Conventional Radioligand Binding Assay Research Articles

Simultaneous Multiple MS Binding Assays for the Dopamine, Norepinephrine, and Serotonin Transporters.

In this work, we present label-free, mass-spectrometry-based binding assays (MS Binding Assays), targeting the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) in simultaneous binding experiments. Using a validated LC-ESI-MS/MS method for quantification of the selective dopamine transporter inhibitor (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol ((R,R)-D-84), the selective norepinephrine transporter inhibitor (S,S)-reboxetine, and the selective serotonin reuptake inhibitor (S)-citalopram, binding affinities at the three monoamine transporters could be characterized simultaneously in a single binding experiment. The performed simultaneous saturation and competition experiments yielded results that are in good accordance with those determined in MS Binding Assays addressing the monoamine transporters individually. The results obtained from this study underscore the potential of MS Binding Assays for simultaneous affinity determination at different targets, which is difficult to accomplish with conventional radioligand binding assays.

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D1 and D2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D1 and D2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D1 and D2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays.

Budded baculovirus particles as a source of membrane proteins for radioligand binding assay: The case of dopamine D1 receptor

IntroductionG-protein-coupled receptors have become very important drug targets and therefore ligand binding assays for these receptors are an essential part of drug discovery. Among a variety of experimental systems, the radioligand binding assay has remained as one of the main methods in this field for decades. Usually cell membranes or tissues are used in these experiments, however in this article we demonstrate that baculoviruses produced in Sf9 cells display recombinant receptors on their surface and therefore can be used in radioligand binding assay. MethodsWe have used baculoviruses with dopamine D1 receptors as a model system to validate the usage of this receptor preparation in radioligand binding experiments. In order to collect membrane receptors and separate free radioligand, we have applied FilterMate Harvester with Filtermat B. ResultsUsing baculoviruses with dopamine D1 receptors as a model system, we have shown that this is a suitable preparation for conventional radioligand binding assay. Here, all the experiments were performed using the dopamine D1-like receptor specific radioligand [3H]SCH23390. There were no significant differences between binding parameters determined in membranes of Sf9 cells and baculovirus particles. Similar pIC50 and Ki values were also determined in competition binding assays with HEK293 cell membranes. DiscussionAll the results obtained with baculovirus preparation were in good agreement with the data obtained in parallel experiments with membrane preparations from Sf9 and HEK293 cells expressing dopamine D1 receptors. Shape uniformity, homogeneous distribution and slow sedimentation of the membranes are some of the advantages of baculovirus preparations, which prove them as promising source of membrane proteins for routine and high throughput analysis.

Development and validation of an LC-ESI-MS/MS method for the triple reuptake inhibitor indatraline enabling its quantification in MS Binding Assays.

We herein present the first LC-MS/MS quantification method for indatraline, a highly potent nonselective inhibitor of the three monoamine transporters (for dopamine, DAT; norepinephrine, NET; serotonin, SERT), and its application to MS Binding Assays. For HPLC, an R18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L(-1), pH 10.0) in a ratio of 90:10 (v/v) at a flow rate of 600 μL min(-1) was used. Recording indatraline at m/z 292.2/261.0 and ((2)H(7))-indatraline, employed as internal standard, at m/z 299.2/268.0 allowed reliable quantification from 5 pmol L(-1) (LLOQ) to 5 nmol L(-1) in biological matrices without additional sample preparation. Validation of the developed quantification method showed that selectivity, calibration standard curve, accuracy, as well as precision meet the criteria of the CDER guideline. Applying this method to mass spectrometry (MS) Binding Assays, a label-free MS-based alternative to conventional radioligand binding assays, binding of indatraline's eutomer, (1R,3S)-indatraline, towards NET could be characterized directly for the first time, revealing an equilibrium dissociation constant (K d) of 805 pmol L(-1). Additionally, it could be shown that the established MS Binding Assays enable characterization of test compounds in competition experiments. As the established setup is based on a 96-well format and an LC MS/MS method with a short chromatographic cycle time (1.5 min), the developed MS Binding Assays enable considerable throughput and are therefore well suited as substitute for corresponding radioligand binding assays.

