Affinity Labeling of & Opiate Receptors Using [ o - Ala 2 , Leu 5 , Cys 6 ] Enkephalin COVALENT ATTACHMENT VIA THIOL - DISULFIDE EXCHANGE

[~-Ala~,Leu~,Cys~]Enkephalin (DALCE) is a synthetic enkephalin analog which contains a sulfhydryl group. DALCE binds with high affinity to &receptors, with moderate affinity to preceptors, and with negligible affinity to K-receptors. Pretreatment of rat brain membranes with DALCE resulted in concentrationdependent loss of &binding sites. Using 2 nM ['H][DPen2,~-Pen5]enkephalin (where Pen represents penicillamine) to label 6-sites, 50% loss of sites occurred at about 3 MM DALCE. Loss of sites was not reversed by subsequent incubation in buffer containing 250 mM NaCl and 100 PM guanyl-S'-yl imidodiphosphate (Gpp(NH)p), conditions which cause dissociation of opiate agonists. By contrast, the enkephalin analogs [~-Ala~,~-Leu~]enkephalin, [~-Ser~,Leu~,Thr']enkephalin, [D-Pen2,D-Pen6]enkephalin, and [D-Ala2,DLeu',Lys']enkephalin were readily dissociated by NaCl and Gpp(NH)p, producing negligible loss at 3 WM. This suggests that DALCE binds covalently to the receptors. Pretreatment of membranes with the reducing agents dithiothreitol and B-mercaptoethanol had no effect on opiate binding. Thus, loss of sites required both specific recognition by opiate receptors and a thiol group. The irreversible effect of DALCE was completely selective for &receptors. Pretreatment with DALCE had no effect on binding of ligands to Nor Kreceptors. The effect of DALCE on &binding was: 1) markedly attenuated by inclusion of dithiothreitol in the preincubation buffer, 2) partially reversed by subsequent incubation with dithiothreitol, 3) slightly enhanced when converted to the disulfide-linked dimer, and 4) prevented by blocking the DALCE sulfhydryl group with N-ethylmaleimide or iodoacetamide. These results indicate that DALCE binds covalently to 6receptors by forming a disulfide bond with a sulfhydryl group in the binding site. The mechanism may involve a thiol-disulfide exchange reaction.