Affinity labeling of mu and kappa receptors with naloxone benzoylhydrazone

KM Standifer, LR Murthy, K Kinouchi, L Steele and GW Pasternak

George C. Cotzias Laboratory of Neuro-Oncology, Memorial Sloan- Kettering Cancer Center, New York, New York 10021.

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]NalBzoH 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 degrees 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.

Volume 39, Issue 3, pp. 290-298, 03/01/1991
Copyright © 1991 by American Society for Pharmacology and Experimental Therapeutics