Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies.
The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [3H]nicotine binding to the purified material. An enantiomeric analogue of nicotine, (-)-6-hydroxymethyl-nicotine, was used to prepare the affinity column. In addition, with the use of anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [3H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of stereospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[3H]nicotine-binding characteristics.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal; D001921:Brain / Q000276:immunology / Q000378:metabolism; D001923:Brain Chemistry; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D009538:Nicotine / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000276:immunology / Q000302:isolation & purification*; D013329:Structure-Activity Relationship
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007130:Immunoglobulin Idiotypes; D011978:Receptors, Nicotinic; D009538:Nicotine