PURIFICATION TO HOMOGENEITY OF AN ACTIVE OPIOID RECEPTOR FROM RAT-BRAIN BY AFFINITY-CHROMATOGRAPHY

Active opioid binding proteins were solubilized from rat brain membran es in high yield with sodium deoxycholate in the presence of NaCl. Pur ification of opioid binding proteins was accomplished by opioid antago nist affinity chromatography. Chromatography using the delta-opioid an tagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to pi-aminododecyl -agarose (Affi-G) (procedure A) yielded a partially purified protein t hat binds selectively the delta-opioid agonist [H-3]Tyr-D-Ser-Gly-Phe- Leu-Thr ([H-3]DSLET), with a K-d of 19 +/- 3 nM and a B-max of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepha rose 4B chromatography of the Affi-G eluate resulted in isolation of a n electrophoretically homogeneous protein of 58 kDa that binds selecti vely [H-3]DSLET with a K-d of 21 +/- 3 nM and a B-max of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6 -aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selective ly [H-3]DSLET with a K-d of 32 +/- 2 nM and a B-max of 12.4 +/- 0.5 nm ol/mg of protein, and NaDodSO(4)/PAGE revealed a major band of apparen t molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised aga inst the Affi-NAL protein inhibit the specific [H-3]DSLET binding to t he Affi-NAL eluate and to the solubilized membranes. Moreover, the Ant i-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N- methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and nalo xone to homogenates of rat brain membranes with equal potency. Further more, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified pro teins from the Affi-G and Affi-NAL matrices revealed that the Anti-R I gG interacts with a protein of 58 kDa.