Specific Binding Sites for S-100 Protein in Isolated Brain Nuclei

Abstract: Isolated brain nuclei possess binding sites for S-100 protein. The interaction of S-100 with these sites is specific and time-, temperature-, and Ca⁺-dependent. The profile of the ¹²⁵I-labelled S-100 binding inhibition is biphasic, displaying a high-affinity component and a low-affinity component. The S-100 binding to brain nuclei is largely irreversible, probably owing to the formation of a tight complex between the protein and its nuclear binding sites. The S-100 binding to brain nuclei is in most aspects similar to that to synaptosomal membranes. Several lines of evidence indicate, however, that the S-100 binding to nuclei is not due to contamination of these structures with plasma membranes. Isolated liver nuclei do not possess the high-affinity component of S-100 binding.     

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Evidence for the Formation of a Tight Complex Between S-100 and Its Binding Sites

Abstract: The dissociation of the ¹²⁵I-labelled S-100 specifically bound to synaptosomal particulate fractions (SYN) has been studied under a variety of conditions after different association times. The results indicate that after a critical association time of about 20 min at 37°C, the bound protein becomes progressively less accessible to the dissociating agents or conditions employed. These findings support the view that the partial irreversibility of the ¹²⁵I-labelled S-100 binding to SYN could be due to the formation of a tight complex between the protein and its synaptosomal sites. These data are discussed mainly in relation to the particulate-bound fraction of native S-100.     

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under "Physiological" Conditions

Abstract: Methyl 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylate ([¹²³I]β-CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5-HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [¹²³I]β-CIT in vivo binding to DA transporters had a much slower washout than binding to 5-HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [¹²⁵I]β-CIT for DA and 5-HT transporters. We now report [¹²⁵I]β-CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5-HT transporters was compatible with one population of sites. At 22°C, [¹²⁵I]β-CIT showed similar affinity to high-affinity DA (0.39 n M ) and 5-HT transporter sites (0.47 n M ). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5-HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [¹²³ I]β-CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.     

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Modulation of Binding by Fractional Occupancy of Sites and by the Physical State of Membranes

Abstract: The nonlinearity of single components of the Scatchard plot of S-100 binding to synaptosomal particulate fractions (SYN) and the observation that dilution of the ¹²⁵I-labeled S-100 site complex results in a greater extent of dissociation of the tracer in the presence than in the absence of an excess of unlabeled S-100 suggest that sites change their binding behavior depending on fractional occupancy. To study this aspect of the interaction in more detail, ¹²⁵I-labeled S-100 binding experiments were conducted in the presence of, or after preincubation of SYN with various concentrations of, unlabeled S-100. The results indicate that: (a) S-100 synaptosomal sites do change their binding behavior depending on fractional occupancy; and (b) the nonrapid equilibrium between bound S-100 and the medium, which has been referred to as the formation of a tight complex between S-100 and its binding sites, is related to the activation of high-affinity sites. However, no univocal interpretation of these data in terms of binding model can be offered at present, as the binding models currently employed in the analysis of ligand-site interactions can each account for only part of the results described in this report. In any case, data obtained by studying ¹²⁵I-labeled S-100 binding to untreated SYN at 2°C and to prefixed SYN at 37°C indicate that the physical state of membranes influences both the extent of the interaction and the binding behavior of the sites.     

