of the insulin molecule. In the middle, there is a hydrophobic surface with an area of about 150 A2 consisting of many hydrophobic residues; while the polar or charged groups distributing around the hydrophobic surface construct a hydrophilic zone. The hydrophobic surface is usually covered by the extended B-chain C-terminal peptides with great mobility and protected from the solvent molecules. The angle between the amphipathic surface and the surface of dimerization is about 20 degrees. The results from the detailed structural comparison between Al-(L-Trp) insulin and Al-(D-Trp) insulin have provided a very good explanation to their great difference in biological activity, and confirmed our proposed binding interaction model of the insulin molecule with its receptor as well.     

Interplay of chemical microenvironment and redox environment on thiol-disulfide exchange kinetics

The interplay between the chemical microenvironment surrounding disulfides and the redox environment of the media on thiol-disulfide exchange kinetics was examined by using a peptide platform. Exchange kinetics of up to 34 cysteine-containing peptides were measured in several redox buffers. The electrostatic attraction/repulsion between charged peptides and reducing agents such as glutathione was found to have a very pronounced effect on thiol-disulfide exchange kinetics (differences of ca. three orders of magnitude). Exchange kinetics could be directly correlated to peptide charge over the entire range examined. This study highlights the possibility of finely and predictably tuning thiol-disulfide exchange, and demonstrates the importance of considering both the local environment surrounding the disulfide and the nature of the major reducing species present in the environment for which their use is intended (e.g., in drug delivery systems, sensors, etc).     

'Living' controlled in situ gelling systems: thiol-disulfide exchange method toward tailor-made biodegradable hydrogels

A 'living' controlled hydrogel formation method was first reported to create loose and compact in situ biodegradable hydrogels. The method executed under mild reaction conditions can conveniently tailor the hydrogel properties, and it has the potential to develop into a powerful tool for the design, synthesis, and self-assembly of novel tailor-made biomaterials and drug delivery systems.     

The influence of non-specific binding on scatchard analysis used in insulin/receptor binding studies with erythrocytes

Conclusions By equilibrium binding studies it could be demonstrated that erythrocytes have been saturated with specifically bound insulin in the order of 0.10–0.20 nmol/l with 4×10¹² erythrocytes/l. From this an insulin receptor number of 15–30 receptors/normal erythrocyte could be calculated.By the current results we got evidence that in the insulin/erythrocyte receptor system the terminal part of the Scatchard plot was artefactual and might not be used for the calculation procedures because it is strongly influenced by non-specific effects. Because of the fluid change from the specific to the non-specific character of binding we propose to use total ligand concentrations only up to 5 nmol/l with 4×10¹² erythrocytes/l for the investigation of specific binding on erythrocyte insulin receptors.By focussing on the initial part of the Scatchard plot curvilinearity disappeared and the character of this hormone/receptor system was reduced to one binding site with one characteristic affinity constant and a receptor concentration of one characteristic order.

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Der Einfluß der unspezifischen Bindung bei Anwendung des Scatchard Plots zur Untersuchung der Insulin/Receptor-Wechselwirkungen an Erythrocyten

     

Inhibition of the binding of low-density lipoprotein to its cell surface receptor in human fibroblasts by positively charged proteins.

A group of proteins and polyamino acids with positively charged domains were shown to inhibit the binding of 125I-LDL to its receptor on the surface of human fibroblasts. The list of inhibitory proteins included platelet factor 4 (which has a cluster of lysine residues at its carboxyl terminus), two lysine-rich histones, poly-L-lysines of chain length greater than 4, and protamine. These proteins were effective in the concentration range of 5--10 microgram/ml. Two other positively charged proteins, lysozyme and avidin, did not inhibit 125I-LDL binding. Kinetic studies suggested that protamine was not acting simply as a competitive inhibitor with regard to the LDL receptor. In light of previous data showing that polyanions such as heparin and polyphosphates also inhibit 125-I-LDL binding to its cell surface receptor, the current findings suggest that charge interactions are important in this binding reaction. In a related series of studies, a number of glycoproteins and their asialo derivatives as well as a number of sugar phosphates failed to inhibit 125I-LDL binding to its receptor in fibroblasts.     

