REVERSIBLE AFFINITY LABELING OF OPIOID RECEPTORS VIA DISULFIDE BONDING - DISCRIMINATIVE LABELING OF MU-SUBTYPES AND DELTA-SUBTYPES BY CHEMICALLY ACTIVATED THIOL-CONTAINING ENKEPHALIN ANALOGS

The 3-nitro-2-pyridinesulfenyl (Npys) group bound to a mercapto group is a highly activated electrophilic reagent, which only reacts with a free mercapto group to form a disulfide bond via the thio-disulfide ex change reaction. We incorporated the Npys group into enkephalin analog s to affinity label mu and delta opioid receptors. When rat brain memb ranes were incubated with [D-Ala(2),Leu(CH(2)SNpys)(5)] enkephalin, an d assayed for the inhibition of binding of DAGO and DSLET enkephalin a nalogs to opioid receptors, the number of receptors decreased sharply, depending upon the concentration of this SNpys-containing enkephalin. It was found that this enkephalin analog occupies mu receptors highly specifically (EC(50)=51 nM) and almost 100 times more selectively tha n delta receptors. In contrast, [D-Ala(2),Leu(5)] enkephalyl-Cys(Npys) (6) attached covalently to delta receptors (EC(50)=34 nM) about 150 ti mes more selectively than to mu receptors. Although N-ethylmaleimide a lso inhibited the binding of DAGO and DSLET, four to six orders of mag nitude higher concentrations were required as compared to SNpys-contai ning enkephalins. When enkephalin-bound rat membranes were treated wit h dithiothreitol, the loss of receptors was reversed, depending upon t he concentration of and incubation time with dithiothreitol. The recov ery was much faster (about 1,000 times) for delta receptors than for m u receptors, The present results indicated that both mu and delta rece ptors in rat brain consist of a free mercapto group near the enkephali n binding site and that SNpys-containing enkephalins can label these m ercapto groups discriminatively. The disulfide bond between [D-Ala(2), Leu(5)]enkephalyl-Cys(6) and delta receptors appears to be exposed, wh ile that between [D-Ala(2),Leu(CH(2)SNpys)5]enkephalin and mu receptor s is shielded.