Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.     

Comparison of the interaction of mono- and oligovalent ligands with cholera toxin. Demonstration of aggregate formation at low ligand concentrations.

The stimulation by cholera toxin of adenylate cyclase in Chinese hamster ovarian cells could be inhibited by various ligands. The latter have been shown to contain the structural oligosaccharide entities required for binding to cholera toxin, established as Galbeta1 leads to 3GalNAcbeta1 leads to 4Gal3 comes from 2alphaNeuAc. The different inhibitory potency of the ligands thereby correlates with the size of the aggregates formed with the toxin, which in turn depends on the valency of the ligands. The conclusion is drawn from a comparison of the interaction of cholera toxin and its B-protomer with ganglioside II3NeuAc-GgOse4-Cer, the newly synthesized bis-(monosialo-gangliotetraityl)amine and monosialogangliotetraose. In a double diffusion test cholera toxin B-protomer precipitated with the ganglioside II3 NeuAcGgOSE4-Cer and the divalent ligand bis(monosialo-gangliotetraityl)amine, suggesting the formation of high molecular weight aggregates, whereas no precipitation was observed with the monovalent monosialo-gangliotetraose. By ultracentrifugation analysis, aggregate formation of the cholera toxin B-protomer could be demonstrated with the ganglioside II3 NeuAc-GgOse4-Cer and bis(monosialo-gangliotetraityl)amine at a concentration at which the ganglioside was assumed to be monodisperse. Ganglioside/cholera toxin B-protomer complexes sediment faster than those of the toxin and bis(monosialo-gangliotetraityl)amine, suggesting higher aggregation of cholera toxin B-protomer with the former. On the other hand, no sedimentation with monosialo-gangliotetraose was observed. By equilibrium displacement dialysis, however, a comparable high affinity of binding to cholera toxin B-protomer of both the mono- and divalent oligosaccharides was demonstrated. Furthermore, values for the maximal concentration of the bound ligand from these binding experiments with cholera toxin B-protomer established molar ratios of ligand to protein of 4 to 1 and 2 to 1 for monosialo-gangliotetraose and bis(monosialo-gangliotetraityl)amine, respectively. From the results it is concluded that the lipophilic moiety of the ganglioside is not directly involved in the binding process to the toxin protein but leads to an oligovalency of this ligand, due to formation of micellar or submicellar structures.     

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.     

Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces.

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside GM1 ligand.     

Classical ligands interact with native and recombinant tubulin from Onchocerca volvulus with similar rank order of magnitude

The alpha- and beta-tubulin genes from Onchocerca volvulus were individually expressed for the first time in Escherichia coli (DH5alpha). The recombinant tubulins were purified, renatured and reconstituted into oligomers, probably dimers, which were competent to bind three classical tubulin ligands: mebendazole (MBZ), taxol (TAX) and vinblastine (VBN). A new charcoal-dependent binding assay allowed accurate discrimination between specific and non-specific ligand binding in crude cell extracts. To compare the magnitude of binding of both native and recombinant forms of tubulin, we developed an ELISA assay for estimating the amount of tubulin in soluble protein extracts of O. volvulus. Binding assays were performed; both the maximum binding at saturating ligand concentrations (B(max)) and the equilibrium dissociation constants (K(d)) were determined. The B(max) values of the different ligands were significantly different from one another (P<0.05), but the order of the B(max) and K(d) for each drug were VBN > TAX > MBZ for both native and recombinant tubulin. Indeed, B(max) values for MBZ with native and recombinant tubulins were similar. On average, native tubulin had higher or similar binding capacity (B(max)) but a consistently higher affinity (lower K(d)) than the recombinant tubulin. We conclude that at least some of the recombinant molecules form receptors that are similar to those in native tubulin dimers. These data suggest that recombinant tubulin can be used to develop a molecular screen for novel anti-tubulin ligands to develop into drugs against onchocerciasis.     