Chemical synthesis of human selenoprotein F and elucidation of its thiol-disulfide oxidoreductase activity

Selenoprotein F (SelF) is an endoplasmic reticulum-residing eukaryotic protein that contains a selenocysteine (Sec) residue. It has been suggested to be involved in a number of physiological processes by acting as a thiol-disulfide oxidoreductase, but the exact role has remained unclear due to the lack of a reliable production method. We document herein a robust synthesis of the human SelF through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are the use of a mild desulfurization process to protect the side-chain of the Sec residue from being affected and the simultaneous removal of acetamidomethyl and p-methoxybenzyl protection groups by PdCl₂, thus facilitating the synthesis of multi-milligrams of homogenous SelF. The reduction potential of SelF was determined and the thiol-disulfide oxidoreductase activity was further supported by its ability to catalyze the reduction and isomerization of disulfide bonds.

The chemical synthesis of the 134-residue human selenoprotein F (SelF) was accomplished on a multi-milligram scale. The synthetic SelF exhibits typical thiol-disulfide oxidoreductase activity.     

Effect of polyion conformation on dye binding. IV. Fluorescence of auramine O in relation to its binding to synthetic polyacids

The effect of polymer conformation, polymer concentration, and added salt concentration on the quantum yield ? of Auramine O (AuO) is discussed in connection with dialysis equilibrium data. The quantum yield per bound dye molecule (?/q) assumes different values which are related principally to solvation changes in the environment of the binding sites. Binding to globular compact a states results in high ?/q, whereas low ?/q (of the order of magnitude of ? of free AuO) are observed for the binding to expanded solvated b states. The quantum yield of bound AuO is therefore affected by the organic nature of the environment, but shows little or no relation to the amount of bound dye molecules.     

Study of thiol-disulfide equilibrium in the whole blood and its fractions by stripping voltammetric titration

A stripping voltammetric signal is used as the source of information in the new version of electrochemical analysis, stripping voltammetric titration (SVT). The reaction RSH + AgNO₃ = AgSR + HNO₃ is used to substantiate the method. A procedure is developed to study the thiol-disulfide equilibrium in the whole blood and its fractions (hemolysate, plasma, and erythrocyte mass). Disulfides are reduced by sodium sulfite before their determination. The accuracy of the results is verified in the analysis of model solutions and using the added-found method. A version of rapid analysis from a single aliquot portion of a test sample is proposed.     

Role of Ion exchange in permeation processes

The single ion transport of transition metal ions like Cu²⁺, Co²⁺, Ni²⁺ and Zn²⁺ were carried out through the H⁺ and alkali metal ion forms of Nafion membrane. These studies showed that the ion exchange selectivity coefficient of the permeating ion had an effect on its transport process. It was found that the diffusion coefficient values (D) were directly proportional to the selectivity coefficient (K). This shows that the initial stage of permeation is governed by ion exchange process (effect of K on D).     

Evaluation of multivalent dendrimers based on melamine: kinetics of thiol-disulfide exchange depends on the structure of the dendrimer

The rate of thiol-disulfide exchange of dansyl groups mediated by dithiothreitol depends on the structure of the dendrimer. In general, the rate of exchange decreases as the size of the dendrimer increases. Dendrimers with disulfides attached near the core undergo exchange more slowly than dendrimers with disulfides near the periphery. Exchange is a bimolecular (noncooperative) process between dansyl-linked disulfides and dithiothreitol. No evidence for intramolecular macrocylization (cooperative) exchange is observed. Mass spectrometry is used to follow exchange in two dendrimers, providing qualitative and quantitative information about this process. Mathematical models suggest that the rates for exchange for all disulfides of a dendrimer are similar, but increase as the exchange reaction progresses.     

Role of exchange interaction in spin catalysis mechanisms

Possible mechanisms are examined for spin catalysis, in which a reaction is accelerated through the action of a catalyst on the spin parameters of the system. Since the orbital and spin characteristics of electrons are interrelated through permutational symmetry (Pauli principle) by means of exchange interactions, this article examines the role of exchange in catalysis. Spin uncoupling has been found to play a role, not only for paramagnetic catalysts, but also for all types of catalysis.Cherkassy Engineering Technology Institute, 460 Shevchenko Boulevard, Cherkassy 257006, Ukraine. Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 32, No. 1, pp. 1–12, January–February, 1996. Original article submitted April 5, 1995.     

