Receptor proteins that bind nicotine with high affinity and specificity are found in the brain tissue of humans and experimental animals and may be important biochemical mediators of nicotine dependence in tobacco users. The major goals of this proposal are to use monoclonal anti-idiotypic (anti-id) antibodies as molecular probes to study neuronal nicotinic receptor structure and function, and to determine the relationship between cell receptors and active site structures of enzymes involved in nicotine metabolism. The anti-id will be used in immunohistochemical and radioimmunological methods to study the localization and expression of receptors on rat and human brain, and on cultured primary fetal rat cortical cells and PC12 cells, and in immunoaffinity chromatography to isolate receptor by nicotine displacement for gas-phase sequencing and structural analysis. The anti-id complementarity determining regions (CDRs) also will be sequenced and peptides corresponding to the antigen recognition sites synthesized and tested along with the intact anti-id and their F(ab')2 and Fab fragments for their ability to mimic nicotine binding to the receptors and to modulate Ca++ influx into the rat neuronal cells as an expression of nicotine-like functional activity. Computer molecular modeling incorporating id, anti-id, and receptor sequence data and ligand binding specificity, along with similar data for CDR-related peptides will be used to develop a model of the various ligand-acceptor complexes based on energy minimization and molecular dynamics routines. For receptor site mapping, the primary structures and receptor binding activity of the id and anti-id CDR peptides will be compared with binding site sequences derived from immunoaffinity purified or gene-cloned rat and human receptors, and site-directed mutagenesis will be used to make specific amino acid substitutions in the receptor combining site to determine structural requirements for ligand binding. These experimental results will be compared with computer predicted optimal structures for ligand-antibody and ligand-receptor complexes. In similar studies, the ability of anti- nicotine anti-id and anti-id prepared from antibodies to a major metabolite, cotinine, will be used to derive peptide mimics of their respective ligands as hepatic and adrenal cytochrome P-450 inhibitors on the hypothesis that antibody, cell receptor, and enzyme active sites are structurally interrelated through """"""""internal image"""""""" anti-id determinants.
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