[3H]acetylcholine binding sites in brain. Effect of disulfide bond modification

Nicotinic cholinergic receptor recognition sites have been measured in rat brain using [3H]acetylcholine. Modification of these sites in vitro with the disulfide bond reducing agent, dithiothreitol, resulted in a decrease in the density (Bmax) of [3H]acetylcholine binding sites while the affinity of these sites was unaltered. Reoxidation of the reduced sites with 5,5'-dithiobis(2-nitrobenzoic acid) reversed the effects of dithiothreitol. The reoxidation effect was partially prevented by p-chloromercuribenzoic acid, which generates thiol complexes with exposed sulfhydryl groups. Reduction of disulfide bonds had no effect on the ability of nicotinic cholinergic agonists or antagonists to compete for the remaining [3H]acetylcholine binding sites. In addition, pretreatment of cortical homogenates with acetylcholine or nicotine did not alter the effects of dithiothreitol on [3H] acetylcholine binding, suggesting that the disulfide bonds which are critical for [3H] acetylcholine binding are not located directly at the recognition site.