Isolation and Characterization of a Lipid-Embedded Domain of the Acetylcholine Receptor from Torpedo calforrzica

A lipid-embedded domain of the acetylcholine receptor isolated from acetylcholine-rich membrane fragments from the electric organ of Torpedo californica and identified by photolabeling with the lipid-soluble reagent 5-[125I]iodonaphthyl-1 -azide was purified by affinity chromatography and characterized. Four subunits of apparent Mr 40000, 48000, 55000, and 64000 were found after sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified material. Exhaustive trypsinization of the purified acetylcholine receptor yielded two domains : an unlabeled 27000-Mr segment and labeled 13000-Mr lipid-embedded polypeptide(s) which were separable by rechromatography with Sepharose-bound Naju nuju siumensis neurotoxin. Trypsin was separated from the 13000-dalton polypeptide(s) by gel filtration on CMC-Sephadex G-50 or by affinity chromatography with Sepharose-bound soybean trypsin inhibitor. The 13000-dalton fragment behaved as a proteolipid upon butanol extraction and ether precipitation. The ether precipitate, which had a relatively hydrophobic amino acid composition resembling the original protein, yielded only one labeled polypeptide band upon electrophoresis. Immunological studies showed that the lipidembedded domain of the acetylcholine receptor was strongly bound to anti-(denatured receptor) antibodies which are essentially directed against sequential antigenic determinants. Furthermore, antibodies made in rabbits against this lipid-embedded domain recognized the membrane-bound receptor, the detergent (Triton X-100)isolated receptor, and the modified receptor. The availability of this purified lipid-embedded segment of the acetylcholine receptor permits both the determination of its sequence and the study of the cross-reactivity of antibodies specific for this domain with other ionophoric proteins.