Porperties of an α-bungarotoxin-binding cholinergic nicotinic receptor from drosophila melanogaster

α-[¹²⁵I]Bungarotoxin specifically binds to homogenates of Drosophila melanogaster head at levels of 0.3–0.8 pmol/mg protein. The dissociation constant calculated from rates of association and dissociation of toxin · receptor complex, is 0.6 · 10^?9M. Ca²⁺, and to lesser extent Na⁺, inhibit the reaction. α-[¹²⁵I]Bungarotoxin binding is inhibited by low concentrations of unlabelled toxin, nicotinic ligands and eserine, but not by low concentrations of muscarinic ligands, decamethonium or an organophosphate. The receptor is membrane bound and can be partially released into 100 000 × g supernatant by a combination of 1 M NaCl and 1% Triton X-100. Most of the activity in the supernatant sediments after further centrifugation at 200 000 × g for 2 h. Toxin binding sites are distinct from acetylcholinesterase molecules as revealed by pharmacological, biochemical and genetic techniques. The gene for the toxin-binding nicotinic receptor in Drosophila is apparently not located adjacent to the gene for acetylcholinesterase.     

Solubilization of an alpha-bungarotoxin-binding component from rat brain.

Binding of [125I]-alpha-bungarotoxin to rat brain was investigated. Picomole quantities of specific toxin binding sites per gram of fresh tissue were found in particulate preparations as well as detergent extracts of whole brain. The toxin-binding macromolecules can be solubilized in low concentrations of Triton X-100. Specific binding occurs to a single class of sites with a dissociation constant of 5.6 X 10(-11) M. The association rate constant in 10 mM sodium phosphate, pH 7.4, was determined to be 6.8 X 10(5) M-1 s-1; the half-life of the complex was found to be 5.1 h, corresponding to a dissociation rate constant of 3.8 X 10(-5) s-1. The binding macromolecules resemble peripheral nicotinic acetylcholine receptors in toxin binding kinetics, solubility, isoelectric point, and hydrodynamic properties.     

PROPERTIES OF A PUTATIVE MUSCARINIC CHOLTNERGIC RECEPTOR FROM DROSOPHILA MELANOGASTER

The powerful muscarinic antagonist [³H]quinuclidinyl benzilate (QNB) specifically binds to homogenates of Drosophila melanogaster head at a level of 65 ± 6 fmol/mg protein, with an apparent dissociation constant of 0.15–0.7 nM. The half-life of the ligand-receptor complex at 25°C is 30–40 min. Binding is inhibited by low concentrations of muscarinic ligands but not by low concentrations of nicotinic ligands, anticholinesterases or non-cholinergic drugs. Binding-sites are membrane bound and are inactivated by trypsin and by Triton X-100. Part of the activity (<20%) is released into a high speed supernatant by 2 M-NaCI. The gene coding for the putative muscarinic receptor in Drosophila is apparently not located adjacent to the gene for acetylcholinesterase     

Biochemical characterization of two nicotinic receptors from the optic lobe of the chick

We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor.     

Studies on nicotinic acetylcholine receptors in mammalian brain. Interaction of solubilized protein with cholinergic ligands

Binding of alpha-bungarotoxin, labeled with 125I, has been studied in detergent extracts and affinity purified acetylcholine receptor from rat cerebral cortex. Binding to detergent extracts is saturable and appears to be due to one class of binding sites present at a level of 0.27 pmol/mg of protein. The association constant is 2 X 10(7) liters mol-1 . Competition with cholinergic ligands indicates that toxin binding to both detergent solubilized and affinity purified receptor retains its nicotinic nature. Values for the ligand concentrations required to produce 50% inhibition of extent and rate of toxin binding are presented.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization, purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000-6000 pmol per mg protein of alpha-[125I]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius 125 +/- 10 A and on sucrose gradient centrifugation one major peak was observed of 20-22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Postnatal Development of Cholinergic Enzymes and Receptors in Mouse Brain