BINDING CHARACTERISTICS OF 125I-LABELLED TETANUS TOXIN TO PRIMARY TISSUE CULTURES FROM MOUSE EMBRYONIC CNS

Abstract— The interaction of ¹²⁵I-labelled tetanus toxin with cells in tissue cultures derived from embryonic CNS has been studied.
The optimum toxin binding occurs about 2–3 weeks after transfer of the cells to culture conditions. The amount of label bound per culture was doubled at this time in comparison to the fourth day nfter inoculation.
The amount of toxin bound depends on the concentration applied. It reaches its maximum 8 h after application then decreases slowly. Low amounts of radioactivity were still detectable 97 h after washing off the unbound toxin. Up to 80% of the label can be replaced by simultaneous application of'cold'toxin. Fixation of the toxin is higher at 4°C than at 37°C.
Preincubation of the cultures with neuraminidase prevents about 75% of the binding. The presence of cytochalasin B leads to a small but reproducible decrease of binding, whereas colchicine had no measurable effect.
The radioactive (¹²⁵1) material was identified by a double-isotope technique in disc gel electrophoresis before and after reductive cleavage of its disulphide bonds. In every test is was indistinguishable from ¹³¹I-labelled toxin added as standard.
Our results largely parallel those obtained with synaptosomes and other systems. They suggest that gangliosides might be the acceptor molecules, and that the culture system will be suitable for studying the actions of this toxin in vitro.     

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick ¹²⁵I-Tf. When the cells were incubated with ¹²⁵I-Tf on ice, most of the cell-associated ¹²⁵I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.7 × 10³. Dissociation constant was estimated to be 2.6 × 10⁻⁸ M.
The binding of ¹²⁵I-Tf was competitively inhibited by the addition of chick unlabeled Tf. Weaker inhibition was observed when bovine Tf was used as a competitor. Horse Tf had no effect on the binding of chick Tf. This agrees well qualitatively with chick cell growth-promoting activites of these Tfs.
Removal of Fe from Tf affected the affinity for its receptors. About 5- to 10-fold higher concentrations of chick apo–Tf was needed to achieve the same degree of inhibition of ¹²⁵I-Tf binding as that made by chick Fe-Tf.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Ethanol and the γ-Aminobutyric Acid-Benzodiazepine Receptor Complex

Abstract: Ethanol appears to enhance γ-aminobutyric acid (GABA)-mediated synaptic transmission. Using radioligand binding techniques, we investigated the possibility that the GABA-benzodiazepine receptor complex is the site responsible for this effect. Ethanol at concentrations up to 100 m M failed to alter binding of [³H]flunitrazepam (FNZ), [³H]Ro 15-1788, or [³H]methyl-γ-carboline-3-carboxylate (MBCC) to benzodiazepine receptors, or of [³H]muscimol to GABA receptors in rat brain membranes. Scatchard analyses of the binding of these radioligands at 4°C and 37°C revealed no significant effects of 100 m M ethanol on receptor affinity or number. A variety of drugs as well as chloride ion increased binding of [³H]FNZ and/or [³H]muscimol, but these influences were not modified by ethanol. These findings indicate that ethanol probably potentiates GABAergic neurotransmission at a signal transduction site beyond the GABA-benzodiazepine receptor complex.     

The Globin Fragment LVV-Hemorphin-7 Is an Endogenous Ligand for the AT4 Receptor in the Brain

Abstract: Angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) has been reported to interact with specific high-affinity receptors to increase memory retrieval, enhance dopamine-induced stereotypy behavior, and induce c- fos expression in several brain nuclei. We have isolated a decapeptide (Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) from sheep brain that binds with high affinity to the angiotensin IV receptor. The peptide was isolated using ¹²⁵I-angiotensin IV binding to bovine adrenal membranes to assay receptor binding activity. This peptide is identical to the amino acid sequence 30–39 of sheep β^A- and β^B-globins and has previously been named LVV-hemorphin-7. Pharmacological studies demonstrated that LVV-hemorphin-7 and angiotensin IV were equipotent in competing for ¹²⁵I-angiotensin IV binding to sheep cerebellar membranes and displayed full cross-displacement. Using in vitro receptor autoradiography, ¹²⁵I-LVV-hemorphin-7 binding to sheep brain sections was identical to ¹²⁵I-angiotensin IV binding in its pattern of distribution and binding specificity. This study reveals the presence of a globin fragment in the sheep brain that exhibits a high affinity for, and displays an identical receptor distribution with, the angiotensin IV receptor. This globin fragment, LVV-hemorphin-7, may therefore represent an endogenous ligand for the angiotensin IV receptor in the CNS.     