Ubiquitination in the First Cytoplasmic Loop of μ-Opioid Receptors Reveals a Hierarchical Mechanism of Lysosomal Down-regulation

μ-Type opioid receptors (MORs) are members of the large seven-transmembrane receptor family which transduce the effects of both endogenous neuropeptides and clinically important opioid drugs. Prolonged activation of MORs promotes their proteolytic degradation by endocytic trafficking to lysosomes. This down-regulation process is known to contribute to homeostatic regulation of cellular opioid responsiveness, but mechanisms that mediate and control MOR down-regulation have not been defined. We show here that lysosomal down-regulation of MORs is ESCRT (endosomal sorting complex required for transport)-dependent and involves ubiquitin-promoted transfer of internalized MORs from the limiting endosome membrane to lumen. We also show that MOR down-regulation measured by conventional radioligand binding assay is determined specifically by ubiquitination in the first cytoplasmic loop. Surprisingly, we were unable to find any role of ubiquitination in determining whether internalized receptors recycle or are delivered to lysosomes. Instead, this decision is dictated specifically by the MOR C-tail and occurs irrespectively of the presence or absence of receptor ubiquitination. Our results support a hierarchical organization of discrete ubiquitin-independent and -dependent sorting operations, which function non-redundantly in the conserved down-regulation pathway to mediate precise endocytic control. Furthermore, they show that this hierarchical mechanism discriminates the endocytic regulation of naturally occurring MOR isoforms. Moreover, they are the first to reveal, we believe, for any seven-transmembrane receptor, a functional role of ubiquitination in the first cytoplasmic loop.

α1‐Adrenoceptors and muscarinic receptors in voiding function – binding characteristics of therapeutic agents in relation to the pharmacokinetics

In vivo and ex vivo binding of α(1)-adrenoceptor and muscarinic receptors involved in voiding function is reviewed with therapeutic agents (α(1)-adrenoceptor antagonists: prazosin, tamsulosin and silodosin; and muscarinic receptor antagonists: oxybutynin, tolterodine, solifenacin, propiverine, imiafenacin and darifenacin) in lower urinary tract symptoms. This approach allows estimation of the inhibition of a well-characterized selective (standard) radioligand by unlabelled potential drugs or direct measurement of the distribution and receptor binding of a standard radioligand or radiolabelled form of a novel drug. In fact, these studies could be conducted in various tissues from animals pretreated with radioligands and/or unlabelled novel drugs, by conventional radioligand binding assay, radioactivity measurement, autoradiography and positron emission tomography. In vivo and ex vivo receptor binding with α(1)-adrenoceptor antagonists and muscarinic receptor antagonists have been proved to be useful in predicting the potency, organ selectivity and duration of action of drugs in relation to their pharmacokinetics. Such evaluations of drug-receptor binding reveal that adverse effects could be avoided by the use of new α(1)-adrenoceptor antagonists and muscarinic receptor antagonists for the treatment of lower urinary tract symptoms. Thus, the comparative analysis of α(1)-adrenoceptor and muscarinic receptor binding characteristics in the lower urinary tract and other tissues after systemic administration of therapeutic agents allows the rationale for their pharmacological characteristics from the integrated viewpoint of pharmacokinetics and pharmacodynamics. The current review emphasizes the usefulness of in vivo and ex vivo receptor binding in the discovery and development of novel drugs for the treatment of not only urinary dysfunction but also other disorders.

Detection and immunochemical characterization of glutamic acid decarboxylase autoantibodies in patients with autoimmune diabetes mellitus

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)).In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65.Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients.It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, β-cell antigens.

