enkephalin--ala(2)-mephe(4)-gly(5)- and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

The zwitterionic detergent CHAPS was used to solubilize the human mu-opioid receptor (hMOR) from SH-SY5Y neuroblastoma cells and recombinant hMOR-CHO (CHO-T7-hMOR) and hMOR-SH-SY5Y (SH-SY5Y-T7-hMOR) cell membranes. Agonist stimulation and G-protein activation by the mu-selective opioid agonist DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) were recovered after removing of CHAPS after polyethylene glycol (PEG) precipitation. Binding assays show that hMOR solubilized and reconstituted this way was functional and able to interact with both agonist peptides and with G-protein. The effective solubilization and reconstitution of hMOR from mammalian cells, without truncation and extensive modification, represent an essential step toward the purification of a receptor bearing important post-translational modifications.

Topics: Animals; Cell Line, Tumor; CHO Cells; Cholic Acids; Cricetulus; Detergents; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Narcotic Antagonists; Polyethylene Glycols; Protein Binding; Protein Refolding; Receptors, Opioid, mu; Solubility

High-affinity mu-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 mu-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 mM, the solubilized opioid receptor bound [3H][D-Ala2,N-Me-Phe4,Gly-Dl5]-enkephalin([3H]DAMGO), a mu-selective opioid agonist, with high affinity (KD = 1.90 +/- 0.93 nM; Bmax = 629 +/- 162 fmol/mg of protein). Of the membrane-associated [3H]-DAMGO binding sites, 29 +/- 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 microM guanosine 5'-O-(3-thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 +/- 0.39 nM, Bmax = 920 +/- 154 fmol/mg of protein). Guanosine 5'-O-(3-thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 microM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [D-Pen2, D-Pen5]-enkephalin, a delta-selective opioid agonist, and U50488H, a kappa-selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of < 1 microM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two-site than a one-site model with KDhigh = 2.17 +/- 1.5 nM, Bmax = 648 +/- 110 fmol/mg of protein and KDlow = 468 +/- 63 nM, Bmax = 253 +/- 84 fmol/mg of protein. In the presence of 10 microM guanosine 5'-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with KD = 815 +/- 33 nM, Bmax = 965 +/- 124 fmol/mg of protein.

Topics: Animals; Binding, Competitive; Brain; Cholic Acids; Detergents; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guanine Nucleotides; Membranes; Precipitin Tests; Rats; Rats, Inbred BUF; Receptors, Opioid, mu; Solubility

An opioid binding protein (OBP) purified to homogeneity from bovine striatal membranes has been reconstituted in liposomes. The liposomes were produced by PEG-precipitation of OBP in the presence of a CHAPS extract of bovine striatum, devoid of opioid binding. High affinity mu-agonist binding was restored. The binding was selective for mu-agonists, stereospecific and inhibited by GTP gamma S. These results demonstrate that there is recoupling of OBP with G-protein and confirm our earlier evidence that the purified OBP is a mu-opioid binding site.

Topics: Animals; Binding Sites; Binding, Competitive; Cattle; Cell Membrane; Cholic Acids; Corpus Striatum; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Liposomes; Morphine; Naloxone; Narcotics; Receptors, Opioid, mu

High-affinity mu-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 mu-selective and non-selective enkephalins competed with [3H]etorphine for the solubilized binding sites; in contrast, delta- and kappa-opioid enkephalins failed to compete with [3H]etorphine for the solubilized binding sites at concentrations of < 1 microM. The mu-selective ligand [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin also bound with high affinity (KD = 0.79 nM; 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, mu-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.

Topics: Animals; Binding Sites; Binding, Competitive; Brain; Cell Membrane; Cholic Acids; Detergents; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Etorphine; Guanine Nucleotides; Rats; Receptors, Opioid, mu; Solubility

Opioid receptors were solubilized from bovine striatal membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-(CHAPS). High concentrations of NaCl (0.5-1.0 M) were necessary to ensure optimal yields, which ranged from 40 to 50% of membrane-bound receptors. This requirement was found to be specific for sodium, with only lithium able to substitute partially, as previously reported for solubilization with digitonin. Opioid antagonists, but not agonists, were able to bind to soluble receptors with high affinity. High-affinity binding of mu, delta, and kappa agonists was reconstituted following polyethylene glycol precipitation and resuspension of CHAPS extract. Evidence is presented suggesting that this is the result of inclusion of receptors in liposomes. Competition and saturation studies indicate that the three opioid receptor types retain their selectivity and that they exist in the reconstituted CHAPS extract in a ratio (50:15:35) identical to that in the membranes. In reconstituted CHAPS extract, as in membranes, mu-agonist binding was found to be coupled to a guanine nucleotide binding protein (G protein), as demonstrated by the sensitivity of [3H][D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin ([3H]DAGO) binding to guanosine 5'-O-(thiotriphosphate) (GTP gamma S). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTP gamma S was observed, whereas membrane-bound receptors were uncoupled only partially. Treatment with GTP gamma S, at concentrations that uncoupled the mu receptors almost completely, resulted in a fourfold decrease in the Bmax of [3H]DAGO binding with a relatively small change in the KD. Competition experiments showed that the Ki of DAGO against [3H]bremazocine was increased 200-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Benzomorphans; Binding Sites; Binding, Competitive; Cattle; Cholic Acids; Corpus Striatum; Detergents; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Receptors, Opioid; Solubility

Reduced glutathione (L-gamma-glutamyl-L-cysteinylglycine; GSH) is an endogenous tripeptide involved in the formation and maintenance of protein thiol groups as well as in various detoxification reactions. Because multiple receptor types contain thiol groups or disulfide bridges, effects of GSH treatments on mu-opioid, neurokinin-1/substance P, and kainic acid receptor binding sites were investigated and compared with those produced by dithiothreitol (DTT), a potent synthetic reducing agent. GSH inhibited binding more potently than did DTT at all three receptor types in porcine striatal membrane homogenates as well as in CHAPS-solubilized preparations of the mu and neurokinin-1 sites. GSH-induced inhibitory effects were associated with decreases in maximal binding capacity (Bmax) without significant alteration in apparent affinity (KD). Cysteine, the functional moiety of GSH, mimicked GSH effects albeit with lower potencies, whereas oxidized glutathione had no effects at similar concentrations. In CHAPS-solubilized preparations, the combination of low concentrations of GSH and guanylylimidodiphosphate markedly decreased the Bmax values of the binding of [3H][D-Ala2,Gly-ol5]enkephalin and [3H]substance P. This GSH-mediated mechanism may be important to prevent cell overstimulation by accelerating receptor uncoupling, desensitization, and/or internalization. This is in keeping with purported roles of GSH related to the maintenance of cellular integrity.

Topics: Animals; Binding Sites; Cholic Acids; Detergents; Dithiothreitol; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glutathione; Guanylyl Imidodiphosphate; Kainic Acid; Receptors, Kainic Acid; Receptors, Neurokinin-1; Receptors, Neurotransmitter; Receptors, Opioid; Receptors, Opioid, mu; Solubility; Substance P; Swine