Affinity labeling of mu and kappa receptors with naloxone benzoylhydrazone
Standifer, K.M.; Murthy, L.R.; Kinouchi, K.; Steele, L.; Pasternak, G.W.
Molecular Pharmacology 39(3): 290-298
1991
ISSN/ISBN: 0026-895X PMID: 1848652 Document Number: 376309
Naloxone benzoylhydrazone (NalBzoH) labels both .mu. and .kappa. receptors in standard homogenate binding assays. We now report that [3H]NalBzoH can effectively photoaffinity label opioid receptors. By modifying [3H]NaBzoH binding conditions, we can selectively label either .mu. or .kappa.3 receptors in calf striatal membranes or classical U50,488-sensitive .kappa.1 receptors in guinea pig cerebellar membranes. After removal of unbound radioligand, the [3H]NalBzoH-labeled membranes were irradiated with UV light to couple the bound radioligand to its binding site. No specific .mu., .kappa.1, or .kappa.3 binding remained after a 20-hr dissociation at 25.degree. without UV irradiation. In contrast, approximately 45% of .mu. and 40% of .kappa.1 and .kappa.3 binding remained after 2 min of UV exposure. In time course studies, increasing the UV exposure from 30 sec to 3 min produced a progressive increase in radioligand incorporation, which did not increase further with UV exposure times up to 5 min. A portion of the binding in irradiated membranes appeared to be covalently coupled to proteins. Following solubilization of irradiated membranes with sodium dodecyl sulfate, approximately 30-40% of the specifically bound radioactivity precipitated with trichloroacetic acid (TCA). These levels of TCA-precipitable binding corresponded to the amount of dissociation-resistant binding described above. No specific binding could be TCA precipitated in samples that were not exposed to UV. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]NalBzoH-labeled membranes revealed a number of labeled peaks with levels of radioactivity that did not correspond to the intensity of the protein bands, suggesting that this technique was not randomly labeling proteins. This approach may be a useful method to affinity label, characterize, and purify .mu. and .kappa. opiate receptor subtypes.