Recent Advances in Understanding Fundamental Mechanisms of Volatile General Anesthetic Action

Over 20 million patients in the United States alone each year receive a general anesthetic for a surgical procedure. Nevertheless, molecular mechanisms of volatile general anesthetic action remain poorly understood. The favored sites of action in the central nervous system are currently a variety of plasma membrane proteins including the Cys-loop superfamily of ligand-gated ion channels and the N-methyl-D-aspartate receptor, because volatile general anesthetics are able to alter the ion conducting properties of these proteins. Volatile general anesthetics are only capable of relatively weak interactions with macromolecular targets, precluding the use of conventional radioligand binding assays for identifying central nervous system targets. In order to overcome this significant technical obstacle, other approaches to monitor volatile general anesthetic binding have been developed that rely on 19F-nuclear magnetic resonance spectroscopy, photoaffinity labeling with halothane, fluorescence spectroscopy, and isothermal titration calorimetry. These techniques have allowed the determination of volatile general anesthetic dissociation constants for a number of different protein complexes. The effect of a bound volatile general anesthetic on protein stability, flexibility, and overall structure has been investigated in recent years, and the results suggest fundamental mechanisms whereby these important clinical compounds reversibly alter protein function. Keywords: Volatile general anesthetic, ligand-gated ion channel, binding affinity, protein stability, protein flexibility, general anesthetic action

MS‐Binding Assays: Kinetic, Saturation, and Competitive Experiments Based on Quantitation of Bound Marker as Exemplified by the GABA Transporter mGAT1

A new kind of binding assay is described in which the amount of a nonlabeled marker bound to the target is quantified by LC-ESI-MS-MS. This new approach was successfully implemented with nonlabeled NO 711 as marker and the GABA transporter subtype mGAT1 as target. The native marker bound to the target was liberated from the receptor protein by methanol denaturation after filtration. A reliable and sensitive LC-ESI-MS-MS method for the quantitation of NO 711 was developed, and data from mass spectrometric detection were analyzed by nonlinear regression. Kinetic MS-binding experiments yielded values for k+1 and k-1, while in saturation MS-binding experiments, Kd and Bmax values were determined. In competitive MS-binding experiments, Ki values were obtained for various test compounds covering a broad range of affinities for mGAT1. All experiments were performed in 96-well plate format with a filter plate for the separation step which improved the efficiency and throughput of the procedure. The method was validated by classical radioligand-binding experiments with the labeled marker [3H2]NO 711 in parallel. The results obtained from MS-binding experiments were found to be in good agreement with the results of the radioligand-binding assays. The new kind of MS-binding assay presented herein is further adapted to the conventional radioligand-binding assay in that the amount of bound marker is securely quantified. This promises easy implementation in accordance with conventional binding assays without the major drawbacks that are inherent in radioligand or fluorescence binding assays. Therefore, MS-binding assays are a true alternative to classical radioligand-binding assays.

Competitive MS Binding Assays for Dopamine D2Receptors Employing Spiperone as a Native Marker

A competitive MS binding assay employing spiperone as a native marker and a porcine striatal membrane fraction as a source for dopamine D2 receptors in a nonvolatile buffer has been established. Binding of the test compounds to the target was monitored by mass-spectrometric quantification of the nonbound marker, spiperone, in the supernatant of the binding samples obtained by centrifugation. A solid-phase extraction procedure was used for separating spiperone from ESI-MS-incompatible supernatant matrix components. Subsequently, the marker was reliably quantified by LC-ESI-MS-MS by using haloperidol as an internal standard. The affinities of the test compounds, the dopamine receptor antagonists (+)-butaclamol, chlorpromazine and (S)-sulpiride obtained from the competitive MS binding assay were verified by corresponding radioligand binding experiments with [3H]spiperone. The results of this study demonstrate that competitive MS binding assays represent a universally applicable alternative to conventional radioligand binding assays.