Demonstration of the pH sensitive binding of multivalent carbohydrate ligands to immobilized Shiga-like toxin 1 B subunits.

The cytotoxic effects of Shiga-like toxins from enterohemorrhagic Escherichia coli O157:H7 depend on the recognition of carbohydrate determinants by B subunits. As a specific carbohydrate ligand, globotriaosylceramide has been characterized. We developed an alternative binding assay using multivalent carbohydrate ligands. We prepared globotriose-conjugated poly-lysine, and measured their binding to immobilized recombinant B subunits by an ELISA format. The signals representing ligand binding were dependent on the amount of immobilized B subunits as well as on the concentration of the ligands. The ligand binding activity was lost in an acidic environment, in which changes in the local conformation of the B subunits have been reported. Furthermore, pH dependent dissociation of the ligands from the B subunits was observed. We also demonstrate that antiserum from mice immunized with the B subunits specifically interferes with ligand binding. This suggests further potential for an assay to screen for blocking antibodies that could inhibit toxin internalization into host cells.     

Gel-filtration chromatographic method for determining relative binding affinities: rat hepatic estrogen receptor as an example system

We report a gel-filtration-based chromatographic method for separation of specific, nonspecific, and free radioligand in a protein receptor-ligand binding assay for the example of the estrogen receptor ERalpha. This assay affords relative binding affinities (RBAs) without the need for a separate determination of nonspecific binding. The probit method is recommended as the most satisfactory method of evaluating the data. The assay responds to both estrogen agonists and antagonists, mixtures respond additively, and the slopes of the probit plots indicate that all ligands bind to the same site on the estrogen receptor. RBAs obtained with rat and rainbow trout ERalpha were in good agreement, and also with those from other reported assays, consistent with the interspecies conservation of key regions of the ligand binding domain among estrogen receptors.     

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.     

The effect of ligand heterogeneity on the Scatchard plot. Particular relevance to lipoprotein binding analysis

Computer simulations of equilibrium binding studies of a mixture of two labeled ligands binding competitively to a single class of identical and independent sites (receptors) were performed to investigate how ligand heterogeneity affects the observed data in such studies. The simulated data are presented in Scatchard plots. Ligand heterogeneity was generally found to be indistinguishable from the case of a homogeneous ligand when usual experimental conditions applied (that is, Scatchard plots of the data were straight lines). Some factors that increased the probability of recognizing heterogeneity in the system were identified, however. These are 1) a large difference between the dissociation constants of the two ligands, 2) a high concentration of receptors relative to the dissociation constant of the higher-affinity ligand, 3) a high concentration of the lower-affinity ligand relative to that of the higher-affinity ligand, 4) a high specific activity of the lower-affinity ligand relative to that of the higher-affinity ligand, and 5) lack of experimental error. When ligand heterogeneity (under certain conditions) did cause curvilinearity in the Scatchard plot, the curve formed was always concave-downwards. Thus, ligand heterogeneity may occasionally mimic positive cooperativity, but never mimics negative cooperativity or multiple classes of binding sites. Implications of these findings for equilibrium binding studies involving lipoproteins (which are generally isolated as heterogeneous mixtures of particles) are discussed in detail. These findings are also relevant to equilibrium binding studies using ligands which are mixtures of stereoisomers or which contain chemical or radiochemical impurities.     

