Affinity Labeling of the Acetylcholine Receptor in the Electroplax: Electrophoretic Separation in Sodium Dodecyl Sulfate

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Electroplax, single cells dissected from electric tissue of Electrophorus, are labeled in a two-step procedure: reduction by dithiothreitol followed by alkylation by the affinity label 4-(N-maleimido)-α-benzyltri-[methyl-3H]methylammonium iodide, either alone or in combination with [2,3-14C]N-ethylmaleimide. Electrophoresis in sodium dodecyl sulfate on polyacrylamide gel of an extract, prepared with this detergent, of single-labeled or of double-labeled cells results in a major peak of 3H activity, with a mobility corresponding to a polypeptide of molecular weight 42,000. In addition, in the double-labeled samples, there is a unique peak in the ratio of 3H to 14C that is coincident with the 3H peak. The electrophoretic patterns of extracts of cells in which affinity alkylation of the reduced receptor has been suppressed by dithiobischoline, an affinity oxidizing agent, by cobratoxin, an irreversible ligand, or by hexamethonium, a reversible ligand, show a considerably diminished peak of 3H activity in the region of molecular weight 42,000. This is the predominant difference between the electrophoretic patterns of extracts of unprotected and of protected cells. Furthermore, extracts of cells protected with dithiobischoline before labeling with both tritiated affinity label and [14C]N-ethylmaleimide do not show the peak in the 3H to 14C ratio seen in the absence of protection. Thus, by several diverse criteria, the peak of 3H activity corresponding to a molecular weight of 42,000 contains affinity-labeled acetylcholine receptor or receptor subunit.

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