Human Y-79 Retinoblastoma Cells Exhibit Specific Corticotropin-Releasing Hormone Binding Sites

Abstract: In this study we have identified specific binding sites for corticotropin-releasing hormone (CRH) in human Y-79 retinoblastoma cell membranes by using ¹²⁵I-Tyrovine CRH (¹²⁵I-oCRH) as radioligand. Binding at 19°C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The ¹²⁵I-oCRH binding was enhanced by Mg²⁺ and inhibited by the GTP analogue guanosine 5'- O -(3'-thiotriphosphate). Y-79 cell membranes exhibited two populations of binding sites, a high-affinity site with an apparent dissociation constant ( K _D) of 1 n M and a low-affinity site with an apparent K _D of 500 n M . ¹²⁵I-oCRH binding was completely antagonized by human/rat CRH, [Met(O)²¹]oCRH, α-helical CRH_9–41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH-binding sites in retinal cells. The Y-79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.     

GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.     

Binding of vitronectin and plasminogen to Helicobacter pylori

Abstract We have studied how some extracellular matrix proteins, fibronectin, fibrinogen, collagen type I and type IV, plasminogen and vitronectin bind to Helicobacter pylori . Radiolabelled vitronectin and plasminogen bound to the haemagglutinating H. pylori strain 17874 at a high level (53% and 32%, respectively), type IV collagen showed an intermediate level of binding (16%), while binding by ¹²⁵I-labelled fibrinogen, fibronectin and collagen type I remained at a low level (5–7%). Both ¹²⁵I-vitronectin and plasminogen showed a dose-dependent binding to cells of H. pylori 17874. Plasminogen binding by this strain was specific since the binding was inhibited by nonlabelled plasminogen, but not by highly glycosylated glycoproteins such as fetuin and orosomucoid or by a variety of monosaccharides. We have previously shown that ¹²⁵I-vitronectin shows a specific and saturable binding to H. pylori 17874, and that sialic acid-rich glycoproteins such as fetuin and orosomucoid drastically reduced binding. We now report that a simultaneous incubation of ¹²⁵I-vitronectin and ¹²⁵I-plasminogen with cells of H. pylori 17874 showed a total binding approximately similar to the level of binding when either ¹²⁵I-plasminogen, or ¹²⁵I-vitronectin only were incubated with the bacterial cells. Nonlabelled vitronectin inhibited the binding of ¹²⁵I-plasminogen by H. pylori , but nonlabelled plasminogen had no effect on the binding of ¹²⁵I-vitronectin. Our findings suggest that there are different but probably closely localized binding sites for vitronectin and plasminogen on H. pylori 17874.     

Vitronectin and type-I collagen binding by Staphylococcus aureus and coagulase-negative staphylococci

Abstract Binding of ¹²⁵I-labelled type-I collagen and ¹²⁵I-labelled vitronectin (human serum spreading factor or S-protein) was studied using Staphylococcus aureus and coagulase-negative staphylococci of different species. Binding of collagen and vitronectin was time dependent for S. aureus ISP 546, and S. haemolyticus E 2498/86. Co-operative binding of vitronectin and collagen by staphylococcal cells was demonstrated. Binding to S. haemolyticus E 2498/86 was more rapid and was enhanced in vitronectin/collagen mixtures than for either protein separately. Furthermore, pre-incubation of staphylococcal cells with unlabelled collagen enhanced vitronectin binding. When cells of S. haemolyticus E 2498/86 were treated with pronase E, proteinase K, subtilopeptidase A or trypsin, vitronectin-binding was decreased by 50% or more, whereas collagen-binding was protease resistant. For the strains of S. aureus tested, both vitronectin and collagen binding were found to be protease sensitive. Type-I collagen peptides inhibited collagen-binding to S. haemolyticus E 2498/86, whereas vitronectin-binding was not affected perhaps indicating different receptors for these proteins. The binding of both collagen and vitronectin was shown to be reversible, since bound ¹²⁵I-collagen and ¹²⁵I-vitronectin were displaced after adding excess of the homologous protein.     