Affinity characterization-mass spectrometry methodology for quantitative analyses of small molecule protein binding in solution

Affinity characterization by mass spectrometry (AC–MS) is a novel LC–MS methodology for quantitative determination of small molecule ligand binding to macromolecules. Its most distinguishing feature is the direct determination of all three concentration terms of the equilibrium binding equation, i.e., (M), (L), and (ML), which denote the macromolecule, ligand, and the corresponding complex, respectively. Although it is possible to obtain the dissociation constant from a single mixing experiment, saturation analyses are still valuable for assessing the overall binding phenomenon based on an established formalism. In addition to providing the prerequisite dissociation constant and binding stoichiometry, the technique also provides valuable information about the actual solubility of both macromolecule and ligand upon dilution and mixing in binding buffers. The dissociation constants and binding mode for interactions of DNA primase and thymidylate synthetase (TS) with high and low affinity small molecule ligands were obtained using the AC–MS method. The data were consistent with the expected affinity of TS for these ligands based on dissociation constants determined by alternative thermal-denaturation techniques: TdF or TdCD, and also consistent enzyme inhibition constants reported in the literature. The validity of AC–MS was likewise extended to a larger set of soluble protein–ligand systems. It was established as a valuable resource for counter screen and structure–activity relationship studies in drug discovery, especially when other classical techniques could only provide ambiguous results.     

Immobilized receptor- and transporter-based liquid chromatographic phases for on-line pharmacological and biochemical studies: a mini-review

This review addresses the synthesis and characterization of two different types of receptor-based liquid chromatographic supports, one based upon a trans-membrane ligand gated ion channel receptor (the nicotinic acetylcholine receptor) and the other a soluble nuclear receptor (the estrogen receptor). In addition, studies with the P-glycoprotein transporter are also reported. The nicotinic receptor was immobilized via hydrophobic insertion into the interstitial spaces of an immobilized artificial membrane (IAM) stationary phase. the estrogen receptor was tethered to a hydrophilic stationary phase and the membranes containing the Pgp transporter were coated on the surface of the IAM stationary phase. The stationary phases were characterized using known ligands and substrates for the respective non-immobilized proteins. The results from zonal and frontal chromatographic experiments demonstrated that the stationary phases could be used to determine binding affinities (expressed as dissociation constants, Kd,'s) and to resolve mixtures of ligands according to their relative affinities. In addition. competitive ligand binding studies on the P-glycoprotein-based stationary phase have established that this phase can be used to identify and characterize competitive displacement and allosteric interactions. These studies demonstrate that immobilized-receptor phases can be used for on-line pharmacological studies and as rapid screens for the isolation and identification of lead drug candidates from complex biological or chemical mixtures.     

The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry.

We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E. coli. It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs. At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1. The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques. This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.     

Probing multivalent carbohydrate-lectin interactions by an enzyme-linked lectin assay employing covalently immobilized carbohydrates

We report here the synthesis of a series of mono- to trivalent N-acetylglucosamine (GlcNAc) derivatives as ligands for the plant lectin wheat germ agglutinin (WGA). Their WGA binding potencies were determined by an established enzyme-linked lectin assay (ELLA) employing microtiter plates with non-covalently immobilized porcine stomach mucin (PSM) as reference ligand and an ELLA with a new GlcNAc derivative covalently immobilized via a thiourea linkage. Comparison of both assays revealed that the type of presentation of GlcNAc residues on the microtiter plates either as part of a glycoprotein or as a covalently immobilized monosaccharide derivative strongly influences the outcome of the assay. Although the apparent dissociation constants K(D)(ELLA) for the interaction of peroxidase-labeled WGA with the microtiter plates are comparable for both surfaces, IC(50) values obtained with the PSM-free ELLA were substantially lower. Even more strikingly, this ELLA displayed a better differentiation between ligands of different valency leading to significantly higher relative inhibitory potencies of multivalent ligands compared to monovalent. Additionally, problems associated with the use of PSM, such as maximum inhibition at considerably less than 100% and poor reproducibility of IC(50) values could be overcome with this type of ELLA.     

Hydrophobic agaroses: basis for a model of multivalent effector-receptor interactions.

