Competitive Binding Analysis Research Articles

Biological inferences from IgM binding characteristics of recombinant human secretory component mutants

The polymeric immunoglobulin receptor (pIgR) or membrane secretory component (SC) selectively transports polymeric IgA and IgM across secretory epithelial cells to mucosal surfaces. The ligand binding ectodomain consists of five homologous Ig-like domains with domain I being an absolute requirement for binding. The role of DII to V in IgM binding remains unknown. Here, using in vitro refolded non-glycosylated recombinant domain deletion mutants of human SC, we show by biological and biophysical binding assays that DII to V are required for high affinity binding to IgM. Competitive binding analysis, by whole cell ELISA, showed that DII–V significantly increase the affinity of recombinant SC for IgM ( K i = 2.42 nM) as opposed to recombinant DI only ( K i = 44.8 nM). Lastly, we provide qualitative data highlighting the complexity of measuring the IgM/SC interaction using surface plasmon resonance spectroscopy.

Specific Interactions between the Viral Coreceptor CXCR4 and the Biguanide-Based Compound NB325 Mediate Inhibition of Human Immunodeficiency Virus Type 1 Infection

The present studies were conducted to better define the mechanism of action of polyethylene hexamethylene biguanide (PEHMB) (designated herein as NB325), which was shown in previous studies to inhibit infection by the human immunodeficiency virus type 1 (HIV-1). Fluorescence-activated flow cytometric analyses of activated human CD4(+) T lymphocytes exposed to NB325 demonstrated concentration-dependent reductions in CXCR4 epitope recognition in the absence of altered recognition of selected CD4 or CD3 epitopes. NB325 also inhibited chemotaxis of CD4(+) T lymphocytes induced by the CXCR4 ligand CXCL12. However, NB325 did not cause CXCR4 internalization (unlike CXCL12) and did not interfere with CXCL12 binding. Additional flow cytometric analyses using antibodies with distinct specificities for extracellular domains of CXCR4 demonstrated that NB325 specifically interfered with antibody binding to extracellular loop 2 (ECL2). This interaction was confirmed using competitive binding analyses, in which a peptide derived from CXCR4 ECL2 competitively inhibited NB325-mediated reductions in CXCR4 epitope recognition. Collectively, these results demonstrate that the biguanide-based compound NB325 inhibits HIV-1 infection by specifically interacting with the HIV-1 coreceptor CXCR4.

A Homogeneous G Protein-Coupled Receptor Ligand Binding Assay Based on Time-Resolved Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) has emerged as a powerful tool to the study of protein-protein interactions, such as receptor-ligand binding. However, the application of FRET to the study of G protein-coupled receptors (GPCRs) has been limited by the method of labeling receptor with fluorescence probes. Here we described a novel time-resolved (TR)-FRET method to study GPCR-ligand binding by using human complement 5a (C5a) receptor (C5aR) as a model system. Human C5aR was expressed in human embryonic kidney 293 cells with a hemagglutinin (HA) epitope at the N-terminus. Purified human C5a was labeled with terbium chelate and used as the fluorescence donor. Monoclonal anti-HA antibody conjugated with Alexa Fluor 488 was used as the fluorescence acceptor. Robust FRET signal was observed when the labeled ligand and C5aR membrane were mixed in the presence of the conjugated anti-HA antibody. This FRET signal was specific and saturable. C5a binding affinity to C5aR measured by the FRET assay was consistent with the data as determined by competition binding analysis using radiolabeled C5a. The FRET assay was also used to determine affinity of C5aR antagonists by competition binding analysis, and the data are similar to those from radioligand binding studies. Compared to the commonly used radioligand binding assay, this TR-FRET-based assay provides a nonradioactive, faster, and sensitive homogeneous assay format that could be easily adapted to high-throughput screening. The principle of this assay should also be applicable to other GPCRs, especially to those receptors with peptide or protein ligands.

Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor Potential Vanilloid 1 Sensitization

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

Differential Antagonism of Activin, Myostatin and Growth and Differentiation Factor 11 by Wild-Type and Mutant Follistatin

Follistatin binds and neutralizes members of the TGFbeta superfamily including activin, myostatin, and growth and differentiation factor 11 (GDF11). Crystal structure analysis of the follistatin-activin complex revealed extensive contacts between follistatin domain (FSD)-2 and activin that was critical for the high-affinity interaction. However, it remained unknown whether follistatin residues involved with myostatin and GDF11 binding were distinct from those involved with activin binding. If so, this would allow development of myostatin antagonists that would not inhibit activin actions, a desirable feature for development of myostatin antagonists for treatment of muscle-wasting disorders. We tested this hypothesis with our panel of point and domain swapping follistatin mutants using competitive binding analyses and in vitro bioassays. Our results demonstrate that activin binding and neutralization are mediated primarily by FSD2, whereas myostatin binding is more dependent on FSD1, such that deletion of FSD2 or adding an extra FSD1 in place of FSD2 creates myostatin antagonists with vastly reduced activin antagonism. However, these mutants also bind GDF11, indicating that further analysis is required for creation of myostatin antagonists that will not affect GDF11 activity that could potentially elicit GDF11-induced side effects in vivo.

Estrogen binding sites in the sea scallop: Characterization and possible involvement in reproductive regulation

Previous reports have suggested that estrogen is involved in bivalve reproduction and have also hypothesized that its effects are mediated through binding sites on specific receptors. In this study, we provide initial characterization of the estrogen binding sites in the gonads of both female and male sea scallops ( Placopecten magellanicus). Saturation analyses indicated two binding sites in fractions which have classically been used to represent the cytosol and the nucleus. One binding site is characterized by high affinity and limited binding capacity while the other site is characterized by low affinity and high capacity. Competitive binding analyses demonstrated that these sites can bind natural and synthetic estrogens with high affinity but only bind testosterone and progesterone at high concentrations. Comparison of binding capacity in scallops at different sexual maturation stages suggested that these sites may be involved in reproductive regulation in sea scallops.

Cadmium accumulation and binding characteristics in intact Sertoli/germ cell units, and associated effects on stage‐specific functions in vitro: insights from a shark testis model

The increased human use of cadmium (Cd) and its increased occurrence in the environment is of concern. The testis is sensitive to Cd because of the steroid-mediated regulation of spermatogenesis, high levels of DNA synthesis and gene transcription, all of which varies in a stage-related manner. Validated techniques (acridine orange vital staining to detect apoptosis and dextran-rhodamine exclusion to assess blood-testis barrier function) were recently developed and the shark testis was proposed as an alternative model for assessing stage-specific functions in living testicular tissue and to study toxicant actions on spermatogenesis. The present paper shows that 109Cd accumulation and binding in vitro was stage-dependent (premeiotic, PrM > meiotic, M > postmeiotic, PoM), rapid and persisted in spermatocysts (intact germ cell/Sertoli cell units) 49 h after washout. In competitive binding analyses of all three spermatocyst stages, Hg, but not Zn, could replace bound 109Cd, suggesting that Cd binding was specific. These findings were associated with a biphasic apoptotic response in the PrM spermatocysts, which was maximal at 10 microm CdCl2 and 1 microm CdCl2 after 2 and 4 days in culture, respectively. Although Cd uptake in PoM cysts was more than 2-fold less than uptake in PrM cysts, the percentage dextran-rhodamine permeant PoM cysts was approximately 8-fold greater than in controls in the presence of both 10 microm CdCl2 and 30 microm CdCl2 after 4 days culture, indicating that blood-testis barrier function in PoM spermatocysts was compromised. These findings demonstrate that this model has utility for use in screening assays of environmental toxicants.

