Characterization and partial purification of solubilized active opiate receptors from toad brain.

Abstract

Opiate receptors were solubilized from toad brain membranes in active form by using digitonin. Between 40-50% of the stereospecific binding activity present in toad brain membranes is recoverable in the ultracentrifugal supernatant of digitonin extracts. Binding of opiates [diprenorphine, etorphine, naltrexone] to the solubilized receptor is enhanced 4 to 5-fold by decreasing digiton concentration to 0.1% or less prior to binding. The solubilized receptor is similar to the membrane-bound receptor in its affinity for various ligands and its sensitivity to heat, trypsin and N-ethylmaleimide. The sodium effect seen in membrane-bound receptor is retained in the solubilized preparation. Both membrane-bound and soluble toad receptors show weak binding of enkephalins [D-Ala2,D-leu5]enkephalin, suggesting that they are predominantly of the .mu. type. The solubilized opiate receptor has an approximate MW of 350,000-400,000. Purification of up to 20-fold has been achieved by gel filtration on Sepharose CL-6B.