Naloxone‐sensitive, haloperidol‐sensitive, [3H] (+) SKF‐10047‐binding protein partially purified from rat liver and rat brain membranes: An opioid/sigma receptor?

A naloxone‐sensitive, haloperidol‐sensitive, [3H](+)SKF‐10047‐binding protein was partially purified from rat liver and rat brain membranes in an affinity chromatography originally designed to purify sigma receptors. Detergent‐solubilized extracts from membranes were adsorbed to Sephadex G‐25 resin containing an affinity ligand for sigma receptors: N‐(2‐[3,4‐dichlorophenyl]ethyl)‐N‐(6‐aminohexyl)‐(2‐[1‐pyrrolidinyl])ethylamine (DAPE). After eluting the resin with haloperidol, a protein that bound [3H](+)SKF‐10047 was detected in the eluates. However, the protein was not the sigma receptor. [3H](+)SKF‐10047 binding to the protein was inhibited by the following compounds in the order of decreasing potency: (+)pentazocine > (−) pentazocine > (±)cyclazocine > (−)morphine > (−)naloxone > haloperidol > (+)SKF‐10047 > DADLE > (−)SKF‐10047. Further, the prototypic sigma receptor ligands, such as 1,3‐di‐o‐tolylguanidine (DTG),(+)3‐PPP, and progesterone, bound poorly to the protein. Tryptic digestion and heat treatment of the affinity‐purified protein abolished radioligand binding. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of the partially‐purified protein from the liver revealed a major diffuse band with a molecular mass of 31 kDa, a polypeptide of 65 kDa, and another polypeptide of > 97 kDa. This study demonstrates the existence of a novel protein in the rat liver and rat brain which binds opioids, benzomorphans, and haloperidol with namomolar affinity. The protein resembles the opioid/sigma receptor originally proposed by Martin et al. [(1976): J. Pharmacol. Exp. Ther., 197:517–532.]. A high degree of purification of this protein has been achieved in the present study. Synapse 25:117–124, 1997. © 1997 Wiley‐Liss, Inc. This article is a US Government work and, as such is in the public domain in the United States of America.