Importance of receptor flexibility in binding of cyclam compounds to the chemokine receptor CXCR4

We have elucidated the binding sites of four moncyclam and one bicyclam antagonist AMD3100, in the human chemokine receptor CXCR4. Using the predicted structural models of CXCR4, we have further predicted the binding sites of these cyclam compounds. We used the computational method LITiCon to map the differences in receptor structure stabilized by the mono and bicyclam compounds. Accounting for the receptor flexibility lead to a single binding mode for the cyclam compounds, that has not been possible previously using a single receptor structural model and fixed receptor docking algorithms. There are several notable differences in the receptor conformations stabilized by monocyclam antagonist compared to a bicylam antagonist. The loading of the Cu(2+) ions in the cyclam compounds, shrinks the size of the cyclam rings and the residue D262(6.58) plays an important role in bonding to the copper ion in the monocylam compounds while residue E288(7.39) is important for the bicyclam compound.     

Protein side chains facilitate Mg/Al exchange in model protein binding sites.

Among the most frequent protein binding sites served by Mg(II), we identify those which have higher affinity towards Al(III). We also estimate the free energies of metal exchange for all these binding sites taking into account solvent effects explicitly. The obtained results show that thermodynamically favored Mg(II)/Al(III) exchange reactions take place at a number of these metal binding sites, which could possibly be related to some dysfunctions of Mg(II)-dependent biological processes. Additionally, they shed light on the molecular basis of the toxicity of Al(III) in living organisms.     

Supramolecular binding of protonated amines to a receptor microgel in aqueous medium

Polyanionic microgels containing negatively charged tetrazole binding sites show supramolecular binding of various protonated amines (e.g. dibucaine and spermine) in a competitive aqueous medium at millimolar concentration.     

Hormone receptor mobility and catecholamine binding in membranes. A theoretical model.

[3H]-Catecholamine binding to intact cells, isolated cell membranes, and to several isolated macromolecules has been shown by several laboratories to be neither stereospecific nor inhibited by known beta-antagonists. Since additional evidence indicates that this binding is not an artifact (i.e. due neither to the binding of a catecholamine oxidation product nor hormone binding to a catabolic enzyme such as COMT), the question remains as to whether this represents binding to a bona fide membrane receptor. Because all ligands which bind strongly or compete for this binding possess a catechol group, one possible explanation is that the binding affinity is primarily determined by the catechol moiety, whereas the correct stereoisomer of the side chain is necessary to activate the receptor. Thus, although binding is a necessary condition for hormone action, the necessary and sufficient condition for activation of adenyl cyclase is both the catechol group and the correct stereoisomer of the side chain. A theoretical model is developed here to provide a quantitative basis for this hypothesis. This model extends the current concept of distinct subunits in the adenyl cyclase system by separating the receptors from the catalytic sites and placing them at separate locations within the membrane. Utilizing the spare receptor model of Furchgott, and the mobility of macromolecules within a "lipid sea," the appropriate equations to predict both hormone binding and enzyme activation are derived. Using the observed affinity constants from catecholamine binding studies, it is then shown that this model can predict the experimental observation and hence explain the apparent dichotomy arising from binding enzyme activation studies.     

Distance geometry analysis of ligand binding to drug receptor sites

Summary The method known as distance geometry approach for receptor mapping procedures is discussed. In this method a ligand binding to a certain receptor is considered as a collection of ligand points. Binding sites of the receptor are either empty or filled site points; a ligand point might bind to an empty site point; filled site points indicate that at that point no binding is possible. A binding mode of a ligand is a list of which ligand points coincide with which empty binding sites. The applicability of the method for QSAR studies is discussed; as examples are mentioned the dihydrofolate reductase, ₁- and ₂-receptors. Finally, some ideas on future developments in receptor mapping are discussed.DEDICATION This article is dedicated to the late Dr. Teake Bultsma who introduced the distance geometry approach into our department.     

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand‐binding domain

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand‐binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRα LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator‐activated receptor γ (PPARγ) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line‐broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo‐ or hetero‐dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor‐interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRα LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C‐terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line‐broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRα LBD–antagonist–peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRα LBD conformations in favor of a compact state, and that a PPARγ LBD‐agonist complex can prevent the antagonist from enhancing the RXRα LBD‐coactivator binding interaction. Copyright © 2009 John Wiley & Sons, Ltd.