An increase in the density of butyl residues bound to Sepharose 4B leads to an enhancement of the affinity of these gels for phosphorylase b in the presence of 1.1M ammonium sulfate. A Hill coefficient of 2.9 indicates that a minimum of ca. 3 binding sites is involved in the positive cooperative adsorption of this enzyme. Binding studies of phosphorylase b on butyl-Sepharose of a specific degree of substitution demonstrate that the affinity of the gel for this ligand decreases as a function of fractional saturation. A Hill coefficient of 0.44 indicates negative cooperativity as a result of multivalent binding. From these observations a multivalent, mobile receptor model is derived which can explain such characteristics of effector-receptor interactions as: positive and negative cooperativity, high binding constants and low dissociation rate constants. The application of this model to experiments taken from the literature on the binding of the multivalent effectors concanavalin A and cholera toxin to fat cells shows that the postulated mode of interactions is probably realized in nature.     

GM(1)-functionalized liposomes in a microtiter plate assay for cholera toxin in Vibrio cholerae culture samples

Vibrio cholerae, the causative agent for cholera, infects its host by expressing a protein consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB frequently is used as an indicator of the presence of pathogenic V. cholerae and typically is detected using enzyme-linked immunosorbent assays (ELISAs). In lieu of an enzyme-linked detection method, we have developed GM(1) ganglioside-functionalized fluorescent dye-encapsulating liposomes for the detection of CTB produced by V. cholerae in a simple microtiter plate assay. Liposomes were compared with fluorescein-labeled antibodies and enzyme-linked secondary antibodies for quantification of purified CTB. A limit of detection for CTB using the liposomes was 340pg/ml, which was comparable to that using the ELISA but 18 times lower than that using the fluorescein-labeled anti-CTB antibodies for the same purpose. The sensitivity of the assay provided by the liposomes was substantial, and the working range improved when compared with that of the fluorescein-labeled antibodies and the ELISA. In addition, the liposomes required shorter assay times, exhibited greater precision, and were less expensive compared with the ELISA. The liposomes were optimized with respect to phospholipid and ganglioside concentrations. The optimized liposomes were then used to probe culture supernatants from V. cholerae El Tor C6706 grown in Dulbecco's modified Eagle's medium and AKI medium for the presence of CTB.     

Intake of monosaccharides or amino acids induces pituitary gland synthesis of proteins regulating intestinal fluid transport

In cholera diarrhoea, the pituitary gland produces a 60-kDa protein known as antisecretory factor (ASF) which reverses intestinal secretion induced by the cholera toxin. We show here that ASF-like proteins are produced in the rat during intestinal secretion triggered by intake of a 500 mg dose of mannose, sorbitol, glycine or alanine. All the ASF-like proteins reversed cholera secretion, and all were of a similar size. However, they differed in charge: mannose and sorbitol induced a protein with an isoelectric point of 4.5; glycine induced two proteins, one with a pI of 6.3, the other of 7.7; and alanine induced two proteins, one with a pI of 6.3, the other of 9.4. Antibodies against naturally occurring ASF from porcine pituitary gland neutralized ASF induced by cholera toxin and two of the amino acid-induced proteins, while the sugar-induced protein(s) did not cross-react. All the proteins showed affinity to agarose and were dissociated again with methyl alpha-D-glucoside. A single peroral dose of cholera toxin or sorbitol induced antisecretory proteins which persisted in the pituitary gland for only 1-3 days. Seven treatments gave a sustained response, the protein induced by cholera toxin persisting for over 2 months, and that induced by sorbitol about 1 month.     

Binding of nonsubstrate ligands to the glutathione S-transferases.