Characterization of fibronectin binding to Salmonella enteritidis strain 27655R

Abstract The optimal conditions for the binding of fibronectin to Salmonella enteritidis strain 27655R, and the cell-surface components involved in the binding, were identified. Cultivation on colonisation factor antigen (CFA) agar or in CFA broth at 33°C for 24 h were found to be optimal for the expression of fibronectin binding. Such cultures exhibited 88% and 70% binding of ¹²⁵I-labelled fibronectin and its 29-kDa N-terminal domain, respectively. The fibronectin binding was reversed by the addition of unlabelled fibronectin or its 29-kDa fragment. Scatchard plot analysis of the binding showed that the strain possessed one high-affinity ( K _d= 5.8 × 10⁻¹⁰ M) and one low-affinity ( K _d= 2 × 10⁻⁸ M) binding site. The fibronectin-binding could be inhibited by cell surface components of S. enteritidis 27655R released by 30 min treatment at 65°C or 95°C. Inhibition could also be achieved using purified fimbriae. A non-fimbriated mutant of strain 27655R showed a much reduced binding of fibronectin (15%). Electron microscopic analysis showed association of the gold-labelled 29-kDa N-terminal fragment with S. enteritidis 27655R fimbriae. In conclusion, the findings suggest that S. enteritidis (strain 27655R) possesses fibronectin-binding fimbriae.     

Antigenic heterogeneity of strains of Saccharomyces cerevisiae and Candida albicans recognised by serum antibodies from patients with Crohn''s disease

Abstract Vitronectin, a serum and extracellular matrix protein involved in immunological reactions, interacts with Helicobacter pylori strains. Of the 20 H. pylori strains tested three strains bound more than 50% of the vitronectin added, five strains bound 25–40%, nine strains bound 10–20% and three strains bound 5–8% vitronectin. Two strains, one with high- and one with low-binding properties, were selected for further characterization of ¹²⁵I-vitronectin binding. Binding to the urea-activated ¹²⁵I-labelled vitronectin was fast, saturable and reversible when an excess of unlabelled vitronectin was added to the bacteria with bound ¹²⁵I-vitronectin. The binding was heat- and protease-sensitive, suggesting that the binding was mediated by bacterial cell-surface proteins. Since components such as fetuin and orosomucoid but not asialofetuin inhibited the binding, sialic-acid specific proteins, related to H. pylori sialic-acid specific haemagglutinins, were probably involved.     

Calcium Regulation of Agonist Binding to α7-Type Nicotinic Acetylcholine Receptors in Adult and Fetal Rat Hippocampus

Abstract: Quantitative autoradiography was used to compare the binding properties of α7-type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of ¹²⁵I-α-bungarotoxin (¹²⁵I-α-BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of ¹²⁵I-α-BTX binding, as well as α-cobratoxin and nicotine potency to displace ¹²⁵I-α-BTX, did not change with age. Addition of Ca²⁺ to the assay buffer did not alter ¹²⁵I-α-BTX binding, or α-cobratoxin inhibition of ¹²⁵I-α-BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca²⁺ on agonist affinity was dose-dependent, with an EC₅₀ value of 0.25–0.5 m M . Ca²⁺ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal ¹²⁵I-α-BTX binding sites are largely similar across age. Ca²⁺ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the α7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.     

Problems Associated with the Binding of L-Glutamic Acid to Synaptic Membranes: Methodological Aspects

Abstract: Specific L-[³H]glutamate binding was investigated in extensively washed synaptic membrane preparations from rat brain. Mild conditions of ultrasonication effected a significant enhancement of binding, attributable to the marked reduction in membrane vesicle size and the removal of endogenous interfering substances such as glutamate. Preincubation of freshly prepared membranes at 37°C for 30 min followed by further washing resulted in enhanced binding. Addition of supernatant from preincubated membranes effectively inhibited [³H]glutamate binding to control membranes; the possibility of the presence of an endogenous glutamate receptor inhibitor is discussed. Treatment of membranes with low concentrations of Triton X-100, in contrast with the findings for GABA, did not produce any significant enhancement of specific glutamate binding. While binding of [³H]glutamate is almost abolished in frozen or cold-stored membranes, lyophilisation had a remarkable effect, not only affording protection, but actually enhancing the binding properties of the synaptic membrane preparation.     

