A simple quantitative assay of 125I-labeled α-bungarotoxin binding to soluble and membrane-bound acetylcholine receptor protein

Kohanski, Ronald A.; Andrews, John; Wins, Pierre; Eldefrawi, Mohyee E.; Hess, George P.

doi:10.1016/0003-2697(77)90676-5

Cited by 27 publications

(17 citation statements)

References 13 publications

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“…The concentration of gallamine used here was sufficient to block reaction of AChR and a-bungarotoxin under these conditions. Reaction mixtures were incubated for 3 h at room temperature to permit saturation of the receptors with toxin, and the bound and free toxins were then separated by ion-exchange chromatography (Kohanski et al, 1977) as follows; 0.2 mL of the reaction mixture was applied to a minicolumn of cation exchange resin (see below) and the reaction mixture was washed in with 0.2 mL and eluted with 2.4 mL of column buffer (0.01% Triton X-lOO,O.Ol% Thimerosol, 1mM sodium phosphate, pH 7.2). The entire 2.8 mL of eluate was collected in glass scintillation vials and counted in a Packard Auto-Gamma counter at 48% efficiency.…”

Section: Methodsmentioning

confidence: 99%

Testosterone increases acetylcholine receptor number in the “levator ani” muscle of the rat

1982

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The "levator ani" muscle of male rats provides a neuromuscular system in which both the muscle and its motoneurons have high levels of androgen receptors. Two weeks of castration caused a 48% loss of acetylcholine receptors in this muscle. One week of testosterone propionate injections initiated on week after castration increased receptor number by 27% over untreated castrate levels. These changes paralleled changes in muscle protein content. In contrast, castration and testosterone treatments of castrates had no effect on total. Triton X-100-extractable acetylcholinesterase activity. This system may provide a useful model of synaptic plasticity.

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Section: Methodsmentioning

confidence: 99%

Testosterone increases acetylcholine receptor number in the “levator ani” muscle of the rat

1982

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“…The details of the influx assays and the determination of the tracer content of the vesicles by using gramicidin (18) and of the determination of the protein concentration of the membrane vesicles (10,21) have been described. The purification and characterization of a-bungarotoxin and the method used to determine the concentration of toxinbinding sites have been described (22,23). Percoll was obtained from Pharmacia.…”

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confidence: 99%

Specific reaction rate of acetylcholine receptor-controlled ion translocation: a comparison of measurements with membrane vesicles and with muscle cells.

Hess¹,

Aoshima²,

Cash³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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The specific reaction rate (J) of the acetylcholine receptor-controlled ion translocation has been determined. In eel Ringer's solution (pH 7.0) at 10C, j = 3 x 107 M'1 sec-1. j is an intrinsic constant that is characteristic of the receptor and independent of other properties of a receptor-containing cell that also determine the rates of ion translocation. Membrane vesicles (prepared , which allows one to measure elementary steps in the formation of ion channels through the cell membrane.The initial rate of acetylcholine receptor-controlled movement of ions across the membrane of a particular cell determines the initial change in transmembrane potential (1, 2) and therefore also determines whether or not the electrical signal generated by a nerve cell will be propagated (3) or, in muscle cells, whether a contraction will occur. This initial rate is characterized by the rate coefficient, JA The value of JA is determined not only by the intrinsic properties of the receptor but also by certain properties of the cell, such as the number of receptor sites and the internal volume, and also the internal and external concentrations of freely mobile inorganic ions. We have, therefore, determined the specific reaction rate, J, which is characteristic of the receptor-controlled ion translocation and independent of these other properties of nerve and muscle cells. The rate of receptor-controlled ion translocation over a wide range of both carbamoylcholine and acetylcholine concentrations can be measured (12-15). The interpretation of all these measurements has led to the formulation of a minimum mechanism (Fig. 1) that relates the formation of ligand-receptor complexes to ion translocation (13,14). The integrated rate equation (Eq. 1) based on this mechanism predicts the rate of receptor-controlled flux over the concentration ranges of carbamoylcholine (13,14) and acetylcholine that we have investigated (15).JA is the observed rate coefficient associated with the initial fast influx before the onset of inactivation (desensitization) of the receptor. J. is the observed rate coefficient associated with the slow phase that occurs after the inactivation process has gone to completion, and a is the rate coefficient for the inactivation process: JA = Jm ([AL2]

