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Solubilization of High‐Affinity, Guanine Nucjeotide‐Sensitive μ‐Opioid Receptors from 7315c Cell Membranes
Author(s) -
Cote Thomas E.,
Gosse Marilyn E.,
Weems Henri B.
Publication year - 1993
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1993.tb03610.x
Subject(s) - membrane , solubilization , guanine , receptor , chemistry , cell membrane , biophysics , biochemistry , biology , nucleotide , gene
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 [ 3 H]‐etorphine with high affinity (K D = 0.304 ± 0.06 n M ; B max = 154 ± 33 fmol/mg of protein). Of the membrane‐associated [ 3 H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ‐selective and non‐selective enkephalins competed with [ 3 H]etorphine for the solubilized binding sites; in contrast, 5‐ and K ‐opioid enkephalins failed to compete with [ 3 H]etorphine for the solubilized binding sites at concentrations of <1 μ M. The μ‐selective ligand [ 3 H][D‐Ala 2 ,A/‐Me‐Phe 4 ,Gly 5 ‐ol]enkephalin also bound with high affinity (K D = 0.79 rM; B max = 108±17 fmol/mg of protein) to the solubilized material. Of the membrane‐associated [ 3 H][D‐Ala 2 , N ‐Me‐Phe 4 ,Gly 5 ‐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 [ 3 H][ D ‐Ala 2 , N ‐Me‐Phe 4 ,Gly 5 ‐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.