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  • Title: Ligand-induced variations in the reactivity of thio groups of the alpha-subunit of the acetylcholine receptor from Torpedo californica.
    Author: de Souza Otero A, Hamilton SL.
    Journal: Biochemistry; 1984 May 22; 23(11):2321-8. PubMed ID: 6477870.
    Abstract:
    We have studied alkylation of the acetylcholine receptor by N-[3H]ethylmaleimide ([3H]NEM) under various conditions. The radiolabeled preparations were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the receptor complex into subunits, and the incorporation of 3H into each type of chain was determined. We found the following: (i) When cysteines of native receptor in intact membranes were reacted with [3H]NEM, only the beta-subunit was labeled; the extent of alkylation did not change significantly if cholinergic effectors were present during this reaction. (ii) When the disulfide bonds of the receptor were reduced with dithiothreitol (DTT), the alpha- and beta-chains were labeled with [3H]NEM. The presence of receptor agonists and competitive antagonists during alkylation significantly altered the labeling patterns. Gallamine and hexamethonium markedly enhanced, while carbamylcholine and decamethonium markedly lessened, labeling of the alpha-subunit. Choline, d-tubocurarine, and alpha-neurotoxin induced small, but significant decreases in alkylation of the alpha-subunit, while procaine had no effect. (iii) When the same ligands were present during the reduction step, subsequent labeling with [3H]NEM produced patterns similar to those described in (ii). We also investigated the effects of gallamine and hexamethonium on reduction of the disulfide bond located near the acetylcholine binding site by using the affinity alkylating reagent (bromoacetyl)choline (BAC). Gallamine (0.1 mM) was able to increase the rate of reduction of this particular disulfide bond 3-fold in comparison to the control. In these experiments, alkylation by BAC blocked 50% of the toxin binding sites. Hexamethonium (1 mM) had a similar effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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