Radioactive Binding Assay Research Articles

Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Designed ankyrin repeat proteins for detecting prostate-specific antigen expression in vivo.

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with a hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 ± 0.58% ID g-1 (n = 3; 2 h post-administration) and was reduced by ∼50% by competitive binding with a low molar activity formulation (blocking group: 2.47 ± 0.42% ID g-1; n = 3; P value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

Development of a non-radioactive mass spectrometry-based binding assay at the μ-opioid receptor and its application for the determination of the binding affinities of 17 opiates/opioids as well as of the designer opioid isotonitazene and five further 2-benzylbenzimidazoles

Radioactive ligand binding assays are the most commonly applied method for the determination of binding affinities of compounds at a particular receptor. While they are highly sensitive and high-throughput capable they come with major disadvantages due to the radioactive ligands utilized. Here we present the development of a mass-spectrometry-based binding assay for the determination of binding affinities at the human μ-opioid receptor using non-labelled DAMGO ([D-Ala2, N-MePhe4, Gly5-ol]-enkephalin). The runtime of the LC-MS/MS method was 5.5 min per data point and allowed for the highly sensitive detection of 38.5 fg DAMGO on column. The assay shows low non-specific binding and the equilibrium dissociation constant of DAMGO was 0.57 nM. The assay was applied to determine the Ki values of 17 opiates/opioids and the results were in good agreement with the data from radioactive receptor binding assays published in the literature. Additionally, the Ki value of six 2-benzylbenzimidazoles, including the widely abused designer opioid isotonitazene, were determined ranging from 0.654 to 72.9 nM. Consequently, the developed assay provides a suitable alternative to radioactive binding assays as it allows for a reliable and rapid determination of receptor binding affinities of e.g. newly emerging designer opioids.

Expression and Purification of Corticosterone Binding Globulin in E. colifor Quantification

Corticosterone (CORT) is analogous to the mammalian stress hormone cortisol and is involved in the acute stress response in birds. Birds can be used as a model of the stress response due to the evolutionarily conserved activation of the hypothalamic‐pituitary‐adrenal (HPA) axis and sympathetic adrenomedullary (SA) system across vertebrate species. Corticosterone binding globulin (CBG) is a protein that binds and carries approximately 80% of corticosterone in the plasma in both avian and mammalian species. There are limitations in the most common technique for quantification of CBG due to the use of radioactive binding assay. The ultimate goal of this project is to develop a robust and safe method to quantify CBG levels. This abstract reports the first part of that goal – the expression and purification of variously‐tagged zebra finch CBG from E. coli. CBG was expressed either as histidine‐tagged or in fusion with glutathione‐S‐transferase (GST) or maltose‐binding protein (MBP). Purified CBG was confirmed and assessed by SDS‐PAGE and western blot using antiserum to zebra finch CBG. Following purification, circular dichroism will be used to analyze the secondary structure of the recombinant CBG protein. The project aims to develop an ELISA‐based method to measure the amount of CBG in samples.

A PF‐3845‐induced novel lipid fraction in mouse medullary interstitial cells causes diuresis and natriuresis via a CB1‐mediated pathway

The relationship between the endocannabinoid system in the renal medulla and the long‐term regulation of blood pressure is not understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF‐3845. Treatment of MMICs with PF‐3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm‐absorbing peak that showed responsiveness to PF‐3845 treatment. The biologic activities of the PF‐3845‐induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically‐instrumented mice. Intravenous and intramedullary infusions of PIP at low doses resulted in a vasodepressor, diuretic and natriuretic effects. Pharmacological and genetic ablation of cannabinoid 1 receptors resulted in the loss of the vasodepressor, diuretic and natriuretic effect of PIP. Radioactive binding assays showed that PIP competes for binding to CB1 receptors in the presence of a full agonist (CP 55,940). PIP did not elevate urine and sodium excretion in a genetic knockout mouse model of FAAH. A PIP‐induced decrease in blood pressure at high doses of PIP was similar between FAAH WT and FAAH KO mice. These data support a model whereby PF‐3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis via a CB1‐mediated mechanism. The diuretic and natriuretic responses to PIP are eliminated by inhibition of FAAH, suggesting an undergoing sensitization to PIP in FAAH KO mice. Efforts to identify the structure of the PF‐3845‐induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.Support or Funding InformationThis work was supported by the National Institute of Diabetes and Digestive and Kidney Disorders grant [grant R01DK102539] and the National Institute on Drug Abuse [grant P30DA033934]. S.D. was supported by the National Institute on Drug Abuse [training grant T32DA007027].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cytokine-like 1 Chemoattracts Monocytes/Macrophages via CCR2.