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“…The preparation ofmono['"I]iodo-a-bungarotoxin and all other chemicals used have been described (21), as have the methods used to determine the concentrations of protein (22) and of toxin-binding sites (23,24) Abbreviations: AcCho, acetylcholine; AcChoR, acetylcholine receptor; CbmCho, carbamoylcholine.…”

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confidence: 99%

“…Preparations labeled EP were made essentially according to a method that has also been described (20); fraction 1 was 20-30% sucrose and fraction 3 was 38-42% sucrose. Preparations were stored in 20% sucrose in liquid N2.The preparation ofmono['"I]iodo-a-bungarotoxin and all other chemicals used have been described (21), as have the methods used to determine the concentrations of protein (22) and of toxin-binding sites (23,24) Abbreviations: AcCho, acetylcholine; AcChoR, acetylcholine receptor; CbmCho, carbamoylcholine. …”

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confidence: 99%

Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus : Chemical kinetic measurements in the millisecond region

Hess

Pasquale

Walker

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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In earlier studies with the acetylcholine receptor (AcChoR) ofElectrophorus electric the rate and equilibrium constants for a model that relates the ligand binding to ion translocation were determined, and the dependence of these constants on the concentrations ofcarbamoylcholine and acetylcholine, over a 200-and 5000-fold range, respectively, could be predicted. AcChoR-controlled cation flux has now been measured in Torpedo californica vesicles by using a pulsed-quench-flow technique with a 2-msec time resolution. vesicle is the existence of a second, slower, inactivation process in T. californica with a rate coefficient, 6, of 0.12 sec'. The second process leads to undetectable flux activity during the time of observation (30 sec in 10 mM carbamoylcholine). These studies are also significant because fundamental differences may exist between the mechanism of AcChoR-controlled ion flux in synaptic (Torpedo) and conducting (E. electricus) membranes. Nachmansohn suggested (1) that the binding of acetylcholine (AcCho) to the acetylcholine receptor (AcChoR) in nerve and muscle cells induces a conformational change in the protein that results in the formation ofion-conducting channels through the membrane. The important relationship between the concentration of AcCho and the rates of AcChoR-controlled ion translocation has been investigated intensively because it determines the transfer ofinformation between cells in the nervous system. Kasai and Changeux (2) demonstrated AcChoR-controlled ion flux in vesicles prepared from Electrophorus electricus electroplax. Subsequently we developed methods for isolating and characterizing (3, 4) homogeneous vesicle preparations containing active receptors. An adaptation of rapid reaction techniques (5) allowed four phases of the process to be measured in the 2-msec to minute time region (2, 3, 6, 7). The effect of ligand concentration [5000-fold range of AcCho and 200-fold range of carbamoylcholine (CbmCho)] (2-4) on the four phases could be predicted over the entire concentration range for each ligand we investigated, and all the rate and equilibrium constants pertaining to a minimum model (Fig. LA) that relates the ligand-binding steps to ion translocation were determined (6-10).AcChoR-controlled ion translocation has been studied by using vesicles prepared from the electroplax of Torpedo (11-16). Torpedo vesicles may contain, on a weight basis, several hundred times more AcChoR than those from E. electricus, and the maximum influx rates had to be measured indirectly. Published reports differ on several important properties of the Torpedo AcChoR: maximal influx rates, the number of ions transported per AcChoR-formed channel per unit time, and the Hill coefficient (12, 16). Most strikingly, the rate of the CbmCho-induced inactivation of the AcChoR (17) appeared to be about 100 times slower than that observed with E. electricus (11,12). Here we mention some of the problems that have not been considered in previous studies using Torpedo vesicles and may account for the d...

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A simple quantitative assay of 125I-labeled α-bungarotoxin binding to soluble and membrane-bound acetylcholine receptor protein

Cited by 27 publications

References 13 publications

Testosterone increases acetylcholine receptor number in the “levator ani” muscle of the rat

Testosterone increases acetylcholine receptor number in the “levator ani” muscle of the rat

Specific reaction rate of acetylcholine receptor-controlled ion translocation: a comparison of measurements with membrane vesicles and with muscle cells.

Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo californica and Electrophorus electricus : Chemical kinetic measurements in the millisecond region

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