Investigations on the Acetylcholine Receptor

Investigations directed toward isolation of the acetylcholine receptor demonstrated that low molecular weight cholinergic ligands lack sufficient specificity for use as receptor labels. However, a-bungarotoxin, a neurotoxin from B. multicinctus, proved to have the desired characteristics of specificity and irreversibility of binding. Chemical and physiological characterization of the purified toxin revealed that a homogeneous species of ~8000 MW was obtained, and that the iodinated derivative was physiologically and antigenically identical to the native protein. The radioiodide-labeled toxin was used as a specific, irreversible label and showed that the membrane of E. electricus electric tissue contained a unique toxin-binding substance which could be extracted by Triton X-100. The toxin complex had a molecular weight of ~2.5 x 105 by density gradient centrifugation and a pI ~ 5.1; uniqueness was suggested by the symmetry of the electrofocused peak. The toxin-binding component was enzymatically and chemically characterized as a protein with a membrane-supplied phospholipid requirement which could also be supplied by Triton X-100 when the latter was used to solubilize the protein. PCMB treatment of the Triton extract suggested involvement of a sulfhydryl group in toxin binding; this group was insensitive to mild oxidation.