Direct Binding Method Research Articles

Direct Binding Methods to Measure Receptor-Ligand Interactions.

G-protein-coupled receptors (GPCRs) contribute to numerous physiological processes via complex network mechanisms. While indirect signaling assays (Ca2+ mobilization, cAMP production, and GTPγS binding) have been useful in identifying and characterizing downstream signaling mechanisms of GPCRs, these methods lack measurements of direct binding affinities, kinetics, binding specificity, and selectivity that are important parameters in GPCR drug discovery. In comparison to existing direct methods that use radio- or fluorescent labels, label-free techniques can closely emulate the native interactions around binding partners. Surface plasmon resonance (SPR) is a label-free technique that utilizes the refractive index (RI) property and is applied widely in quantitative GPCR-ligand binding kinetics measurement including small molecules screening. However, purified GPCRs are further embedded in a synthetic lipid environment which is immobilized through different tags to the SPR sensor surface, resulting in a non-native environment. Here, we introduced a methodology that also uses the RI property to measure binding interactions in a label-free, immobilization-free arrangement. The free-solution technique is successfully applied in quantifying the interaction of bioactive lipids to cognate lipid GPCRs, which is not purified but rather present in near-native conditions, i.e., in milieu of other cytoplasmic lipids and proteins. To further consider the wide applicability of these free-solution approaches in biomolecular interaction research, additional applications on a variety of receptor-ligand pairs are imperative.

The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation

Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity.

Effect of spacer modification on enzymatic synthetic and hydrolytic activities of immobilized trypsin

A thorough comparison of spacer-mediated covalent and non-covalent immobilization of trypsin on micro-magnetic particles was accomplished in the present study. Trypsin was coupled via diaminoalkanes, aminoalkanoic acids, bovine serum albumin (BSA), and biotin-derivate spacers onto magnetic particles. A comparison of resulting synthetic and hydrolytic activities after immobilization was performed. Whereas hydrolytic trypsin activity was measured employing N-α-benzoyl-dl-arginine 4-nitroanilide (BAPNA) assay, synthetic trypsin activity was measured employing a dipeptide synthesis assay. Within spacer-mediated trypsin immobilization, diaminoalkanes, aminoalkanoic acids and biotin spacers showed up to 40% increased synthetic specific activity of trypsin compared to the spacer-free method. Within the hydrolytic reaction type, coupling of trypsin via diaminoalkanes and biotin spacers resulted in a specific activity increase of up to 30%. BSA-bound trypsin displayed only minor increasing effects on both activities of trypsin. Furthermore, protein loading-dependent specific synthetic and hydrolytic activities were evaluated for 8-aminooctanoic acid, 12-aminododecanoic acid and 1,12-diamonododecane as spacers and compared to the direct covalent binding method. The protein binding capacity of spacer-modified particles was lower compared to the direct binding method. Synthetic activity of 8-aminooctanoic acid-bound trypsin was higher than in the case of the spacer-free method over a broad protein loading range with an up to 10-fold increase. The hydrolytic activity was increased by 64% using 8-aminooctanoic acid as spacer within a lower protein loading range. Spacer-bound trypsin showed a slightly lowered reusability over ten sequential cycles compared to spacer-free covalent binding method.

Biomolecular Interactions of small-molecule inhibitors affecting the YopH protein tyrosine phosphatase.

We have developed competitive and direct binding methods to examine small-molecule inhibitors of protein tyrosine phosphatase activity. Focusing on the Yersinia pestis outer protein H, a potent bacterial protein tyrosine phosphatase, we describe how an understanding of the kinetic interactions involving Yersinia pestis outer protein H, peptide substrates, and small-molecule inhibitors of protein tyrosine phosphatase activity can be beneficial for inhibitor screening, and we further translate these results into a microarray assay for high-throughput screening.

