Isolation and Characterization of a Lipid-Embedded Domain of the Acetylcholine Receptor from <em>Torpedo californica</em>.

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 Naja naja siamensis 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 lipid-embedded 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 anti-bodies specific for this domain with other ionophoric proteins. [ABSTRACT FROM AUTHOR]