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  • Title: Photoaffinity labeling of opiate receptors using intrinsically photoactive 3H-opiates.
    Author: Kooper GN, Levinson NR, Copeland CF, Bowen WD.
    Journal: Mol Pharmacol; 1988 Mar; 33(3):316-26. PubMed ID: 2832721.
    Abstract:
    Opiate receptors in rat and cow brain membranes have been labeled irreversibly using the intrinsic photolability of 3H-opiates. Membranes were incubated with 3H-ligand and then irradiated with UV light of 254 nm. Nonspecific binding was determined in the presence of 10 microM unlabeled levallorphan. Irreversible binding was defined as binding which survived heat or acid denaturation of membranes. Specific incorporation of label into denatured samples was observed only when unbound or loosely bound 3H-ligand was washed free from the membranes prior to irradiation. There was a general correlation between photosensitivity of the 3H-ligand and its ability to photolabel receptors. Hence, photolabeling presumably results by covalent attachment of highly reactive species generated during photochemical decomposition of ligand. With 3H-etorphine, optimal irradiation time was 5 min. In addition to 3H-etorphine, receptors could be labeled irreversibly with 3H-oxymorphone, 3H-dihydromorphine, and 3H-ethylketocyclazocine. Of the specific binding present in irradiated, nondenatured samples, 45-60% remained attached to receptors upon denaturation. 3H-Ethylketocyclazocine exhibited an 86% yield of incorporation. Signal-to-noise levels of 50-80% could be achieved in denatured samples. Therefore, this method provides a means of covalently labeling opiate receptors in high yield and with high signal-to-noise ratios. The opioid peptides, 3-H-D-Ala2,D-Leu5-enkephalin, 3H-D-Ser2,Leu5,Thr6-enkephalin, 3H-D-Ala2,Met5-enkephalin amide, and 3H-D-Ala2,N-MePhe4,Gly-ol5-enkephalin, as well as the benzomorphan, 3H-bremazocine, apparently lack the structural characteristics which allow photolabeling. 3H-Etorphine was incorporated at high specific activity into a glycoprotein fraction of bovine brain membranes which was retained on wheat germ agglutinin affinity columns. Unretained proteins were essentially unlabeled. 3H-Etorphine was also incorporated at high specific activity into proteins of 17,000 and less than 13,700 Da and, at lower specific activity, into a protein of 2.4 X 10(6) Da in guinea pig brain membranes when kappa receptors were selectively labeled. This method therefore allows labeling of specific membrane proteins and will prove useful in molecular characterization of opiate receptors.
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