Cytokine-like 1 (CYTL1) is a novel potential cytokine that was first identified in CD34(+) cells derived from bone marrow and cord blood, and it was also found using our immunogenomics strategy. The immunobiological functions of CYTL1 remain largely unknown, and its potential receptor(s) has not been identified. A previous proposed hypothesis suggested that CYTL1 had structural similarities with CCL2 and that CCR2 was a potential receptor of CYTL1. In this study, we verify that CYTL1 possesses chemotactic activity and demonstrate that its functional receptor is CCR2B using a series of experiments performed in HEK293 cells expressing CCR2B or CCR2B-EGFP, including chemotaxis, receptor internalization, and radioactive binding assays. CYTL1 chemoattracts human monocytes but not PBLs, and its chemotactic activity toward monocytes is dependent on the CCR2B-ERK pathway. Furthermore, both human and mouse recombinant CYTL1 protein have chemotactic effects on macrophages from wild-type mice but not from Ccr2(-/-) mice. Furthermore, the chemotactic activity of CYTL1 is sensitive to pertussis toxin. All of the above data confirm that CCR2B is a functional receptor of CYTL1.

How to measure the affinity of aptamers for membrane proteins expressed on the surface of living adherent cells

Recently, an increasing number of aptamers have been selected against biomarkers that are expressed at the surface of cells. This class of targets, mostly membrane proteins, is in close contact with the intra- and extra-cellular matrixes and their three-dimensional structures are inextricably linked to their inclusion in lipid bilayers. Therefore, although binding studies can be performed on the isolated form of these proteins, it remains crucial to measure the affinity of these aptamers in a more physiological environment, i.e., directly on living cells. Here, we describe a procedure for radioactive binding assays that can be adapted for measuring the affinity of aptamers against different cell lines. This method has been semi-automated using a liquid handling robot in order to reproducibly measure the apparent dissociation constant Kd and the apparent number of targets per cell. Relevant issues are discussed including the labeling of aptamers, the cells preparation, the incubation, the washings, the use of non-specific competitors, the data analysis and finally the reporting.

The immunomodulatory effects of barettin and involvement of the kinases CAMK1α and RIPK2

Background: Barettin is a marine natural compound with reported anti-inflammatory and antioxidant properties. The combination of these effects led us to explore barettin further as an inhibitor of atherosclerosis development.Methods: The effect of barettin on MCP-1 and IL-10 secretion from activated immune cells was detected by ELISA. Determination of cell viability of oxidized low-density lipoprotein (oxLDL) and barettin exposed HUVEC cells were investigated by using CellTiter 96® AQueous One Solution. The kinase inhibition assays were performed using a radioactive (33P-ATP) filter binding assay at the University of Dundee, UK.Results: Barettin reduces the secretion of monocyte chemotactic protein-1 (MCP-1) from LPS-stimulated monocytes, but was not able to prevent oxLDL-induced cell death in HUVEC. Barettin has inhibitory activity against two protein kinases related to inflammation, namely the receptor-interacting serine/threonine kinase 2 (RIPK2) and calcium/calmodulin-dependent protein kinase 1α (CAMK1α). We also demonstrate that barettin reduce the production of the anti-inflammatory cytokine interleukin-10 (IL-10) in a dose and time-dependent manner, possibly by inhibiting CAMK1α.Conclusions: The anti-inflammatory activity of barettin is exerted through the regulation of inflammatory mediators such as MCP-1 and IL-10, possibly via inhibition of kinases.

Portraying G protein-coupled receptors with fluorescent ligands.

