Purification of acetylcholine receptor from Torpedo californica by affinity chromatography is known to result in the conversion of a majority of the high affinity acetylcholine binding sites, as determined by equilibrium binding assays, to a state of lower affinity. In this report we demonstrate that specific interaction between phospholipids and acetylcholine receptor appears to exist even in 1% nonionic detergent solutions, and we find a definite relationship between the amount of endogenous phospholipid remaining in the purified rcceptor and its retention of high affinity acetylcholine sites. Phospholipase A2 treatment or delipidation of the receptor resulted in the loss of 69% and over 80% of the high affinity sites, respectively. The addition of phosphatidylinositol and, to a lesser extent, phosphatidylcholine to the delipidated receptor results in a partial recovery of the high affinity state. By limiting the extent of delipidation with minimum volumes and concentrations of detergent during purification, the original high affinity sites for acetylcholine in the crude extract could be preserved. The dissociation constant associated with the high affinity sites of the H-form (13S) receptor in such preparations was 4 ± 1 × 10-9 M. Positive cooperativity was indicated in acetylcholine binding and was characterized by a Hill coefficient of about 2.0 for the crude receptor extract, and this coefficient decreased to 1.7 for the purified receptor, suggesting that some structural alteration is still occurring on purification. The amount of phospholipid tightly associated with this purified receptor was 20 ± 2 relative to one α-bungarotoxin site. Receptor purified under these limited delipidation conditions contained 4 to 5 sulfhydryl groups per a-bungarotoxin site. These groups comprised two classes that could be distinguished by their rate of reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. The first class displayed a pseudo-first-order rate constant k1= 2.0 min-1 associated with 2-3 sulfhydryls, while the second class showed a rate constant k2 = 0.52 min-2 associated with 2 sulfhydryls. Delipidated receptor contained only 2 sulfhydryls per α-bungarotoxin site, one from each of the two classes. In the presence of these endogenous phospholipids, receptor sulfhydryl groups appear to be partially protected from intramolecular oxidation. The protection of these sulfhydryl groups presumably minimizes modification of receptor structure that leads to the irreversible loss of the high acetylcholine affinity state. These results suggest that acetylcholine receptor protein requires a highly specific hydrophobic environment, perhaps defined by certain phospholipids, in order to retain native structure. © 1979, American Chemical Society. All rights reserved.