Persistent binding of ligands to the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. While HAHs are several orders of magnitude more potent in producing AhR-dependent biochemical effects than PAHs or other AhR agonists, only the HAHs have been observed to produce AhR-dependent toxicity in vivo. Here we have characterized the dissociation of a prototypical HAH ligand ([(3)H] 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and PAH-like ligand ([(3)H] beta-naphthoflavone [betaNF]) from the guinea pig, hamster, mouse, and rat hepatic cytosolic AhR in order to elucidate the relationship between the apparent ligand-binding affinities and the divergent potency of these chemicals. Both compounds dissociated very slowly from the AhR with the amount of specific binding remaining at 96 h ranging from 53% to 70% for [(3)H]TCDD and 26% to 85% for [(3)H] betaNF, depending upon the species examined. The rate of ligand dissociation was unaffected by protein concentration or incubation temperature. Preincubation of cytosol with 2,3,7,8-tetrachlorodibenzofuran, carbaryl, or primaquine, prior to the addition of [(3)H]TCDD, shifted the apparent IC(50) of these compounds as competitive AhR ligands by approximately 10- to 50-fold. Our results support the need for reassessment of previous AhR ligand-binding affinity calculations and competitive binding analysis since these measurements are not carried out at equilibrium binding conditions. Our studies suggest that AhR binding affinity/occupancy has little effect on the observed differences in the persistence of gene expression by HAHs and PAHs.

ETS-1 AND P300 SYNERGISTICALLY UP-REGULATE NATRIURETIC PEPTIDE RECEPTOR A GENE (NPR1) TRANSCRIPTION.

Activation of guanylyl cyclase/natriuretic peptide receptor A (GC-A/NPRA) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. Studies to elucidate the molecular regulation of Npr1 (coding for NPRA) gene transcription are limited. The present study was conducted to elucidate the molecular mechanism governing Npr1 gene transcription, which would provide new insights into strategies to control NPRA-dependent signaling in pathophysiology of hypertension and cardiovascular disorders. Npr1 promoter-reporter deletion constructs were transiently transfected in mouse mesangial cells (MMCs) and transcriptional activity was measured by dual luciferase assay. To determine the effect of transcription factors, cotransfections were performed along with the promoter constructs. Functional deletion analysis of Npr1 promoter defined a ≈400 base pairs (bp) region as a basal promoter having Sp1, Lyf-1, p300, c-ETS, and GATA 1/2 transcription factor binding sites. Further deletion of the basal promoter defined a 90 bp fragment as core promoter, having two Ets-1 and a p300 binding motifs, which was found to be essential in controlling transcription. Electrophoretic mobility shift assay, competition binding, and supershift analyses confirmed that endogenously expressed Ets-1 binds to Ets-1 consensus sites. Site-directed mutagenesis of the two Ets-1 and p300 binding sites significantly reduced the functional activity (p

Induction of Porphyromonas gingivalis GroEL signaling via binding to Toll‐like receptors 2 and 4

Heat shock protein 60 (HSP60) has been recognized as an important molecule in infectious and autoimmune diseases. Although Porphyromonas gingivalis GroEL, a homologue of HSP60, is a potent stimulator of inflammatory cytokines, its receptor and signaling mechanisms are not yet understood in detail. In this study, we investigated whether the Toll-like receptor (TLR) family plays a functional role as a P. gingivalis GroEL receptor. Human macrophage-like THP-1 cells were used and the nuclear factor-kappaB (NF-kappaB) activity of cells stimulated with a recombinant P. gingivalis GroEL was measured with a luciferase assay. Flow cytometry analysis was used to determine the binding to THP-1 cells of fluorescein isothiocyanate (FITC)-labeled GroEL. In addition, anti-human TLR (anti-hTLR)2 and anti-hTLR4 monoclonal antibodies were used to assess the functional role of TLR2 and TLR4 as the receptors for GroEL. We observed by luciferase assay that the purified recombinant GroEL was able to stimulate NF-kappaB transcriptional activity in THP-1 cells. Flow cytometry analysis showed that the FITC-labeled GroEL bound to THP-1 cells in a dose-dependent fashion. Our binding competition analysis with FITC-labeled and unlabeled GroEL showed that it bound to the cells as a specific mode of action. On the other hand, GroEL-stimulated NF-kappaB transcriptional activity was significantly inhibited by anti-hTLR2 and anti-hTLR4 antibodies and was inhibited more strongly by a combination of both antibodies. Our present study demonstrates that P. gingivalis GroEL induces its intracellular signaling cascade in THP-1 cells via TLR2 or TLR4 and via a combination of both receptors.

