This 5-year renewal application is directed at using 14beta-(bromoacetamido)-7,8-dihydromorphine (H2BAM) to affinity label and purify the mu opioid receptor. In the absence of a disulfide bond reducing reagent, this affinity ligand bound reversibly to the mu opioid binding site in rat brain membranes. However, after reduction of a disulfide bond at the mu opioid binding site, H2BAM bound irreversibly to this binding site, as determined by the inhibition of opioid binding to the mu site, but not to the delta or kappa site. Protection experiments demonstrated that only opioids that bind to the mu opioid binding site were able to block the alkylation of this site. [3H]H2BAM was synthesized to a high specific activity. The affinity and specificity of [3H]H2BAM to bind reversibly and irreversibly to the mu opioid binding site in rat and bovine brain membranes will be determined by radioreceptor binding assays in the presence and absence of the disulfide bond reducing reagent dithiothreitol. Experimental conditions will be optimized for the specific alkylation of the mu opioid binding site with [3H]H2BAM. The molecular weight of protein(s) specifically labeled with [3H]H2BAM will be determined by separating [3H]H2BAM-labeled membranes on polyacrylamide gels under denaturing and reducing conditions, followed by fluorography. Specificity studies will determine which opioids can block the irreversible labeling of specific protein(s). The possibility of multiple mu opioid binding sites will be examined. Cell lines, specific brain regions, and other tissues will be tested to further characterize the specificity of [3H]H2BAM alkylation of membranes. These studies will result in a profile of the affinity labeling of the mu opioid binding site. Proteins, specifically labeled with [3H]H2BAM, will be purified from bovine striatal membranes under denaturing conditions, including affinity chromatography, high pressure liquid chromatography (HPLC), using ion exchange and molecular sieve columns, and gel electrophoresis. Peptide fragments from the purified [3H]H2BAM-labeled protein will be generated by enzyme or chemical digestion. Peptides will purified by reverse-phase HPLC, and partial amino acid sequences of the peptides will be determined. Based on the amino acid sequences, oligonucleotide probes will be synthesized, and used to screen cDNA libraries, in order to ultimately clone the mu opioid receptor.
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