Purification and characterization of the glycine receptor of pig spinal cord.
A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.
Graham
D
D
Pfeiffer
F
F
Simler
R
R
Betz
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D000939:Epitopes / Q000032:analysis; D005998:Glycine / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D018009:Receptors, Glycine; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism*; D013331:Strychnine / Q000378:metabolism; D013552:Swine; D014316:Tritium
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D018009:Receptors, Glycine; D017981:Receptors, Neurotransmitter; D014316:Tritium; D013331:Strychnine; D005998:Glycine