Purification by Affinity Chromatography

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutininSepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethano1 revealed three glycine receptor-associated polypeptides of M, = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the M, = 48,000 polypeptide upon W-illumination. The dissociation constant (&) of [3H]strychnine binding to the purified glycine receptor was 9.3 f 0.6 n” The glycine receptor agonists glycine, /3-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/HzO and sucrose/DzO gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 f 0.005 ml/g and a sedimentation coefficient S ~ O , ~ of 8.2 f 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 & 6,000 was calculated for the glycine receptor protein.