Affinity labeling and purification of the opiate receptor from rat brain

Affinity labeling of the opiate receptor has been performed on neural membranes from rat brain utilizing[125I]-14-bromoacetamidomorphine, an opiate agonist, and [125I]-14-chloracetylmorphine, an antagonist. With the use of SDS gel electrophoresis it could be shown that the agonist labeled three proteins with molecular weights of 43,000, 35,000 and 23,000, whereas the antagonist only labeled the 23,000 component. The preferential labeling of the 23,000 protein by the antagonist suggests that this component may be a primary recognition site for opiate antagonists. Calcium was stimulatory to the affinity labeling of all three proteins while sodium was inhibitory. With the use of affinity columns prepared by conjugating either ligand to omega-aminohexyl Sepharose, a receptor complex was obtained consisting all three proteins. Stereospecific opiate binding was demonstrable in the complex prepared from either column.