Photoaffinity Labeling with 2-[2-(4-Azido-3-[125I]-Iodophenyl)ethylamino]adenosine and Autoradiography with 2-[2-(4-Amino-3-[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain

Abstract: The A_2a adenosine receptor agonist 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [¹²⁵I]APE, discriminates between high- and low-affinity conformations of A_2a adenosine receptors. In this study, [¹²⁵I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [¹²⁵I]APE binding is consistent with that expected of an A_2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [¹²⁵I]APE binding to these brain regions is abolished by 1 µ M 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 n M 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [¹²⁵I]APE to the corresponding arylazide results in [¹²⁵I]AzPE. The rank-order potency of compounds to compete for [¹²⁵I]AzPE binding in the dark is CGS-21680 > d -( R )- N ⁶-phenylisopropyladenosine > N ⁶-cyclopentyladenosine, indicating that it also is an A_2a-selective ligand. Specific photoaffinity labeling by [¹²⁵I]AzPE of a single polypeptide (42 kDa) corresponding to A_2a adenosine receptors is reduced 55 ± 4% by 100 µ M guanosine 5'- O -(3-thiotriphosphate) and 91 ± 1.3% by 100 n M CGS-21680. [¹²⁵I]APE and [¹²⁵I]AzPE are valuable new tools for characterizing A_2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.     

Taurine Interactions with Chick Retinal Membranes

Abstract: Binding of [³H]taurine to whole retinal membranes and to membranes obtained from retinal subcellular fractions was studied. [³H]Taurine bound to chick retinal membranes with high affinity and specificity. Two types of [³H]taurine binding associated to retinal membranes were observed, one with a K_D= 0.68 μM and the other one with a K_D,= 9.32 μM. Both types of binding were highly Na⁻-dependent. The Na⁺-dependent taurine binding was antagonized by strychnine. Bound [³H]taurine was effectively displaced by β-alanine but not by GABA or glycine. Taurine binding was preferentially localized in membranes obtained from the crude synaptosomal fraction, although it is also present in substantial amounts in all retinal membranes. A Na⁺-independent [³H]taurine binding exhibiting properties which might represent interaction with postsynaptic receptor sites could not be demonstrated in the chick retina.     

CHOLINERGIC SITES IN SKELETAL MUSCLE: INTERACTION WITH CONCANAVALIN A

Abstract– The interaction of normal and denervated skeletal muscle cholinergic sites with the lectin concanavalin A and concanavalin A-Sepharose are detailed. Concanavalin A blocks the binding of ¹²⁵I-α-bungarotoxin to both the high and low affinity sets of cholinergic sites described previously. The characteristics of the block of ¹²⁵I-α-bungarotoxin binding to the high affinity set (acetylcholine receptor) is not competitive. The data suggest that the concanavalin binds multivalently to the macro-molecular complex containing the ACh receptor site and sterically prevents the α-bungarotoxin binding. The interaction of both sets of cholinergic sites with concanavalin A-Sepharose was also studied. The macromolecule(s) containing both the high and low affinity sets of sites bind to the concanavalin A-Sepharose. The data indicate a multivalent association with the affinity resin. Following the affinity procedure, a partial purification in both sets of sites is effected. The equilibrium binding of ¹²⁵I-diiodo-α-bungarotoxin to the preparations from the affinity procedure (both normal and denervated muscle) was examined. The K_D of the α-bungarotoxin binding to the high affinity sets of sites (acetylcholine receptor) in both normal and denervated preparations changes from ∼10⁻⁹mol/l to ∼ 10⁻¹⁰ mol/l following purification. No change in the K_D of the α-bungarotoxin binding to the low affinity set of sites was observed following purification. The ¹²⁵l-α-bungarotoxin binding to the partially purified acetylcholine receptor was blocked by unlabelled α-bungarotoxin, concanavalin A, d-tubocurarine and carbamylcholine.