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Solubilization of High‐Affinity, Guanine Nucleotide‐Sensitive μ‐Opioid Receptors from Rat Brain Membranes
Author(s) -
Weems Henri B.,
ChaleckaFranaszek Elzbieta,
Côté Thomas E.
Publication year - 1996
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 - 10.1046/j.1471-4159.1996.66031042.x
Subject(s) - diprenorphine , damgo , chemistry , agonist , enkephalin , opioid receptor , receptor , opioid , (+) naloxone , stereochemistry , biochemistry
High‐affinity μ‐opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane‐associated receptors with morphine during solubilization in the detergent 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate appeared to stabilize the μ‐opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate concentration to 3 m M , the solubilized opioid receptor bound [ 3 H][ d ‐Ala 2 , N ‐Me‐Phe 4 ,Gly‐ol 5 ]‐enkephalin ([ 3 H]DAMGO), a μ‐selective opioid agonist, with high affinity ( K D = 1.90 ± 0.93 n M; B max = 629 ± 162 fmol/mg of protein). Of the membrane‐associated [ 3 H]‐DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [ 3 H]DAMGO binding was completely abolished in the presence of 10 µ M guanosine 5′‐ O ‐(3‐thiotriphosphate). The solubilized receptor also bound [ 3 H]diprenorphine, a nonselective opioid antagonist, with high affinity ( K D = 1.4 ± 0.39 n M, B max = 920 ± 154 fmol/mg of protein). Guanosine 5′‐ O ‐(3‐thiotriphosphate) did not diminish [ 3 H]diprenorphine binding. DAMGO at concentrations between 1 n M and 1 µ M competed with [ 3 H]diprenorphine for the solubilized binding sites; in contrast, [ d ‐Pen 2 , d ‐Pen 5 ]‐enkephalin, a δ‐selective opioid agonist, and U50488H, a κ‐selective opioid agonist, failed to compete with [ 3 H]diprenorphine for the solubilized binding sites at concentrations of <1 µ M . In the absence of guanine nucleotides, the DAMGO displacement curve for [ 3 H]diprenorphine binding sites better fit a two‐site than a one‐site model with K Dhigh = 2.17 ± 1.5 n M, B max = 648 ± 110 fmol/mg of protein and K Dlow = 468 ± 63 n M, B max = 253 ± 84 fmol/mg of protein. In the presence of 10 µ M guanosine 5′‐ O ‐(3‐thiotriphosphate), the DAMGO displacement curve better fit a one‐ than a two‐site model with K D = 815 ± 33 n M, B max = 965 ± 124 fmol/mg of protein.