Poly(glycidyl methacrylate)-Polystyrene Diblocks Copolymer Grafted Nanocomposite Microspheres from Surface-Initiated Atom Transfer Radical Polymerization for Lipase Immobilization: Application in Flavor Ester Synthesis

Functional hairy poly(styrene-b-glycidylmethacrylate) (P(S-GMA)) brushes were generated by grafting from bromoacetylated poly(styrene-divinylbenzene) (P(S-DVB)) microspheres via surface-initiated atom transfer radical polymerization (SI-ATRP). Two different approaches for the covalent immobilization of lipase onto microspheres were studied for the first time: (1) direct immobilization of lipase to the polymer brushes via their epoxy groups, and (2) immobilization of lipase via glutaraldehyde coupling after attachment of a spacer arm (hexamethylendiamine (HMDA)) to the polymer brushes. The covalent immobilization of the lipase on microspheres after spacer-arm attachment and glutaraldeyhde coupling was found to be the more effective than the direct binding method. In this case, a maximum value of the immobilized enzyme activity 498.5 U g−1 was found with an enzyme loading of 27.6 mg per gram of support. Thermal and storage stabilities increase upon immobilization on the P(S-DVB)-g-P(S-GMA)-HMDA-GA microsphe...

Comparison of non-digitalis binding properties of digoxin-specific Fabs using direct binding methods

Digibind and DigiFab are commercial formulations of polyclonal, ovine, digoxin-specific Fabs in clinical use for treatment of digoxin intoxication. Of interest for extending its use to other clinical indications, Digibind has also been reported to neutralize the effect of endogenous digoxin-like molecules, including ouabain, that are linked to clinical disorders ranging from preeclampsia to congestive heart failure. Although Digibind and DigiFab are equivalent in their digoxin-binding activity, the antigens used to produce these Fabs are different. We therefore explored, using native (3)H-digoxin and (3)H-ouabain in four different types of solution-phase binding methods, whether they might exhibit different profiles with respect to ouabain and other digoxin-like factors. Consistent with previous results, both Fab preparations bound digoxin with the same affinities and capacities. However, (3)H-ouabain was found to bind with high affinity only to Fab sub-populations present in both products. Interestingly, this sub-population was twice as large for Digibind compared to DigiFab. Competition experiments also showed differences in specificity within Fab sub-populations. Therefore, the equivalence in digoxin-binding activity of the two Fab preparations does not extend to ouabain-binding capacity and Fab specificity, with implications for clinical differentiation between the preparations in treatment of disorders related to control of non-digoxin cardenolides. The existence of a small but perhaps clinically relevant sub-population of antibodies was detected using specific radioligands. This sub-population could not have been detected nor quantified using standard cross-reactivity in an ELISA assay.

Metal-ion-mediated allosteric triggering of yeast pyruvate kinase. 2. A multidimensional thermodynamic linked-function analysis.

A role has been proposed for the free divalent metal in triggering the allosteric responses of yeast pyruvate kinase based upon a kinetic linked-function analysis [Mesecar, A. D., & Nowak, T. (1997a) (preceding paper in this series)]. The major conclusion from the analysis is that the allosteric activator, fructose 1,6-diphosphate (FDP), does not directly communicate with the substrate, phosphoenolpyruvate (PEP), at the active site of the enzyme: it is Mn2+ that mediates the allosteric communication between the PEP and FDP sites in an allosteric relay mechanism. Assumptions were necessary to treat kinetic parameters as thermodynamic parameters, and the presence of the substrate ADP was necessary for the kinetic analysis. In this study, the influence of FDP on the interactions of PEP and Mn2+ and the influence of PEP and Mn2+ on the interaction of FDP with YPK were measured, where possible, by direct binding methods in the absence of ADP. Direct binding data were then subjected to a thermodynamic linked-function analysis for a heterotropic, three ligand coupled system in order to ascertain the two and three ligand coupling free energies. The two ligand coupling free energies deltaG(Mn-PEP), deltaG(Mn-FDP), and deltaG(PEP-FDP) are -3.88, -1.09, and -0.22 kcal/mol, respectively. These values indicate that positive, heterotropic interactions exist between each of these ligand pairs. The three ligand coupling free energy term, deltaG(Mn-PEP-FDP), indicates that simultaneous binding of Mn2+, PEP, and FDP is considerably favored over the sum of their independent binding free energies by -6.6 kcal/mol. These results demonstrate the key role of the metal in the modulation of ligand binding and are consistent with the values and the relationships of the kinetic parameters obtained from the kinetic linked-function analysis.

