Purification by affinity chromatography of the glycine receptor of rat spinal cord.
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 agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 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.
D000345:Affinity Labels; D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D018009:Receptors, Glycine; D013116:Spinal Cord / Q000032:analysis*; D013331:Strychnine / Q000378:metabolism
D000345:Affinity Labels; D011956:Receptors, Cell Surface; D018009:Receptors, Glycine; D013331:Strychnine