Isolation of a nicotine binding site from rat brain by affinity chromatography.

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Abstract
With the use of affinity chromatography, a [3H]-nicotine binding site was purified almost 1000-fold from a Triton X-100-solubilized extract of rat brain neural membranes. The affinity column was prepared by conjugation of (R,S)-6-(2-hydroxyethyl)nicotine to epoxy-activated Sepharose. Further purification of the material from the affinity column was resolved by using another column of the same affinity gel, resulting in the isolation of a major protein (about 95% purity) that had a MW 56,000, as determined by NaDodSO4 [sodium dodecyl sulfate]/polyacrylamide gel electrophoresis, with very minor components ranging in MW from 47,00-83,000. With various nicotine analogs, the purified material exhibited nearly identical binding characteristics to rat brain membrane preparations, including stereoselectivity for the nicotine enantiomers. The Kd of the purified site, 3.5 .times. 10-9 M, was similar to that observed with membrane and Triton X-100-soluble preparations, whereas the binding capacity was > 25 pmol/mg of protein, as compared to 0.07 pmol/mg of protein in the starting material. The results are discussed in relation to the purified nicotinic cholinergic receptor from electroplax. Evidently the nicotine site in rat brain was different from the cholinergic receptor of electroplax or calf skeletal muscle.