Characterization and Application of a Novel RNA Aptamer against the Mouse Prion Protein

In order to isolate RNA aptamers against the mouse prion protein (mPrP), we carried out in vitro selection from RNA pools containing a 30-nucleotide randomized region. Aptamer 60-3 was found to have a high affinity for mPrP (K(d) = 5.6 +/- 1.5 nM), and 2'-fluoro-pyrimidine modifications for RNase resistance did not abolish its binding activity (K(d) = 22 +/- 4 nM). Following 5' biotinylation, aptamer 60-3 specifically detected PrP in mouse brain homogenate in a Northwestern blotting assay. To determine the mPrP-aptamer binding region, we performed protein-deletion-mutant analysis and competition-binding analysis using heparin. The results showed that aptamer 60-3 appears to have binding sites located between amino acids 23-108.

Structural Basis for Reduced Staphylocoagulase-mediated Bovine Prothrombin Activation

Staphylocoagulase (SC) is a protein secreted by the human pathogen, Staphylococcus aureus, that activates human prothrombin (ProT) by inducing a conformational change. SC-bound ProT efficiently clots fibrinogen, thus bypassing the physiological blood coagulation pathway. The crystal structure of a fully active SC fragment, SC-(1-325), bound to human prethrombin 2 showed that the SC-(1-325) N terminus inserts into the Ile(16) pocket of prethrombin 2, thereby inducing expression of a functional catalytic site in the cognate zymogen without peptide bond cleavage. As shown here, SC-(1-325) binds to bovine and human ProT with similar affinity but activates the bovine zymogen only very poorly. By contrast to the approximately 2-fold difference in chromogenic substrate kinetic constants between human thrombin and the SC-(1-325).human (pro)thrombin complexes, SC-(1-325).bovine ProT shows a 3,500-fold lower k(cat)/K(m) compared with free bovine thrombin, because of a 47-fold increase in K(m) and a 67-fold decrease in k(cat). The SC-(1-325).bovine ProT complex is approximately 5,800-fold less active compared with its human counterpart. Comparison of human and bovine fibrinogen as substrates of human and bovine thrombin and the SC-(1-325).(pro)thrombin complexes indicates that the species specificity of SC-(1-325) cofactor activity is determined primarily by differences in conformational activation of bound ProT. These results suggest that the catalytic site in the SC-(1-325).bovine ProT complex is incompletely formed. The current crystal structure of SC-(1-325).bovine thrombin reveals that SC would dock similarly to the bovine proenzyme, whereas the bovine (pro)thrombin-characteristic residues Arg(144) and Arg(145) would likely interfere with insertion of the SC N terminus, thus explaining the greatly reduced activation of bovine ProT.

Chiral recognition mass spectrometry: remarkable effects observed from the relative ion abundances of ternary diastereomeric complexes using electrospray ionization

The relative abundances of ternary diastereomeric complexes (composed each from a cinchonane-type chiral selector, a model chiral acid, and an alkali cation) are shown to change remarkably and fortuitously with variation in concentration and type of alkali metal using electrospray ionization-mass spectrometry and competitive binding analysis.

