Kinetics and physical parameters of rat brain opioid receptors solubilized by digitonin and CHAPS.
Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35-37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation.
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002793:Cholic Acids; D002850:Chromatography, Gel; D004072:Digitonin; D007202:Indicators and Reagents; D007700:Kinetics; D009270:Naloxone / Q000378:metabolism; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D012995:Solubility
D002793:Cholic Acids; D007202:Indicators and Reagents; D011957:Receptors, Opioid; D009270:Naloxone; D004072:Digitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate