The acetylcholine receptor from Torpedo californicu electric tissue consisting of polypeptide chains of molecular weight 42 000 (k 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270 000 ( 2 30 000). Besides the receptor subunit (a-chain), at least three further classes of polypeptide chains are part of the complex : ( M , 48 000), y ( M , 62 000) and 6 ( M , 68 000).This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent : From the 270 000 molecular weight particle the four predominant polypeptide chains of the membrane, a, p, y, and 6, can be obtained.The &polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.The nicotinic acetylcholine receptor protein is believed to be involved in the ion permeability changes of the postsynaptic membrane of cholinergic synapses induced by the transmitter acetylcholine. Elucidation of the mechanism of the events in the membrane during opening and closing of ion gates is expected from the analysis of the structure and function of the molecules comprising the membrane. Relatively few protein species appear to be present. Their intensive investigation in recent years has provided a large amount of information (for a collection of recent reviews see [l]), but most of the basic questions remain unanswered. It is still unknown if the receptor, i.e. the transmitterbinding protein, is simultaneously the ion pore and if its conformation determines the ion permeability of the membrane. Most laboratories tackled this question by means of reconstitution experiments with the purified binding protein [2-51 but the results are equivocal. Another approach is to investigate the function of the various components by means of affinity labeling [6,7]. This approach has shown that not all components contain binding sites for cholinergic ligands. A polypeptide chain of molecular weight 40 000 contains the binding sites for compounds with quaternary ammonium groups [6] and for a-neurotoxin from cobra venom [7]. This polypeptide chain appears to represent A preliminary communication has been prescnted at a meeting of the Gesell,sc.hqft ,fur Biologische Chemir [34]. the acetylcholine receptor, but its relationship to the other chains detectable in the membrane remains unclear.The acetylcholine receptor from the electric organ of Electroplzorus electricus or Torpedo culifornicu appears to be part of an oligomeric protein complex of several different polypeptide chains [7 -141. After extraction with non-ionic detergents and purification by affinity chromatography, the protein from Electrophorus has been shown to be possibly a pentamer of two different polypeptide chains [9]. Other authors obtained preparations with three different chains [16]. For the protein from Torpedo, one [ 15, 171, two [ 1 1 , 181, three [ 191, and four [ l6,20,7] different polypeptide chains hav...

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...

A novel rapid purification method for the nicotinic acetylcholine receptor from Torpedo electric tissue was developed.It allows preparation of 10 mg quantities of pure and stabile receptor protein within 2 days. This protein is used for crystallization attempts. Conditions are described which reproducibly yield crystals.

The nicotinic acetylcholine receptor (nAChR) from Torpedo californica and T. marmorata electric tissue polymerises irreversibly when DTE and CaZ+ are added to receptor-rich membranes. The polymerisation is time-dependent and complete within 3 h at 30°C. It can be completely prevented by EGTA or the transglutaminase inhibitor cystamine. Transglutaminase activity can also be monitored with the exogenous substrates [3H]putrescine and dimethylcasein. This assay can also be inhibited by EGTA or cystamine.Nicotinic acetylcholine receptor Ca2'

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