The thermodynamics of ligand–receptor interactions at the surface of living cells represents a fundamental aspect of G protein-coupled receptor (GPCR) biology; thus, its detailed elucidation constitutes a challenge for modern pharmacology. Interestingly, fluorescent ligands have been developed for a variety of GPCRs in order to monitor ligand–receptor binding in living cells. Accordingly, new methodological strategies derived from noninvasive fluorescence-based approaches, especially fluorescence resonance energy transfer (FRET), have been successfully developed to characterize ligand–receptor interactions. Importantly, these technologies are supplanting more hazardous and expensive radioactive binding assays. In addition, FRET-based tools have also become extremely powerful approaches for visualizing receptor–receptor interactions (i.e., GPCR oligomerization) in living cells. Thus, by means of the synthesis of compatible fluorescent ligands these novel techniques can be implemented to demonstrate the existence of GPCR oligomerization not only in heterologous systems but also in native tissues. Finally, there is no doubt that these methodologies would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Overall, herein, we provide a thorough assessment of all technical and biological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied to the study of GPCR biology at the plasma membrane of living cells.

From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEX of an Anti-Annexin A2 Aptamer Targeting Tumors

BackgroundCell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged.Methodology/Principal FindingsCell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence.Conclusions/SignificanceAlthough aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application.

Secretoneurin Induces Airway Mucus Hypersecretion by Enhancing the Binding of EGF to NRP1

Aims: Secretoneurin(SN), a neuropeptide, has been considered a reliable marker of allergenic stimulation. However, the relationship between SN and the secretion of airway mucin remains unclear. In this study, we aimed to examine the in vitro relationship between SN and airway mucin over synthesis, as well as the signaling pathways involved. Methods and Results: Exogenous SN was added to two human airway epithelial cell lines (16HBE and NCI-H292). Measured by real-time quantitative polymerase chain reaction (qPCR) and enzyme-linked immuno sorbent assay (ELISA) respectively, the intracellular mucin(MUC)5AC mRNA and MUC5AC protein of culture supernates exhibited a time- and dose-dependent increase after stimulation of SN. Based on the evidence of an increased phosphorylation of ERK1/2 induced by exogenous SN, we performed the radioactive binding assay. We failed to find direct binding of SN to either epidermal growth factor receptor (EGFR) or Neuropilin-1(NRP1), the co-receptor of EGFR. But we detected an enhanced binding of EGF to NRP1 in the two airway epithelial cell lines induced by exogenous SN. Either EGF neutralizing antibody or MEK specific inhibitor (PD-98059) could attenuate the over synthesis of MUC5AC induced by exogenous SN, indicating an endogenous EGF dependent mechanism in MUC5AC over synthesis induced by SN. Conclusions: We conclude that SN induces MUC5AC hypersecretion in a dose- and time-dependent manner; moreover, the MUC5AC over synthesis induced by SN is strongly associated with the enhanced binding of EGF to NRP1 and the activation of EGFR and ERK1/2 subsequently.

Functional evidence for physiological mechanisms to circumvent neurotoxicity of cardenolides in an adapted and a non-adapted hawk-moth species

Because cardenolides specifically inhibit the Na(+)K(+)-ATPase, insects feeding on cardenolide-containing plants need to circumvent this toxic effect. Some insects such as the monarch butterfly rely on target site insensitivity, yet other cardenolide-adapted lepidopterans such as the oleander hawk-moth, Daphnis nerii, possess highly sensitive Na(+)K(+)-ATPases. Nevertheless, larvae of this species and the related Manduca sexta are insensitive to injected cardenolides. By radioactive-binding assays with nerve cords of both species, we demonstrate that the perineurium surrounding the nervous tissue functions as a diffusion barrier for a polar cardenolide (ouabain). By contrast, for non-polar cardenolides such as digoxin an active efflux carrier limits the access to the nerve cord. This barrier can be abolished by metabolic inhibitors and by verapamil, a specific inhibitor of P-glycoproteins (PGPs). This supports that a PGP-like transporter is involved in the active cardenolide-barrier of the perineurium. Tissue specific RT-PCR demonstrated expression of three PGP-like genes in hornworm nerve cords, and immunohistochemistry further corroborated PGP expression in the perineurium. Our results thus suggest that the lepidopteran perineurium serves as a diffusion barrier for polar cardenolides and provides an active barrier for non-polar cardenolides. This may explain the high in vivo resistance to cardenolides observed in some lepidopteran larvae, despite their highly sensitive Na(+)K(+)-ATPases.