Characterization of Opioid and ORL1 Receptors

AbstractThis unit describes methods for studying activity at opioid receptor subtypes and the ORL1 receptor using conventional radioligand binding assay methods as well as procedures for quantitating functional activity of these receptors by measuring agonist‐induced G protein activation with [35S]GTPγS binding in a manner analogous to traditional receptor‐binding methods. These procedures permit the assessment of the agonist or antagonist character of compounds in a rapid manner that, like most radioligand binding methodologies, is also amenable to high throughput technologies.

Autoantibodies to IA‐2 in Type 1 Diabetes

The tyrosine phosphatase-like protein IA-2 is an important islet autoantigen in type 1 diabetes. Although the radioligand binding assay with in vitro synthesized (35)S-labeled antigen has been used extensively for measuring autoantibodies to IA-2, disadvantages of both assays with respect to synthesis and handling of the radioactive antigen limit their use in routine laboratories. Therefore, we attempted to develop a nonradioactive ELISA for the simple detection of IA-2 autoantibodies. The biotinylated cytoplasmic domain of IA-2 expressed in Escherichia coli was used as an antigen. We evaluated two kinds of ELISA: ELISA with biotin-IA-2 directly captured on streptavidine-coated plates (solid phase) and ELISA with antigen-antibody preincubation in solution in which serum samples were reacted first with biotin-IA-2 and the mixture was transferred to streptavidine-coated plates (liquid phase). We compared their disease sensitivity and specificity with a conventional radioligand binding assay in 52 patients with recent-onset type 1 diabetes and 138 normal individuals. The radioligand binding assay had 61.5% sensitivity and 99.3% specificity. The liquid-phase ELISA showed relatively higher sensitivity (55.8%) and specificity (99.3%) than the solid-phase ELISA (sensitivity 53.8% and specificity 97.1%). Furthermore, the mean SD score in IA-2 autoantibody-positive serum samples measured by liquid-phase ELISA was significantly higher than the SD score obtained by solid-phase ELISA (P < 0.0001). We concluded that this liquid-phase ELISA is suitable for detecting IA-2 autoantibodies in patients with type 1 diabetes with a similar sensitivity and specificity to those of conventional radioligand binding assay.

A new enzyme-linked immunosorbant assay for the measurement of human vitamin D receptor

The hormonal actions of 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] are mediated by its cognate receptor protein, the vitamin D receptor (VDR). Despite the growing importance of the VDR system as a modulator of cell growth and differentiation, convenient assays for quantitative measurement of VDR are not readily available, and [ 3H]1,25(OH) 2D 3 ligand binding assays remain the standard method. In this paper, we present data to validate and characterize the usefulness of a new VDR enzyme-linked immunosorbant assay (ELISA) kit developed for the measurement of VDR in biological samples. In this assay, samples are added to microtitration wells coated with anti-VDR antibody and incubated with a second anti-VDR antibody that is biotinylated. The antibody receptor complex is then detected with streptavidin-labeled horseradish peroxidase followed by incubation with a chromogenic substrate, tetramethylbenzidine. The assay was found to be sensitive and accurate for measurements of VDR and compared favorably with the conventional radioligand binding assay (RBA). The interassay variation ranged from 5% to 25% and the intraassay variation was less than 5%. The ELISA presents several advantages over existing methodology, including the use of nonradioactive detection systems, lower protein and sample volume requirements, as well as convenience and speed. The assay can be completed in as short a time as 3 h, avoiding overnight incubations. Data are also presented to demonstrate the ability of the ELISA to detect both occupied and unoccupied VDR, making it a valuable research tool in settings where 1,25(OH) 2D 3 is present. However, the ELISA, as currently formulated, is only useful for the detection of human VDR.

