Solubilization of High‐Affinity, Guanine Nucjeotide‐Sensitive μ‐Opioid Receptors from 7315c Cell Membranes

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Abstract
High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [3H]-etorphine with high affinity (KD= 0.304 ± 0.06 nM; Bmax= 154 ± 33 fmol/mg of protein). Of the membrane-associated [3H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of M.The μ-selective ligand [3H][D-Ala2,A/-Me-Phe4,Gly5-ol]enkephalin also bound with high affinity (KD= 0.79 rM; Bmax= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [3H][D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′-O-(3-thiotriphosphate), GTP, and guanosine 5′-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin binding in a concentration-dependent manner. Finally, μ-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate.