Isolation of opiate binding components by affinity chromatography and reconstitution of binding activities.

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Abstract
Rat brain membranes exhibiting stereospecific opiate binding activity were solubilized by sonication and detergent treatment. The active material could be bound to an affinity column containing 6-succinylmorphine but could not be eluted with free agonist. Although 2 protein peaks could be eluted with NaCl, neither possessed binding activity; 1 of the peaks (A) in combination with an acidic lipid fraction, eluted subsequently from the column, showed stereospecific binding. Opiate ligands [morphine, etorphine, diprenorphine, buprenorphine, naloxone, naltrexone, nalbuphine, nalorphine] of the .mu. type bound to this protein/lipid mixture with an order of affinities closely correlating with those of the original membrane but 1 to 2 orders of magnitude lower; binding of .delta., .kappa. and .sigma. prototype opioids was considerably less. The protein/lipid mixture also competed with the membranes for .mu. ligands. The isolated protein-lipid complex may be a component of the opiate receptor and, specifically, the .mu. receptor or binding site. Because of the lower affinities of .mu. opiates for this complex, some essential membrane component still may be missing. Preliminary analysis of peak A indicates that it contains a broad spectrum of protein bands, but it remains to be seen which of these are essential for activity.