Labeling of dopamine D 3 and D 4 receptor subtypes in human peripheral blood lymphocytes with [ [formula omitted]]7-OH-DPAT: a combined radioligand binding assay and immunochemical study

Molecular biology studies have demonstrated that human peripheral blood lymphocytes express dopamine D 2-like receptors belonging to the D 3 and D 4 receptor subtypes, whereas the characterization of these receptors using radioligand binding assay techniques provided conflicting results. The preferential dopamine D 3 receptor agonist [ 3 H ]7-hydroxy- N, N-di- n-propyl-2-aminotetralin ([ 3 H ]7-OH-DPAT) was used recently for labeling lymphocyte dopamine D 3 receptor. However, the selectivity of this compound for the D 3 receptor was questioned. In this study we have investigated human peripheral blood lymphocyte dopamine receptor subtypes labeled by [ 3 H ]7-OH-DPAT using a conventional radioligand binding assay technique and antibodies against dopamine D 2-like receptor subtypes. [ 3 H ]7-OH-DPAT was specifically bound to intact human peripheral blood lymphocytes with a dissociation constant ( K d) value of 0.32+0.03 nM and a maximum density of binding sites ( B max) of 18.2+0.8 fmol/2×10 6 cells. [ 3 H ]7-OH-DPAT binding was unaffected by antibodies against dopamine D 2 and D 2S receptors. Anti-dopamine D 3 and D 4 receptor antibodies reduced [ 3 H ]7-OH-DPAT binding by about 53% and 32% respectively. Combination of anti D 3 and D 4 receptor antibodies reduced remarkably [ 3 H ]7-OH-DPAT binding. The above results suggest that the dopamine receptor agonist [ 3 H ]7-OH-DPAT labels dopamine D 3 and D 4 receptor subtypes in human peripheral blood lymphocytes. The use of antibodies raised against dopamine receptor subtypes in combination with radioligand binding assay may contribute to define receptor subtypes expressed by human peripheral blood lymphocytes in health and disease.

Usefulness of flow cytometric detection of cell surface interleukin-6 receptors in human myeloma cell lines.

A flow cytometric procedure was used to analyse the cell surface interleukin-6 receptor (IL-6R) based on the principle of detecting the binding of IL-6 to IL-6R. The bound IL-6 was visualized by reacting with anti-IL-6 antibody, a second biotinylated antibody to immunoglobulins, fluorescein-conjugated avidin and biotinylated, fluorescein-conjugated bovine serum albumin. Studies with a number of human myeloma cell lines showed that the fluorescence signal from cellular binding of IL-6, and thus IL-6R, could be specifically discerned from the background. The specific IL-6R signal could be semi-quantitatively expressed as the mean channel number of the fluorescence histogram or as relative IL-6R density in relation to a reference cell line, such as U266-BL cells. The relative IL-6R density of various myeloma cell lines thus determined was found to correlate with their sensitivity to the growth inhibitory effect of glucocorticoids in vitro. Quantitatively, calibration of the staining procedure with microbeads that have a defined binding capacity for anti-IL-6 antibody allowed calculation of cellular IL-6R density that yielded results close to that reported with conventional radio-ligand binding assay. Similarly, the cytometric method was applied to the studies of kinetics of IL-6/IL-6R interaction and the cellular IL-6R changes after ligand-binding to provide estimates of IL-6R binding affinity and IL-6R turnover rate. It is suggested that flow cytometric measurement of cellular IL-6R is useful in providing biologically relevant information on myeloma cells.

Adrenergic Receptor Autoradiography and in Situ Hybridization

The distribution of α- and β-adrenergic receptors in slices of anatomically complex tissues can be delineated autoradiographically with great precision and resolution. This brief review highlights methodological aspects of adrenergic receptor autoradiography and in situ hybridization at the light microscopic level of resolution. It focuses on technical differences between autoradiographic analysis in tissue sections and conventional radioligand binding assays in membranes prepared from tissue homogenates. it emphasizes strategies for characterizing quantitatively the distribution of specific receptor subtypes and classes using autoradiography and ways of detecting naturally occurring low-abundance adrenergic receptor mRNAs using in situ hybridization.

Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo.

The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P less than 0.01, t test), and this impairment of von Willebrand factor-dependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von Willebrand factor (vWF) levels. We hypothesized that heparin may inhibit vWF-dependent platelet hemostatic functions by directly binding vWF in solution and interfering with vWF-GpIb binding. Using the in vitro techniques of ristocetin-induced platelet agglutination, fluorescent flow cytometric measurement of vWF-platelet binding, and conventional radioligand binding assays we observed that heparin inhibited both vWF-dependent platelet function and vWF-platelet binding in a parallel and dose-dependent manner. Heparin also inhibited platelet agglutination induced by bovine vWF and inhibited the binding of human asialo-vWF to platelets in ristocetin-free systems. The inhibitory potency of heparin was not dependent upon its affinity for antithrombin III, but was molecular weight dependent: homogeneous preparations of lower molecular weight were less inhibitory. Heparin impairment of vWF function may explain why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.

Characterization of Canine Renal Receptors for the Parathyroid Hormone-like Protein Associated with Humoral Hypercalcemia of Malignancy

Parathyroid hormone-like proteins (PTHLP) display actions in the kidney which are similar to those of parathyroid hormone (PTH). We compared the binding properties of PTHLP and PTH in canine renal cortical membranes to determine if they interacted with the same or different receptors. Radioiodination to high specific activity (greater than 400 microCi/micrograms) of [Nle8,18,Tyr34]human PTH-(1-34)amide and [Tyr36]PTHLP-(1-36)amide was performed using the lactoperoxidase method. Complete enzymatic digestion of both radioligands demonstrated that the peptides were monoiodinated. Both radioligands retained full biological activity in the renal adenylate cyclase assay, and neither was significantly degraded during incubation with highly purified canine renal membranes under binding assays conditions. Specific binding reached equilibrium by 20 min at 20 degrees C. Competition binding studies using unlabeled [Nle8,18,Tyr34]human PTH-(1-34)amide, [Tyr36] PTHLP-(1-36)amide, and bovine PTH-(1-34) with either radioligand revealed similar binding affinities for all three peptides. Biologically inactive PTHLP fragments did not show significant displacement. In contrast to its similar binding affinity, [Tyr36]PTHLP-(1-36)amide was 6-15-fold less potent than bovine PTH-(1-34) in the renal adenylate cyclase assay, suggesting less efficient receptor-effector coupling. Photoaffinity cross-linking using either radioligand in canine renal membrane labeled indistinguishable 70,000-dalton proteins. In the presence of multiple protease inhibitors, binding to an 85-kDa component was observed. Labeling of both receptor forms was specifically abolished by an excess of either cold peptide and dose-response curves using affinity cross-linked membranes corroborated the apparent binding affinities determined by conventional radioligand binding assays. We conclude that PTHLP-(1-36) and amino-terminal PTH analogues bind to indistinguishable receptors in canine renal cortical membranes, but display differential coupling to post-receptor events.

Identification of serotonin-binding proteins using a photoaffinity labeling probe.

Two types of serotonin-binding proteins from rat hypothalamus and spinal cord have been isolated and purified in our laboratory.1 Antibodies to both types of purified serotonin-binding proteins were obtained, and their interactions with LSD were studied2. Unfortunately, the low yield of purified serotonin-binding proteins prevented thorough biochemical characterizations. We are now reporting a new approach to the study of serotonin-binding proteins. An arylazide derivative of ssr tonin has been synthesized as a photoaffinity labeling probe.3,4 This derivative, [3H]-nitroarylazidophenyl-serotonin ([3 H]-NAP-serotonin), binds to protein components of brain homogenates and upon irradiation becomes covalently attached to protein through an active nitrene group. The radioactively labeled proteins can then be separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis5. This technique provides more specific information than do conventional radioligand binding assays with membrane preparations because the covalent binding of the photoaffinity labeling probe allows individual serotonin-binding proteins to be identified.KeywordsCovalent BindingRadioligand Binding AssayLysergic AcidSerotonin BindingPostsynaptic SerotoninThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.