FACS binding assay for analysing GDNF interactions

Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

Effects of Extracellular Calcium on the Modulation of Metabotropic Glutamate Receptor 1 Alpha (mGluR1α) by L-Quis, (S)-Mcpg, Cpccoet and Ro 67-4853

Metabotropic glutamate receptor 1α (mGluR1α), known as a member of the family C GPCRs, couples to Gq and modulates consequent PLC activity, IP3 accumulation and intracellular Ca2+ release from ER lumen. mGluR1α is abundantly expressed in the central nervous system and has been shown to be responsible for the slow phase of the action potential in post-synaptic neurons, and to be involved in chronic neuronal degenerative diseases, like Parkinson's disease, Huntington's disease and Alzheimer's disease. We have predicted a potential Ca2+ binding site adjacent to the binding site previously reported for the endogenous agonist, glutamate, and receptor antagonists. In this study, we have applied single cell imaging and a radioactive binding assay to probe the effects of extracellular calcium in modulating various drugs modulating mGluR1α such as agonists, antagonists and allosteric modulators. We have shown that extracellular Ca2+ enhances the activation of the the receptor by its agonists and positive allosteric modulators by interacting with the Ca2+ binding site. In addition, extracellular Ca2+ differentially reduces the inhibition of the receptor by antagonists and negative allosteric modulators. Our studies open a new avenue for modulating drug effects and developing novel drugs against neurodegenerative diseases.

Glucocorticoid treatment increases density of serotonin 5-HT2A receptors in humans

Interactions between the serotonergic system and the hypothalamic-pituitary-adrenal axis have been suggested, albeit the details for such interactions have yet to be established. Animal studies have shown that the density of serotonin 5-HT2A receptors is increased after administration of exogenous glucocorticoids. The objective of this study was to explore possible changes in the pattern of density and affinity of 5-HT2A receptors in humans after treatment with glucocorticoids. Using a radioactive binding assay, the density and affinity (measured as Bmax and Kd) of 5-HT2A serotonin receptors were measured in blood samples drawn from 27 individuals diagnosed with polymyalgia rheumatica and/or giant cell arteritis before and after start of an oral treatment with prednisolone. For each patient Bmax and Kd at baseline before prednisolone treatment were compared with Bmax and Kd in samples drawn at a first and second follow-up clinic visit at an average of 8.8 (±2.5) days and 33.6 (±6.8) days, respectively. The density of 5-HT2A receptors increased after treatment in 23 individuals. The mean Bmax value at baseline for all patients was 45.2 fmol/mg protein compared with 64.9 fmol/mg protein in the corresponding samples drawn at the second follow-up visit (p=0.001). There also was an association between individuals accumulated prednisolone dose and the magnitude of change in Bmax between baseline and the first follow-up visit. Erythrocyte sedimentation rate, platelet count or gender had no influence on the results. There were no significant differences in Kd during the treatment period. However, a low Kd value at baseline was a predictor for an increase in Bmax following treatment. The results of this study showed that the density of 5-HT2A serotonin receptors in man is increased after a subchronic treatment with glucocorticoids. The magnitude of the increase appears to be associated with the affinity of 5-HT2A receptors before treatment and the accumulated dose of glucocorticoid early in the treatment period.

Abstract 2817: An RNA aptamer is a potential biomarker for pancreatic cancer

Abstract Introduction: The symptoms of pancreatic cancer are usually non-specific and late. Existing tumor markers (such as CA19-9 and CEA) are neither sensitive nor specific enough to serve as useful screening tests. Tools for earlier detection could increase the proportion of patients who are cured with surgical resection. Aptamers are oligonucleotide ligands that are identified from large libraries of RNA or DNA molecules using an iterative selection process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers bind their specific targets with high affinity and specificity and can be used for the dual purpose of identifying novel biomarkers and detecting them in biological specimens. Methods: In order to select for aptamers against molecules that are selectively secreted by cancerous cells but not by normal cells, “positive/negative” SELEX was performed using the conditioned media (the “secretome”) of the MiaPaCa2 human pancreatic ductal adenocarcinoma cell line (positive selection) and the immortalized, non-cancerous human pancreatic ductal cell line HPDE (negative selection). Standard radioactive filter-based binding assays were used to measure binding. Results/Conclusion: We have successfully selected several nuclease resistant (2′-fluoro-modified) RNA aptamers that differentially bind the secretome of pancreatic cancer cells compared to non-cancerous cells. One of the aptamers (M9-5) binds pancreatic cancer patient sera with high affinity compared to control sera collected from healthy donors. So far, the aptamer has demonstrated specific binding in 20 of 22 patients with histologically-proven pancreatic cancer and none of 20 healthy donors. In addition, paired serum samples obtained from two patients pre- and post-chemoradiation therapy and one patient pre- and two months post-resection have demonstrated a decrease in M9-5 binding with decreased tumor burden. Notably, the serum CA19-9 and CEA levels in several of these patients were normal, indicating that our biomarker might be more sensitive than these existing serum biomarkers for pancreatic cancer. M9-5 binding to the MiaPaCa2 “secretome” is sensitive to Proteinase K treatment indicating that the aptamer target is a protein. Currently, we are using a biotinylated version of the aptamer to affinity purify the target and identify it by mass-spectrometric techniques. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2817. doi:10.1158/1538-7445.AM2011-2817

Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a

The growth hormone secretagogue receptor type 1a (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FRET signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. K i values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening.

Kinetics of albumin homocysteinylation measured with matrix‐assisted laser/desorption ionization mass spectrometry versus with a radioactive tracer

Homocysteinylation is a post-translational protein modification which involves homocysteine-thiolactone and may be responsible for many pathophysiological changes secondary to hyperhomocysteinemia. Therefore, methods to measure protein homocysteinylation in intact biological samples are required. We tested whether matrix assisted-laser/desorption ionization mass spectrometry (MALDI-MS) can detect time- and dose-dependent changes in in vitro homocysteine-thiolactone binding to human serum albumin. We have compared this method with a 35S-thiolactone radioactive binding assay. Incubations with and without dithiothreitol allowed measurement of the amide-linked and disulfide-linked thiolactone-protein adducts, respectively. A good correspondence in time- and dose-dependent protein-thiolactone formation was observed between the two methods. A maximum of 9 to 12 thiolactone residues were bound to each albumin molecule. The 35S-thiolactone bound albumin tightly, particularly at the lowest concentrations, with approximately 70% of the binding amide-linked. Although the results of the two methods were rather similar, the radioactive method appears to be more sensitive than the MALDI-MS technique.

Transactivation within the AT1 angiotensin receptor homodimer: the role of the conserved DRY motif

Methods In this study we examined the possible functional consequence of transactivation within angiotensin AT1 receptor homodimers. To do this, we used the S109Y mutant of the AT1 receptor, in which the binding site of the nonpeptid AT1 receptor antagonist, candesartan, was destroyed. Expressing this mutant receptor together with the wildtype in CHO cells, in the presence of candesartan, we could examine the interaction between them by stimulating the S109Y mutant receptor, and following the activation of the wild-type receptor. To monitor the signaling of the receptor two parameters were measured: the conformational change of the receptor using an intramolecular sensor, and the binding of β-arrestin-2 to the activated receptors. In both cases the highly sensitive method of bioluminescence resonance energy transfer (BRET) was applied. Additionally, we performed a radioactive ligand binding assay to examine the cooperativity between receptor monomers.

Crucial Role of the CB3-Region of Collagen IV in PARF-Induced Acute Rheumatic Fever

Acute rheumatic fever (ARF) and rheumatic heart disease are serious autoimmune sequelae to infections with Streptococcus pyogenes. Streptococcal M-proteins have been implicated in ARF pathogenesis. Their interaction with collagen type IV (CIV) is a triggering step that induces generation of collagen-specific auto-antibodies. Electron microscopy of the protein complex between M-protein type 3 (M3-protein) and CIV identified two prominent binding sites of which one is situated in the CB3-region of CIV. In a radioactive binding assay, M3-protein expressing S. pyogenes and S. gordonii bound the CB3-fragment. Detailed analysis of the interactions by surface plasmon resonance measurements and site directed mutagenesis revealed high affinity interactions with dissociation constants in the nanomolar range that depend on the recently described collagen binding motif of streptococcal M-proteins. Because of its role in the induction of disease-related collagen autoimmunity the motif is referred to as “peptide associated with rheumatic fever” (PARF). Both, sera of mice immunized with M3-protein as well as sera from patients with ARF contained anti-CB3 auto-antibodies, indicating their contribution to ARF pathogenesis. The identification of the CB3-region as a binding partner for PARF directs the further approaches to understand the unusual autoimmune pathogenesis of PARF-dependent ARF and forms a molecular basis for a diagnostic test that detects rheumatogenic streptococci.