Metal-Ion-Mediated Allosteric Triggering of Yeast Pyruvate Kinase. 1. A Multidimensional Kinetic Linked-Function Analysis

Regulation of the glycolytic pathway is considered to be primarily achieved by the carbon metabolites resulting from glucose metabolism [e.g., fructose 1,6-diphosphate (FDP), phosphoenolpyruvate (PEP), and citrate] and by the ATP charge of the cell. The divalent cations (e.g., Mg2+ and Mn2+) have not been considered as having regulatory roles in glycolysis, although they are involved in almost every enzyme-catalyzed reaction in the pathway. Using a kinetic linked-function analysis of steady-state kinetic data for the interactions of PEP, FDP, and Mn2+ with yeast pyruvate kinase (YPK), we have found that the divalent metal is the principal trigger of the allosteric responses observed with this enzyme. The interaction of Mn2+ to YPK enhances the interaction of FDP by -1.6 kcal/mol and the interaction of PEP by -2.8 kcal/mol. The simultaneous interaction of all three of these ligands to YPK is favored by -4.3 kcal/mol over the sum of their independent binding free energies. Surprisingly, the binding of the allosteric activator FDP does not directly influence the binding of the substrate PEP since a coupling free energy near zero was calculated for these two ligands. Thus, communication between the PEP and FDP sites occurs structurally through the metal by an allosteric relay mechanism. These conclusions are supported by results of a thermodynamic linked-function analysis of direct binding data for the interactions of PEP, FDP, and Mn2+ with YPK [Mesecar, A. D., & Nowak, T. (1997) Biochemistry (following paper in this series)]. Our findings raise important questions as to the possible roles of divalent metals in modulating multiligand interactions with YPK and in the regulation of the glycolytic pathway.

A TCR Binds to Antagonist Ligands with Lower Affinities and Faster Dissociation Rates Than to Agonists

T lymphocyte activation is mediated by the interaction of specific TCR with antigenic peptides bound to MHC molecules. Single amino acid substitutions are often capable of changing the effect of a peptide from stimulatory to antagonistic. Using surface plasmon resonance, we have analyzed the interaction between a complex consisting of variants of the MCC peptide bound to a mouse class II MHC (E k) and a specific TCR. Using both an improved direct binding method as well as a novel inhibition assay, we show that the affinities of three different antagonist peptide–E k complexes are ∼10–50 times lower than that of the wild-type MCC–E k complex for the TCR, largely due to an increased off-rate. These results suggest that the biological effects of peptide antagonists and partial agonists may be largely based on kinetic parameters.

Fluorometric Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane Filter Hybridization in which Radioisotopes Are Used To Determine Genetic Relatedness among Bacterial Strains

Fluorometric hybridization in microdilution wells was developed to determine genetic relatedness among microorganisms. Total chromosomal deoxyribonucleic acid (DNA) for hybridization reactions was labeled with photoreactive biotin (photobiotin). The biotinylated DNA was hybridized with single-stranded unlabeled DNAs which had been immobilized on the surfaces of microdilution wells. After hybridization, biotinylated DNA was quantitatively detected with beta-D-galactosidase and a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactopyranoside. Homology values obtained with this fluorometric direct binding method were compared with values obtained with two membrane filter methods, one in which photobiotin labeling was used and one in which radioisotope labeling was used. The results showed that the fluorometric direct binding method in which microdilution wells are used could be an alternative to radioisotope and membrane filter hybridization methods.

Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances.

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.

Interaction of ADP and magnesium with the active site of myosin subfragment-1 observed by reactivity changes of the critical thiols and by direct binding methods at low and high ionic strength.

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg X ADP and uncomplexed ADP to the active site of myosin subfragment-1. Additionally, the binding parameters for Mg2+ in the ternary complex protein X Mg X ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg X ADP just one mole of ligand binds/mole subfragment-1. The affinity of Mg X ADP at low ionic strength is 2.1 X 10(6) M-1 and only five-times lower in the absence of Mg2+. In the ternary complex Mg2+ has a low affinity of 4.1 X 10(4) M-1. At high ionic strength the uncomplexed ADP binds with a 43-times-lower affinity than Mg X ADP, whose affinity is 6.9 X 10(5) M-1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 X 10(5) M-1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.

Role of Cyclic Nucleotides in Secretory Mechanisms and Actions of Parathyroid Hormone and Calcitonin

This chapter discusses the role of cyclic nucleotides in secretory mechanisms and actions of parathyroid hormone (PTH) and calcitonin ( CT).The development of more purified preparations of PTH-secreting cells has made it feasible to assess directly intracellular cyclic nucleotide content and its relationship to hormonal secretion. The separation of distinct functional and morphologic classes of bone cells has made possible analysis of the sites of action and mechanistic basis of hormonal effects on bone. Labeled ligands are used to analyze by direct binding methods for hormone receptors on the several isolated cell types. The elegant microdissection techniques and the application of ultrasensitive histochemical bioassays help to describe the action of PTH and CT on the kidney. The chapter also reviews the progress in these areas with emphasis on advances in methods, regulation of cyclic nucleotides by hormones and other agents, and mechanisms for cyclic nucleotide control of cellular functions. The development of a radioimmunoassay for PTH made it feasible to determine directly the effects of diverse secretagogues on PTH secretion. The development of in vitro parathyroid slice and fragment preparations made it possible to explore indirectly cyclic nucleotide involvement in secretion.

Identification of beta-adrenergic receptors in human lymphocytes by (-) (3H) alprenolol binding.

Human lymphocytes are known to posessess a catecholamine-responsive adenylate cyclase which has typical beta-adrenergic specificity. To identify directly and to quantitate these beta-adenergic receptors in human lymphocytes, (-) [3H] alprenolol, a potent beta-adrenergic antagonist, was used to label binding sites in homogenates of human mononuclear leukocytes. Binding of (-) [3H] alprenolol to these sites demonstrated the kinetics, affinity, and stereospecificity expected of binding to adenylate cyclase-coupled beta-adrenergic receptors. Binding was rapid (t1/2 less than 30 s) and rapidly reversible (t1/2 less than 3 min) at 37 degrees C. Binding was a saturable process with 75 +/- 12 fmol (-) [3H] alprenolol bound/mg protein (mean +/- SEM) at saturation, corresponding to about 2,000 sites/cell. Half-maximal saturation occurred at 10 nM (-) [3H] alprenolol, which provides an estimate of the dissociation constant of (-) [3H] alprenolol for the beta-adrenergic receptor. The beta-adrenergic antagonist, (-) propranolol, potently competed for the binding sites, causing half-maximal inhibition of binding at 9 nM. beta-Adrenergic agonists also competed for the binding sites. The order of potency was (-) isoproterenol greater than (-) epinephrine greater than (-)-norepinephrine which agreed with the order of potency of these agents in stimulating leukocyte adenylate cyclase. Dissociation constants computed from binding experiments were virtually identical to those obtained from adenylate cyclase activation studies. Marked stereospecificity was observed for both binding and activation of adenylate cyclase. (-)Stereoisomers of beta-adrenergic agonists and antagonists were 9- to 300-fold more potent than their corresponding (+) stereoisomers. Structurally related compounds devoid of beta-adrenergic activity such as dopamine, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine did not effectively compete for the binding sites. (-) [3H] alprenolol binding to human mononuclear leukocyte preparations was almost entirely accounted for by binding to small lymphocytes, the predominant cell type in the preparations. No binding was detectable to human erythrocytes. These results demonstrate the feasibility of using direct binding methods to study beta-adrenergic receptors in a human tissue. They also provide an experimental approach to the study of states of altered sensitivity to catecholamines at the receptor level in man.

Construction and study of electrodes using cross-linked enzymes.

The construction of electrodes, the behavior of which could be accurately analyzed and optimized is described. Two methods were used to construct such enzyme electrodes. A direct binding method, where a cation electrode was dipped into a solution of enzyme, albumin, and glutaraldehyde: a thin layer of cross-linked protein coated the electrode bulb. A two-step method, where an active membrane was first made by cross-linking these proteins by glutaraldehyde on a glass plate, then fitting with a silicone membrane onto a gas electrode (O2 or CO2). Both methods were used to construct glucose and urease electrodes measuring either NH4+ ions or CO2. Glucose oxidase and L-amino acid oxidase-pO2 electrodes were also developed. The theoretical and experimental behavior of these enzymatic membrane electrodes were compared using the analysis of product concentrations resulting from the association of diffusion with enzyme reaction in the active layer, and the optimum operating conditions were determined.