The developmental profiles for the cholinergic enzymes acetylcholinesterase and choline acetyltransferase, and the muscarinic and nicotinic receptors were determined in whole mouse brain. The enzyme activities (per milligram of protein) increased steadily from birth, reaching adult levels at 20 days of age. These increases were primarily due to increases in Vmax. Muscarinic receptor numbers, measured by [3H]quinuclidinyl benzilate binding, also increased from birth to 25 days of age. Brain nicotinic receptors were measured with the ligands L-[3H]nicotine and alpha-[125I]-bungarotoxin. Neonatal mouse brain had approximately twice the number of alpha-bungarotoxin binding sites found in adult mouse brain. Binding site numbers rose slightly until 10 days of age, after which they decreased to adult values, which were reached at 25 days of age. The nicotine binding site was found in neonatal brain at concentrations comparable to those at the alpha-bungarotoxin site followed by a steady decline in nicotine binding until adult values were reached. Thus, brain nicotinic and muscarinic systems develop in totally different fashions; the quantity of muscarinic receptors increases with age, while the quantity of nicotinic receptors decreases. It is conceivable that nicotinic receptors play an important role in directing the development of the cholinergic system.     

Effects of Monovalent and Divalent Cations on 3-(+)[125I]Iododizocilpine Binding to the N-Methyl-d-Aspartate Receptor of Rat Brain Membranes

We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.     

Nicotinic cholinergic receptor in brain detected by binding of α-[3H]bungarotoxin

α-[³H]Bungarotoxin was prepared by catalytic reduction of iodinated α-bungarotoxin with tritium gas. Crude mitochondrial fraction from rat cerebral cortex bound 40 · 10^?15 ?60 · 10^?15 moles of α-[³H]bungarotoxin per mg of protein. This binding was reduced by 50% in the presence of approx. 10^?6 M d-tubocurarine or nicotine, 10^?5 M acetylcholine, 10^?4 M carbamylcholine or decamethonium or 10^?3 M atropine. Hexamethonium and eserine were the least effective of the drugs tested. Crude mitochondrial fraction was separated into myelin, nerve endings, and mitochondria. The highest binding of toxin per mg of protein was found in nerve endings, as well as the greatest nhibition of toxin binding of d-tubocurarine. Binding of α-[³H]bungarotoxin to membranes obtained by osmotic shock of the crude mitochondrial fraction indicates that the receptor for the toxin is membrane bound. ¹²⁵I-Labeled α-bungarotoxin, prepared with Na ¹²⁵I and chloramine T, was highly specific for the acetylcholine receptor in diaphragm, however, it was less specific and less reliable than α-[³H]bungarotoxin in brain. We conclude that a nicotinic cholinergic receptor exists in brain, and that α-[³H]bungarotoxin is a suitable probe for this receptor.     

Kinetic analyses demonstrate that the equilibrium assumption does not apply to [125I]endothelin-1 binding data.

The kinetics of [125I]Endothelin-1 ([125I]ET-1) binding were studied using membranes from rat heart, rat lung, rat brain, and porcine vascular smooth muscle at 37 degrees C in 0.05M Tris-HCl buffer (pH = 7.4). The dissociation half-life (t1/2, diss.) for bound [125I]ET-1 was in excess of 30 hours for each tissue studied. Equilibrium-time requirements for proper Scatchard analysis of [125I]ET-1 were also far in excess of 30 hours for each tissue. These data suggest that determination of dissociation constants, Kd, and receptor concentrations, Bmax, by conventional Scatchard analysis is not feasible with [125I]ET-1. Kinetic analyses may provide a more accurate means for determining [125I-ET-1] binding characteristics including Kd and Bmax.     

Different modes of ligand binding to the hepatic galactose/N-acetylgalactosamine lectin on the surface of rabbit hepatocytes

A study of the binding of three different 125I-labeled, galactose-terminated ligands to the hepatic galactose/N-acetylgalactosamine-specific lectin found on the surface of rabbit hepatocytes revealed that the different ligands manifest different physical parameters of binding. Asialoorosomucoid (125I-ASOR) binding was best described as involving two independent classes of binding sites on rabbit hepatocytes, with 161 000 sites/cell with a dissociation constant of 0.44 nM and 292 000 sites/cell with a Kd of 9.7 nM. Asialotriantennary glycopeptide purified from human alpha-1 protease inhibitor and modified with tyrosine at the N-terminus to permit radioiodination (TRI) [Lee, Y. C., Townsend, R. R., Hardy, M. R., L?nngren, J., Arnarp, J., Haraldsson, M., & L?nn, H. (1983) J. Biol. Chem. 258, 199-202] was also found to bind to two apparent classes of binding sites but with different binding parameters: 292 000 sites/cell of Kd = 1.47 nM and 982 000 sites/cell of Kd = 25.3 nM. A synthetic ligand, alpha,beta-diaspartamide of tris[(beta-lactosyloxy)methyl](6-aminohexanamido)methane (di-tris-lac) containing six nonreducing galactose residues [Lee, R. T., Lin, P., & Lee, Y. C. (1984) Biochemistry 23, 4255-4261], was found to bind to 817 000 sites/cell of Kd = 0.63 nM and 1.23 X 10(6) sites/cell of Kd = 25.3 nM. Thus, there were many more total binding sites for TRI or di-tris-lac on the surface of rabbit hepatocytes than there were for asialoorosomucoid, although the dissociation constants were similar for all three ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

