Solubilization and purification of opioid receptor molecules of rat brain

A P2 membrane preparation of rat brain (without cerebellum) was solubilized with CHAPS in Tris containing DTT and trypsin inhibitor. Two opiate ligands, 10b and 10cd, prepared by Liu et al, were employed consecutively in affinity chromatography, from which OPR's were eluted with Nx. The eluate was subsequently passed through a WGA affinity column and the OPR's eluted with N-GluNAc. This eluate was further purified and concentrated by preparative granular gel isoelectric focusing on SG200. Two protein peaks appeared separately at pH 5 and 7.8. The eluates from both peaks were examined for protein contents using the silver staining method, and binding activity was measured by RRA with 3H-etor. The results revealed that both samples contained active OPR purified to over 80,000 fold. The Mr was estimated by gel filtration to be 52 kD and 42 kD for OPR in the pH 5 and pH 7.8 samples respectively. OPR in the pH 5 sample have been determined to be of mu-type by their binding activity with 3H-ohm.