Affinity partitioning and centrifugal counter-current distribution of membrane-bound opiate receptors using naloxone-poly(ethylene glycol).
Crude synaptic membranes isolated from calf brain cortex were subjected to an aqueous two-phase system and the partition of the various membrane constituents and activities between the phases were studied. These constituents were phosphate, cholesterol and protein. The activities measured were acetyl-cholinesterase, succinate dehydrogenase, 2',3'-cyclicnucleotide-3'-phosphohydrolase and stereospecific opiate-binding. The successful fractionation of the membranes was achieved by the use of an aqueous two-phase system in a counter-current distribution process. A ligand bound to poly(ethylene glycol) with an affinity for opiate receptors was synthesized by reacting 6-aminonaloxone with tresylpoly(ethylene glycol). The ligand-polymer was used to extract membrane-bound opiate receptors into the upper, poly(ethylene glycol)-rich phase. This use of affinity partitioning resulted in membrane fractions with a 3-4 fold higher ability to bind stereospecifically etorphine than the original preparations of synaptic membranes.
D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D003377:Countercurrent Distribution; D009270:Naloxone / Q000378:metabolism*; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D011957:Receptors, Opioid / Q000032:analysis*; D013570:Synaptic Membranes / Q000032:analysis*; D014020:Tissue Extracts / Q000032:analysis; D014461:Ultracentrifugation
D011957:Receptors, Opioid; D014020:Tissue Extracts; D009270:Naloxone; D011092:Polyethylene Glycols; D011189:Potassium Chloride