Purification of opioid receptor in the presence of sodium ions.
Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.
Li
L Y
LY
Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710.
Zhang
Z M
ZM
Su
Y F
YF
Watkins
W D
WD
Chang
K J
KJ
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D019204:GTP-Binding Proteins / Q000737:chemistry; D009270:Naloxone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000302:isolation & purification*; D017450:Receptors, Opioid, mu; D012964:Sodium / Q000494:pharmacology*; D014909:Wheat Germ Agglutinins
D009292:Narcotic Antagonists; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; D014909:Wheat Germ Agglutinins; D009270:Naloxone; D012964:Sodium; D019204:GTP-Binding Proteins