Fluorescence spectroscopy and inhibition kinetics were used to quantitate the affinity of nonsubstrate ligands for the rat liver glutathione S-transferases AA, A, B, and C in the presence of glutahione. The dissociation constants KD, for ligands such as bilirubin, indocyanine green, and hematin were determined by measuring the decrease in the intrinsic fluorescence of the proteins attendant on the addition of ligand. A second technique, used for compounds which absorb strongly at the excitation maxima of tryptophan, was to utilize 8-anilinonaphthalen sulfonate in the formation of protein complex fluorescing at a higher wavelength. The quenching of this complex allowed the determination of the dissociation constants for ligands such as 3,6-dibromosulfophthalein and cephalothin. These data indicate that all four proteins bind these ligands but do so with different affinities. The bilirubin-induced decrease in fluorescence was used to estimate the stoichiometry of binding as 1.2 mol of bilirubin bound/mol of transferase B and 0.7 mol/mol of transferase C. All of the ligands examine are inhibitors of catalytic activity, as tested in a standard assay with GSH and 1-chloro-2,4-dinitrobenzene as substrates. From these studies we conclude that these proteins have a broad specificity not only for their substrates, but for the binding of nonsubstrate ligands as well.     

Grafting protein ligand monolayers onto the surface of microparticles for probing the accessibility of cell surface receptors.

Coupling of a specific ligand to vaccines or drugs can be a powerful aid to route these compounds to a certain target cell population. However, if the targeted receptor is buried in a glycocalyx, binding of the ligand may be sterically hindered or even abolished, especially when the ligand is attached to bulky payloads. The antigen-transporting M cells that cover the gut-associated lymphoid tissue have a less pronounced glycocalyx than neighboring enterocytes. Such architectural differences might provide a possibility for targeting micro- or nanoparticulate vaccines to the mucosal immune system. To investigate the influence of the glycocalyx on the accessibility of cell surface receptors, we developed a system where a monolayer of ligand molecules is coupled in spatially aligned manner onto the surface of microparticles. On the basis of fluorescent carboxylate-modified particles of 1 micron diameter, different synthetic strategies were tested. Particles were first modified to display aldehyde functions on their surface, then protein ligands were coupled via Schiff base formation. The performance of the particles was tested on cultured mouse fibroblasts using the B subunit of cholera toxin as ligand and the plasma membrane glycolipid ganglioside G(M1) as receptor. Cholera toxin B subunit-coated microparticles generated by one of our synthetic pathways exhibited specific binding to fibroblasts which could be blocked with soluble cholera toxin B subunit. As particles as small as 50 nm and any proteinaceous ligand may be used, this system provides a versatile means for monitoring receptor accessibilities in vitro and in vivo.     

Zinc environment in sheep liver sorbitol dehydrogenase

The extended X-ray absorption fine structure (EXAFS) associated with the zinc K-absorption edge has been recorded for sorbitol dehydrogenase. It is interpreted in terms of one cysteine sulfur among the ligands to the active site zinc atom. Simulations of the EXAFS based on the presence of two such sulfurs are less satisfactory, and comparison with the EXAFS of such systems points to the presence of only one sulfur ligand in sorbitol dehydrogenase. These results provide evidence that sorbitol dehydrogenase does not have the characteristic one water, one His, two Cys arrangement of ligands to the active site zinc found in the homologous alcohol dehydrogenases and are consistent with the one water, one His, one Cys, one Glu ligand arrangement of the proposed model of sorbitol dehydrogenase [Eklund, H., Horjales, E., J?rnvall, H., Br?ndén, C.-I., & Jeffery J. (1985) Biochemistry 24, 8005-8012]. Evidence for the correctness of the model is also evidence for validity of predictive techniques used in constructing the model, i.e., computer graphics fitting of the amino acid sequence to the crystallographically derived structure of a different but homologous protein.     

A new approach for quantitation of receptors and their ligands

A novel and sensitive method for quantitating receptors and their ligands is described, using the calf uterine estrogen receptor and estradiol as a model system. When ligand conjugated to malate dehydrogenase is incubated with the estrogen receptor, the enzyme is inhibited proportionately to the concentration of receptor. However, receptor saturated with free ligand has almost no effect on the ligand-conjugated enzyme. The assay can detect as little as three femtomoles of receptor and one femtomole of estradiol and can, in principle, be applied to any receptor-ligand interaction.