α-Bungarotoxin Binds to Low-Affinity Nicotine Binding Sites in Rat Brain

Reported differences in the pharmacology and distribution of [3H]nicotine and [125I]alpha-bungarotoxin binding sites in mammalian brain suggest that these ligands label separate receptor sites. Affinity purification of an alpha-bungarotoxin binding protein from rat brain failed to copurify the high-affinity nicotine binding site, which remained in the nonbound soluble fraction after the affinity chromatography step. This confirms the independence of these putative receptor sites. Nevertheless, the binding of [125I]alpha-bungarotoxin to P2 membranes was inhibited by (-)-nicotine (Ki = 9 X 10(-6) M), and this sensitivity was preserved after affinity purification. It is proposed that alpha-bungarotoxin binds to a population of low-affinity nicotine binding sites. Comparison of the enantiomers of nicotine in competition studies at both radioligand binding sites revealed an 80-fold preference for the (-) form at the high-affinity [3H]nicotine binding site, whereas the site labelled by [125I]alpha-bungarotoxin displayed little stereoselectivity. In this respect, the brain alpha-bungarotoxin binding site resembles the nicotinic acetylcholine receptor from Torpedo electric organ.     

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.     

Interactions of cathepsin-D and insulin-like growth factor-II (IGF-II) on the IGF-II/mannose-6-phosphate receptor in human breast cancer cells and possible consequences on mitogenic activity of IGF-II

The lysosomal enzyme cathepsin-D (cath-D) and insulin-like growth factor-II (IGF-II), which share a common IGF-II/mannose-6-phosphate (M6P) transmembrane receptor, are both synthesized and secreted by breast cancer cells, upon which they might exert an intracrine/autocrine control on proliferation. We have evaluated the binding of 125I-immunopurified human cath-D in different breast cell membrane preparations. The concentration of high affinity M6P reversible binding sites (mean Kd, 0.85 nM) varied among the different breast cancer cells (0-0.82 pmol/mg membrane protein), but there was no correlation between the presence of steroid receptor and M6P-dependent binding. Cross-linking experiments with [125I]cath-D and [125I]IGF-II showed the formation of complexes with the 270,000 mol wt IGF-II/M6P receptor molecule which migrated, respectively, at 330,000 and 270,000 mol wt in 3-10% gradient sodium dodecyl sulfate-polyacrylamide gels. [125I]IGF-II cross-linking was increased by M6P (20% above control), whereas cath-D strongly inhibited IGF-II interaction by 80%. Conversely, IGF-II reduced [125I]cath-D cross-linking by 55%. Direct ligand binding on receptors transferred onto nitrocellulose sheets by Western blotting confirmed the interaction of both ligands on the same receptor molecule. By studying IGF-II's growth-promoting activity in these cells in a wide range of concentrations, we show that IGF-II triggers its mitogenic response via IGF-II/M6P receptor at low concentrations, whereas it is mainly acting via IGF-I receptor at high concentrations. Three lines of evidences lead us to that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding sites for [3H]-acetylcholine and 125I-alpha-bungarotoxin in the optic ganglion of Loligo pealii

1. In the optic ganglion of Loligo pealii, binding sites for [3H]-acetylcholine (KD: 5.2 x 10(-7) M; Bmax: 1.7 x 10(-11) mol/g tissue) and 125I-alpha-bungarotoxin (KD: 3.3 x 10(-9) M; Bmax: 9.7 x 10(-11) mol/g tissue) were observed. 2. Both sites are blocked by nicotinic compounds, but differ significantly in their affinity for individual ligands, with the acetylcholine site preferentially binding agonists, and the toxin site, antagonists. 3.The acetylcholine site is substantially more thermolabile than the toxin site. 4. A partial separation of the two binding activities is accomplished by sucrose density centrifugation. 5. These observations and a comparison with other tissues (Torpedo californica electroplaque; chick optic lobe; rat brain) suggest the presence, in the squid, of more than one kind of neuronal nicotinic receptor.     

Characterization of the beta-adrenergic receptor of the rat peritoneal macrophage

The beta-adrenergic receptor was characterized on BCG-activated rat peritoneal macrophage membranes by radio-ligand binding studies. Saturable binding with [125I]iodocyanopindolol (125I-ICYP) was demonstrated. With Scatchard analysis, rat macrophages demonstrate approximately 1000 receptors per cell with a Kd of 5 X 10(-11) M for 125I-ICYP. Competition curves with (-) and (+) propranolol at concentrations below 10(-6) M confirmed stereospecificity. The potency of various ligands to compete for 125I-ICYP binding sites followed the order: propranolol greater than isoproterenol greater than epinephrine greater than norepinephrine with apparent Kd of 2.0 X 10(-9), 3.9 X 10(-7), 1.0 X 10(-5), and 2.5 X 10(-5) M, respectively. Isoproterenol-stimulated adenylate cyclase activity was two-fold above basal activity. The potential physiologic significance of a beta-adrenergic receptor on rat peritoneal macrophages was suggested by a dose-dependent decrease in phagocytosis of soluble, model immune complexes (aggregated gamma-globulin) by macrophages incubated with metaproterenol. We conclude that the rat macrophage has a beta-adrenergic receptor and that catecholamines may thereby modulate macrophage function.     

Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

Comparative Analysis of Nicotine-Like Receptor-Ligand Interactions in Rodent Brain Homogenate

The effects of different variables such as incubation time, temperature, tissue protein content, and pH on the interactions of various labelled nicotinic ligands with nicotine-like binding sites in vitro were studied in rodent brain preparations. The ligands tested were alpha-[3H]bungarotoxin (alpha-[3H]BTX), [3H]tubocurarine ([3H]TC), and [3H]nicotine ([3H]NIC). The regional distribution of the labelled nicotinic ligand binding was also studied and affinity constants and maximal binding (Bmax) values for the equilibrium [3H]NIC binding are given. Association kinetics for [3H]NIC and [3H]TC binding to brain homogenate were similar, with maximal binding within 5-10 min of incubation, followed by a continuous decrease. In contrast, the binding of alpha-[3H]BTX to brain homogenate was much slower, reaching equilibrium after 30-60 min of incubation. Scatchard analysis of equilibrium binding data for [3H]NIC in the hippocampus indicated two binding sites: a high-affinity site (Bmax, 60 pmol/g protein; KD, 6 nM) and a low-affinity site (Bmax, 230 pmol/g protein; KD, 125 nM). The data for the high-affinity [3H]NIC binding site are very similar to previously found data for the high-affinity binding site of [3H]TC and the binding site of alpha-[3H]BTX. Each ligand showed regional differences in binding, and the binding pattern also differed between the ligands.     

Regulation of Brain Nicotinic Receptors by Chronic Agonist Infusion

Several studies have demonstrated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. To determine whether this effect is elicited by other nicotinic agonists found in tobacco, the effects of chronic infusion with nicotine on brain nicotinic receptors were compared with those after anabasine and lobeline. C57BL/6 mice were infused with saline or equimolar doses (18.5 mumol/kg/h) of nicotine, anabasine, or lobeline for 8 days. Nicotinic receptors, quantified by the binding of [3H]nicotine and [125I]iodo-alpha-bungarotoxin (alpha-[125I]BTX), and muscarinic receptors, quantified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB), were then assayed in eight brain regions. An increase in [3H]nicotine binding was observed in all regions except cerebellum following chronic infusion with nicotine and anabasine, whereas lobeline did not alter the number or affinity of these binding sites. This increase was due to changes in Bmax and not in the affinity of the receptor for the ligand (KD). A slight increase in alpha-[125I]BTX binding was observed in cortex following chronic anabasine infusion. [3H]QNB binding sites were largely unaltered following chronic infusion with any of the nicotinic analogs. The levels of the agonists in the brain were also determined after chronic treatment, and the amounts of lobeline and anabasine were found to be higher than that of nicotine. Thus, the failure of lobeline to elicit changes in nicotine binding is not due to reduced brain concentrations.     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.