Affinity labeling of the acetylcholine receptor in the electroplax

The receptor for acetylcholine in the electroplax of Electrophorus electricus is affinity labeled in situ in a two-step process consisting of reduction by dithiothreitol followed by alkylation with tritiated 4-(N-maleimido)-α-benzyltrimethylammionium iodide. From the effect of these and other reagents on the response of the electroplax to cholinergic agents it has been inferred that dithiothreitol reduces an S-S group in the vicinity of the acetylcholine binding site on the receptor and that MBTA alkylates one of the SH-groups thus produced three orders of magnitude faster than other SH groups present. Furthermore, the alkylation by MBTA is specifically blocked either by prior affinity oxidation of the reduced receptor with dithiobischoline or by the simultaneous presence of hexamethonium. Consequently, the portion of the labeling of the dithiothreitol-treated electroplax by [³H]MBTA, which is eliminated by prior treatment with dithiobischoline and by the presence of hexamethonium, is considered to be specific for the receptor. This portion is 10 to 20% of the total labeling. The asymptotic limit of the specific labeling yields an estimate of the quantity of receptor binding sites. The quantity of acetylcholinesterase catalytic sites is fourfold to sevenfold as great. The average surface-density of receptor binding sites in the synaptic area of the electroplax membrane is approximately 3000 sites per μm².