Purification of opiate receptors has lagged behind that of other receptors for neurotransmitters and hormones. Thus, little is known of opiate receptor structure and its relationship to function. We propose to combine a number of approaches in order to achieve purification of kappa opiate receptors. These include affinity chromatographic, affinity labeling, and immunologic techniques. Novel, kappa-selective affinity chromatography matrices will be synthesized by attaching amine derivatives of U50-488H to activated solid supports. These columns will then be used to purify kappa receptors which retain ability to bind opiate agonists and antagonists. These columns will also be used to resolve putative U50-488H-sensitive and U50-488H-insensitive kappa receptor subtypes. Successful separation of these activities would support existence of separate kappa receptor entities. Novel electrophilic and photolabile ligands have been developed for covalent, site-directed labeling of kappa receptor proteins. These affinity probes are also based on the structure of the kappa selective agonist, U50-488H. After covalent attachment of radiolabeled probe, affinity labeled proteins will be purified by chromatographic techniques. In an alternative approach, antibodies will be raised against the appropriate kappa affinity ligand. These antibodies will then be used to isolate the corresponding covalent ligand-receptor complex. Verification that purified affinity labeled proteins are in fact kappa receptors will be obtained using an immunologic approach. Partial N-terminal sequences of the purified complexes will be obtained. Antibodies will then be raised against synthetic peptides corresponding to the sequences. These antibodies will then be tested for ability to recognize active kappa receptors. Cross-reacting antibody would confirm kappa-relatedness of the purified affinity labeled protein. Characteristics of interest in purified kappa receptors are molecular weight, isoelectric point, amino acid composition, peptide map, and sequence. Any sequence information obtained for kappa opiate receptors will be compared to known sequences of other neurotransmitter and hormone receptors.
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