Reversible affinity labeling of opioid receptors via disulfide bonding: Discriminative labeling of μ and δ 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 thiol-disulfide exchange reaction. We incorporated the Npys group into enkephalin analogs to affinity label μ and δ opioid receptors. When rat brain membranes were incubated with [D-Ala²,Leu(CH₂SNpys)⁵] enkephalin, and assayed for the inhibition of binding of DAGO and DSLET enkephalin analogs 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 μ receptors highly specifically (EC₅₀, = 51 nM) and almost 100 times more selectively than δ receptors. In contrast, [D-Ala²,Leu⁵] enkephalyl-Cys(Npys)⁶ attached covalently to δ receptors (EC₅₀ = 34 nM) about 150 times more selectively than to μ receptors. Although N-ethylmaleimide also inhibited the binding of DAGO and DSLET, four to six orders of magnitude higher concentrations were required as compared to SNpys-containing enkephalins. When enkephalin-bound rat membranes were treated with dithiothreitol, the loss of receptors was reversed, depending upon the concentration of and incubation time with dithiothreitol. The recovery was much faster (about 1,000 times) for δ receptors than for μ. receptors. The present results indicated that both μ and δ receptors in rat brain consist of a free mercapto group near the enkephalin binding site and that SNpys-containing enkephalins can label these mercapto groups discriminatively. The disulfide bond between [D-Ala²,Leu⁵]enkephalyl-Cys⁶ and δ receptors appears to be exposed, while that between [D-Ala²,Leu(CH₂SNpys)⁵] enkephalin and μ receptors is shielded.