Purification to apparent homogeneity of a mu-type opioid receptor from rat brain.

Cho, Tae Mook; Hasegawa, Junichi; Ge, Bang-Lun; Loh, Horace H.

doi:10.1073/pnas.83.12.4138

Cited by 77 publications

(22 citation statements)

References 25 publications

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“…This hypothesis is not without precedent since it has recently been demonstrated that the/ztype opiate receptor binding site has an apparent molecular mass of 58 kDa [19,20]. In addition, we suggest that crosslinked, purified receptor binds SUPERFIT more efficiently than noncrosslinked receptor.…”

Section: Discussionmentioning

confidence: 72%

Immunoaffinity‐purified opiate receptor specifically binds the δ‐class opiate receptor ligand, cis‐(+)‐3‐methylfentanylisothiocyanate, SUPERFIT

Carr

DeCosta²,

Jacobson³

et al. 1987

FEBS Letters

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Using an antibody generated against the opiate receptor on NG108‐15 cells, we recently purified the putative receptor from this hybrid cell line. We herein report that the purified receptor complex specifically binds tritiated cis‐(+)‐3‐methylfentanylisothiocyanate (SUPERFIT), with the predominant binding associated with a 58 kDa polypeptide chain. Consistent with these findings is the in situ labeling of a 58 kDa protein with [3H]SUPERFIT on NG108‐15 cells.

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Section: Discussionmentioning

confidence: 72%

Immunoaffinity‐purified opiate receptor specifically binds the δ‐class opiate receptor ligand, cis‐(+)‐3‐methylfentanylisothiocyanate, SUPERFIT

Carr

DeCosta²,

Jacobson³

et al. 1987

FEBS Letters

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“…Opioid receptors have proven to be difficult to solubilize from cell membranes in a high-affinity, agonist-binding state. In most solubilization studies, the solubilized material was able to bind opiate alkaloids but failed to bind enkephalin peptides (Simonds et a!., 1980;Gioannini et al, 1985;Maneckjee et al, 1985;Cho et al, 1986;Ueda et a!., 1988;Li et al, 1992;Ofri et al, 1992). Furthermore, the solubilization process frequently resulted in the loss of guanine nucleotide influence on opioid agonist binding.…”

mentioning

confidence: 99%

Solubilization of High‐Affinity, Guanine Nucleotide‐Sensitive μ‐Opioid Receptors from Rat Brain Membranes

Weems

Chalecka-Franaszek

Côté

1996

Journal of Neurochemistry

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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 mM, the solubilized opioid receptor bound [3H][d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO), a μ‐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 µM 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 µM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [d‐Pen2,d‐Pen5]‐enkephalin, a δ‐selective opioid agonist, and U50488H, a κ‐selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. 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 µM 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.

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“…K d can also be determined by measuring the amount of drug bound to the protein at different drug and lysate/protein concentrations. 24 In fact, receptors for the majority of known drugs were identified using this method, including adrenergic receptors 25–27 , opioid receptors, 28, 29 cyclophilins, 30 and FKBP12. 31 In many of these studies, affinity chromatography was used side by side with binding assays as one of the purification steps.…”

Section: Target Identificationmentioning

confidence: 99%

Identification and validation of protein targets of bioactive small molecules

Titov

Liu

2012

Bioorganic & Medicinal Chemistry

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Identification and validation of protein targets of bioactive small molecules is an important problem in chemical biology and drug discovery. Currently, no single method is satisfactory for this task. Here, we provide an overview of common methods for target identification and validation that historically were most successful. We have classified for the first time the existing methods into two distinct and complementary types, the “top-down” and “bottom-up” approaches. In a typical top-down approach, the cellular phenotype is used as a starting point and the molecular target is approached through systematic narrowing down of possibilities by taking advantage of the detailed existing knowledge of cellular pathways and processes. In contrast, the bottom-up approach entails the direct detection and identification of the molecular targets using affinity-based or genetic methods. A special emphasis is placed on target validation, including correlation analysis and genetic methods, as this area is often ignored despite its importance.

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Purification to apparent homogeneity of a mu-type opioid receptor from rat brain.

Cited by 77 publications

References 25 publications

Immunoaffinity‐purified opiate receptor specifically binds the δ‐class opiate receptor ligand, cis‐(+)‐3‐methylfentanylisothiocyanate, SUPERFIT

Immunoaffinity‐purified opiate receptor specifically binds the δ‐class opiate receptor ligand, cis‐(+)‐3‐methylfentanylisothiocyanate, SUPERFIT

Solubilization of High‐Affinity, Guanine Nucleotide‐Sensitive μ‐Opioid Receptors from Rat Brain Membranes

Identification and validation of protein targets of bioactive small molecules

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