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

Reed, K.; Vandlen, Richard; Bode, Jürgen; Duguid, John R.; Raftery, Michael A.

doi:10.1016/0003-9861(75)90449-x

Cited by 91 publications

(32 citation statements)

References 14 publications

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“…After they were killed, the electric organs were excised, frozen in liquid nitrogen, and then stored at -90'C until use. AcChoR-enriched membrane fragments were prepared as described (2,3) or by a modification of these methods. In the latter case, centrifugation on a discontinuous sucrose gradient in a Beckman VTi-50 reorienting tube rotor replaced the zonal centrifugation step.…”

Section: Methodsmentioning

confidence: 99%

Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.

Blanchard¹,

Raftery²

1979

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

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Procaine amide azide, a derivative of the local anesthetic procaine amide, was prepared and its interactions with acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax were studied. Procaine amide azide was radiolabeled by a method that may be of general use for the preparation of other radioactive tertiary amines. When low concentrations of 3H-labeled procaine amide azide were photolyzed in the presence of receptor-rich membrane preparations, a simple pattern of incorrated radioactivity was seen after electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. Only two major labeled bands were seen, corresponding to apparent M, of about 43,000 and 90,000. When the length of the gels was increased, the labeled band of lower Mr was resolved into two labeled proteins, one major and one minor, with apparent M, of 43,000 and 40,000, respectively. The radioactivity incorporated into the protein of M, 40,000 could be attributed to interaction of [3H]procaine amide azide with cho-

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Section: Methodsmentioning

confidence: 99%

Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.

Blanchard¹,

Raftery²

1979

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

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“…If the receptor-rich membrane fragments prepared as described (15,8) originate unaltered from the postsynaptic membrane, the structure of this membrane appears to be relatively simple. Possibly only four different types of polypeptide chains comprise its integral proteins and are responsible for the functions of the membrane, e.g., transmitter binding and ion translocation.…”

Section: Discussionmentioning

confidence: 99%

“…In accordance with ref. 8, the NaDodSO4 gel ( Fig. la) revealed four components which we name (bottom to top) a, ,B, y, and 6.…”

Section: Methodsmentioning

confidence: 99%

“…The protein extracted from the electric tissue of Electrophorus electricus has been shown to be an oligomer (1-5), possibly a pentamer (2) consisting of two different types of polypeptide chains. Two to four different polypeptide chains have been reported for receptor purified from Torpedo californica (6)(7)(8). The function of the different chains is unclear, because only one of them binds the neurotoxin from Naja naja siamensis (9).…”

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confidence: 99%

“…It has been shown by electron microscopy of negative stained and freeze-etched membranes that the postsynaptic membrane contains doughnut-like particles believed to represent receptor molecules (13,14). Receptor-rich membrane fragments can be purified (15,7,8), revealing a lattice organization of similar particles and containing acetylcholine receptor up to 20-40% of the total protein (14). By refining the purification procedures and by analyzing the receptor-rich membrane fragments we come to the conclusion that possibly up to 75% of the total protein is receptor protein and that the structure of the membrane is relatively simple.…”

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confidence: 99%

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Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica.

Hucho¹,

Layer²,

Kiefer³

et al. 1976

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

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Membrane fragments from electric tissue of Torpedo californica containing nicotinic acetylcholine receptor are composed of four different polypeptide chains with molecular weights of 40,000 (a), 48,000 (a), 62,000 ('y), and 66,000 (6). The a and a chains are still present in all and y ahd 6 in some of the receptor preparations after Triton X-100 extraction and purification by affinity chromatography. All components of the receptor react covalently with the photoaffinity label 4-azido-2-nitrobenzyltrimethylammonium fluoroborate, the 6 chain incorporating less of the reagent as compared to the a and # chains. Agonists and antagonists containing a quaternary ammonium group protect all chains against the label; the principal neurotoxin from Naja naja siamensis protects the a chain only. We conclude that the a chain binds the neurotoxin from Naja naja, the a and fi chains are involved in the binding of ligands with quaternary ammonium groups, and the function of the y and 6 chains remains to be determined. The quaternary structure of the nicotinic acetylcholine receptor appears to be complex. The protein extracted from the electric tissue of Electrophorus electricus has been shown to be an oligomer (1-5), possibly a pentamer (2) consisting of two different types of polypeptide chains. Two to four different polypeptide chains have been reported for receptor purified from Torpedo californica (6)(7)(8). The function of the different chains is unclear, because only one of them binds the neurotoxin from Naja naja siamensis (9). After reduction with dithiothreitol only the component having a molecular weight of 38,000 binds the affinity label 4-(N-maleimido)benzyltri[3H]methylammonium iodide (6, 10). In this paper experiments with the photoaffinity label 4-azido-2-nitrobenzyltrimethylammonium fluoroborate are reported. This arylazide inactivates membrane-bound erythrocyte acetylcholine esterase and frog satorius muscle acetylcholine receptor after irradiation (11). Since nonspecific reaction with other proteins of the membrane occurred and could not be selectively prevented by appropriate nitrene scavengers, it was concluded that the reaction might not represent a true photoaffinity labeling but an ordinary affinity labeling by a long-lived intermediate generated by photolysis (12). In our experiments reported here with detergent-solubilized and purified receptor the arylazide turned out to be a valuable tool for the investigation of the receptor structure.A central question remains: what is the structure of the acetylcholine receptor in the postsynaptic membrane? Does the molecule extracted by detergent and purified by affinity chromatography represent the true entity or is it a preparative artifact? Finally, what is the structure of the functional postsynaptic membrane? It has been shown by electron microscopy of negative stained and freeze-etched membranes that the postsynaptic membrane contains doughnut-like particles believed to represent receptor molecules (13,14). Receptor-rich membrane fragments can be puri...

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A monoclonal anti‐idiotypic antibody against anti‐receptor antibodies from myasthenic sera

Lefvert¹,

James

Alliod

et al. 1982

Eur J Immunol

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

Cited by 91 publications

References 14 publications

Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.

Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.

Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica.

A monoclonal anti‐idiotypic antibody against anti‐receptor antibodies from myasthenic sera

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