Purification of an active opioid-binding protein from bovine striatum.
We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003342:Corpus Striatum / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007526:Isoelectric Point; D037102:Lectins; D008970:Molecular Weight; D009271:Naltrexone / Q000031:analogs & derivatives / Q000378:metabolism; D009294:Narcotics / Q000378:metabolism*; D011957:Receptors, Opioid / Q000302:isolation & purification*; D012685:Sepharose; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014909:Wheat Germ Agglutinins
D000345:Affinity Labels; D037102:Lectins; D009294:Narcotics; D011957:Receptors, Opioid; D014909:Wheat Germ Agglutinins; C040902:beta-naltrexyl-6-ethylenediamine; D009271:Naltrexone; D012685:Sepharose