Michael A. Raftery

A simple assay for the study of solubilized acetylcholine receptors

Abstract Detergent-solubilized acetylcholine receptors from the electric organs of Electrophorus electricus and Torpedo californica are rapidly, quantitatively and specifically assayed by exposure to 125I-α-bungarotoxin and subsequent adsorption onto DEAE-cellulose paper disks.

Specificity of α-Bungarotoxin binding to Torpedo californica electroplax

Abstract Binding of [ 125 I]α-Bungarotoxin to Triton X-100 extracts of Torpedo californica electroplax membranes has been studied. Major and minor binding components have been observed; these differ in their apparent Stokes radii and in their behaviour in sucrose density gradients. The concentrations of both toxin binding components agree with previous studies of cholinergic ligand binding to membrane particles of the related species Torpedo marmorata . Some physical parameters of the major binding component compare with those previously determined for a toxin-binding macromolecule from Electrophorus electricus .

Identification of a large molecular weight peptide associated with a tetrodotoxin binding protein from the electroplax of Electrophoruselectricus

Abstract We have previously described the purification of a tetrodotoxin binding protein from the electroplax of Electrophorus electricus . The preparation consisted of three peptides of M r ∼ 46,000, 59,000 and ∼ 300,000 daltons. Further investigation has now shown that the large peptide of M r ∼ 260,000 daltons is part of the tetrodotoxin binding component of the voltage-sensitive sodium channel.

Use of affinity chromatography for acetylcholine receptor purification

Abstract An acetylcholine receptor (AChR) affinity resin was prepared by covalently linking [N-(ϵ-aminohexanoyl)-3-aminopropyl]trimethyl ammonium bromide hydrobromide to agarose. A partially purified membrane protein preparation from the electroplax of Narcine was resolved on this resin into a peak of inactive protein and a peak of protein displaying α-bungarotoxin binding activity. Analysis of the purified toxin binding material by SDS polyacrylamide electrophoresis shows a major band of apparent molecular weight 28,000 and two minor bands of approximately 45,000 and 38,000.

Immunological comparison of acetylcholine receptors and their subunits from species of electric ray.

Abstract Rabbit antibodies were produced against purified acetylcholine receptor and each of the four acetylcholine receptor subunits from Torpedo californica. Using the technique of double diffusion in agar, cross reactivities were observed between these antibodies and purified acetylcholine receptor and receptor subunits from Torpedo marmorata , Torpedo nobiliana , and Narcine brasiliensis , as well as from Torpedo californica. The specificity of each of the four anti-subunit antibodies and the conservation of subunit antigenic determinants in the four electric rays studied are demonstrated.

Demonstration of a specific α-bungarotoxin binding component in Electrophorus electricus electroplax membranes

Abstract Studies of the binding of 125I-α-Bgt to membranes from the electroplax of Electrophorus electricus have demonstrated a high degree of specificity. The association is due to complex formation with a unique macromolecular membrane bound component.

Characterization of acetylcholine receptor-rich and acetylcholinesterase-rich membrane particles from Torpedo californica electroplax.

Abstract Large-scale purification of acetylcholinesterase-rich and acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax is described. Electron microscopy studies reveal structural differences in the two types of particles and the results are discussed in terms of structural aspects of the postsynaptic cleft. Polyacrylamide gel electrophoresis of receptor-rich fragments reveals that the fragments contain the same polypeptide components observed in receptor preparations purified from the same electroplax membranes, indicating that purified Torpedo receptor is not composed of species degraded by proteolysis. Results obtained from fluorescence studies of a cholinergic analog allow conclusions to be reached regarding species differences in electroplax acetylcholine receptor preparations.

Binding of acetylcholine and related compounds to purified acetylcholine receptor from Torpedocalifornica electroplax

Abstract The binding properties of the purified acetylcholine receptor from Torpedo californica were investigated. One type of binding was observed for acetylcholine (KD = 2.3 μM), dimethyl tubocurarine (KD = 6.2 μM), and decamethonium (KD = 55 μM). No cooperativity was observed in ligand binding. By virtue of its ligand binding properties, the purified receptor is nicotinic in nature.

Use of a fluorescent probe for the study of ligand binding by the isolated cholinergic receptor of Torpedo californica

Summary The binding of a fluorescent dye, bis-(3-aminopyridinium) 1, 10-decane to a highly purified isolated acetylcholine receptor from Torpedo californica has been studied. Inhibition of dye binding by various cholinergic agonists and antagonists yields inhibition constants for these compounds. It has been observed that various cations bind to the isolated receptor with high affinity and in a manner consistent with previously observed in vivo effects.

Cholinergic ligand-induced affinity changes in Torpedo californica acetylcholine receptor.

Abstract The binding of cholinergic ligands to Torpedo californica acetylcholine receptor has been studied in vitro by inhibition of the time course of 125 I-labeled α-bungarotoxin-receptor complex formation. The extent of inhibition was dependent on the duration of exposure to the ligand, the apparent affinity for ligand increasing with time, and was reversible upon removal of ligand. Ligand concentration, temperature, and Ca 2+ ions influenced this effect which is reminiscent of receptor desensitization in vivo . Such effects were observed both for a cholinergic agonist, carbamylcholine, and for an antagonist, bis(3-aminopyridinium)-1,10-decane diiodide. A minimal model is discussed which can account for these effects and for receptor ligand association leading to postsynaptic depolarization.