The R6A-1 peptide binds to switch II of Gα i1 but is not a GDP-dissociation inhibitor

Heterotrimeric G-proteins are molecular switches that convert signals from membrane receptors into changes in intracellular physiology. Recently, several peptides that bind heterotrimeric G-protein α subunits have been isolated including the novel Gα i1 · GDP binding peptides R6A and KB-752. The R6A peptide and its minimized derivative R6A-1 interact with Gα i1 · GDP. Based on spectroscopic analysis of BODIPYFL-GTPγS binding to Gα i1, it has been reported that R6A-1 has guanine nucleotide dissociation inhibitor (GDI) activity against Gα i1 [W.W. Ja, R.W. Roberts, Biochemistry 43 (28) (2004) 9265–9275]. Using radioligand binding, we show that R6A-1 is not a GDI for Gα i1 subunits. Furthermore, we demonstrate that R6A-1 reduces the fluorescence quantum yield of the Gα i1–BODIPYFL-GTPγS complex, thus explaining the previously reported GDI activity as a fluorescence artifact. We further show that R6A-1 has significant sequence similarity to the guanine nucleotide exchange factor peptide KB-752 that binds to switch II of Gα i1. We use competitive binding analysis to show that R6A-1 also binds to switch II of Gα subunits.

Insulin-Like Growth Factor Binding Protein-2: Contributions of the C-Terminal Domain to Insulin-Like Growth Factor-1 Binding

Signaling by the insulin-like growth factor (IGF)-1 receptor (IGF-1R) has been implicated in the promotion and aggressiveness of breast, prostate, colorectal, and lung cancers. The IGF binding proteins (IGFBPs) represent a class of natural IGF antagonists that bind to and sequester IGF-1/2 from the IGF-1R, making them attractive candidates as therapeutics for cancer prevention and control. Recombinant human IGFBP-2 significantly attenuated IGF-1-stimulated MCF-7 cell proliferation with coaddition of 20 or 100 nM IGFBP-2 (50 or 80% inhibition, respectively). We previously identified IGF-1 contact sites both upstream and downstream of the CWCV motif (residues 247-250) in human IGFBP-2 (J Biol Chem 276:2880-2889, 2001). To further test their contributions to IGFBP-2 function, the single tryptophan in human IGFBP-2, Trp-248, was selectively cleaved with 2-(2'nitrophenylsulfenyl)-3-methyl-3 bromoindolenine (BNPS-skatole) and the BNPS-skatole products IGFBP-2(1-248) and IGFBP-2(249-289) as well as IGFBP-2(1-190) were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an approximately 20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-2(1-248) = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-2(1-190) EC50 = 9.2 nM). In kinetic assays, IGFBP-2(1-248) and IGFBP-2(1-190) exhibited more rapid association and dissociation rates than full-length IGFBP-2. These results confirm that regions upstream and downstream of the CWCV motif participate in IGF-1 binding. They further support the development of full-length IGFBP-2 as a cancer therapeutic.

Evolutionarily divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway

The herpesvirus entry mediator (HVEM), a member of the TNF receptor (TNFR) superfamily, can act as a molecular switch that modulates T cell activation by propagating positive signals from the TNF-related ligand LIGHT (TNFR superfamily 14), or inhibitory signals through the Ig superfamily member B and T lymphocyte attenuator (BTLA). Competitive binding analysis and mutagenesis reveals a unique BTLA binding site centered on a critical lysine residue in cysteine-rich domain 1 of HVEM. The BTLA binding site on HVEM overlaps with the binding site for the herpes simplex virus 1 envelope glycoprotein D, but is distinct from where LIGHT binds, yet glycoprotein D inhibits the binding of both ligands, potentially nullifying the pathway. The binding site on HVEM for BTLA is conserved in the orphan TNFR, UL144, present in human CMV. UL144 binds BTLA, but not LIGHT, and inhibits T cell proliferation, selectively mimicking the inhibitory cosignaling function of HVEM. The demonstration that distinct herpesviruses target the HVEM-BTLA cosignaling pathway suggests the importance of this pathway in regulating T cell activation during host defenses.

