ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
Abstract: 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 μ, δ, and κ 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, μ-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) (GTPγS). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTPγS was observed, whereas membrane-bound receptors were uncoupled only partially. Treatment with GTPγS, at concentrations that uncoupled the μ 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. This indicates that the observed decrease in Bmax is due to a reduction in affinity of the uncoupled receptors to a level too low to be measurable, whereas the residual coupled receptors retain high affinity for μ agonists. The methods described should prove useful for opioid receptor purification and reconstitution with G proteins and second messenger systems.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1471-4159.1992.tb09764.x
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