Generation of a Yeast Two-Hybrid Strain Suitable for Competitive Protein Binding Analysis

The yeast two-hybrid (Y2H) system is one of the most common applied genetic systems utilized for the identi-fication of protein-protein interactions in vivo (1). Over the years this system has been systematically exploited for analyzing the physiological function of proteins via the generation of huge protein interaction networks (2). It is based on the principle that different transcription factors can be split into a DNA binding and a polymerase II activation domain. When these independently functional domains are brought into close proximity, the activity of the transcription factor is restored. If an interaction of a protein fused to the DNA binding domain (bait protein) and another protein fused to the activation domain (prey protein) occurs, a functionally transcription factor is reconstructed, and transcription of certain reporter genes involved in amino acid biosynthesis is initiated. The colorimetric reporter LacZ is often exploited as well, because it allows the relative quantification of its activity and, consequently, the relative strength of protein-protein interactions in vivo (3–5).Since proteins are multifunctional and interact with many factors, it would be obviously advantageous and in particular less time-consuming to characterize the interplay between proteins in vivo by the Y2H system in comparison to existing in vitro approaches (6,7). Therefore, the Y2H technology has been broadened and applied for further characterization of protein-protein interactions. Reverse approaches have been developed allowing the identification of particular protein mutations, peptides, or agents that inhibit the protein-protein inter-action of interest (8,9). Furthermore, Y2H systems have been generated in which the interaction between proteins is enhanced or depends upon expression of a third protein or ligand (5,10,11). However, the simultaneous expression of a third protein or factor to analyze competitive protein binding in the Y2H background can be difficult, because often the auxotrophic marker genes available have been exhausted in the course of generating the strains for a particular Y2H system. Moreover, many Y2H systems are incompatible to each other because of the different promoters and the auxotrophic markers that have to be used. Thus, switching between the available Y2H systems and strategies is often impossible or very time-consuming due to required cloning efforts for the creation of different bait and prey plasmids needed.Hence, to facilitate competitive protein binding approaches in a quite frequent Y2H system, we aimed to create a strain still exploitable for Y2H screens, but additionally allowing the simultaneous expression of a third protein (Figure 1A). Given that most yeast expression vectors are based on auxothropic markers, we decided to replace the URA3 gene in the standard strain L40c [MATa his3∆200 trp1–901 leu2–3,112 ade2 lys2–801am can1 URA3::(lexAop)

Cooperative Interaction of Human XPA Stabilizes and Enhances Specific Binding of XPA to DNA Damage

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected Sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K(1) = 1.4 x 10(6) M(-)(1) and K(2) = 1.8 x 10(7) M(-)(1). When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.

Neuromedin Elicits Cytokine Release in Murine Th2-Type T Cell Clone D10.G4.1

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for (125)I-labeled NmU (K(D) 364 pM and B(max) 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca(2+) concentration (EC(50) 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.

Analysis of competitive binding of ligands to human serum albumin using NMR relaxation measurements

The competitive binding of two ligands, ibuprofen (IBP) and salicylic acid (SAL), to human serum albumin (HSA) was studied by using nuclear magnetic resonance (NMR) relaxation measurements. When the concentration of one ligand was increased in the solution containing IBP, SAL and HSA, the fractions of free IBP and SAL were increased because of the competitive binding. The 1 H relaxation rates ( R 1) of both ligands were subsequently decreased. If a ligand is in fast exchanging between the free and bound forms, the observed 1 H relaxation rate is a weighted average of that for the free ligand and the protein–ligand complex. The concentrations of the free and bound ligands can be quantitatively derived from the relaxation rates. The results presented in this work revealed that IBP and SAL shared certain low-affinity binding sites on the HSA molecule, in addition to the same high-affinity binding site of AIII.