Abstract

Modification of membrane groups by N-ethylmaleimide (NEM) differentially inactivates the binding of the [³H]-labeled opioid peptide agonist [D-Ala²-Met⁵]-enkephalinamide (Dala²met⁵amide) and the opiate antagonists [³H]naltrexone and [³H]naloxone to NG108-15 opiate receptors. There appear to be at least two different NEM-sensitive groups involved. Alteration of one group by NEM prevents equally the binding of opiate agonists and antagonists and is seen as a reduction in the number of opiate binding sites. Furthermore, this process is first-order, and alteration of one of these groups is therefore sufficient to inactivate the receptor completely. The second group affected by NEM is at least 4-fold more sensitive to NEM, and in its altered state causes a selective reduction in the affinity of opiate agonists for these receptors. The affinity of at least one of the two antagonists tested (i.e., naloxone) actually is increased by 50% after modification of this group. With the opioid agonist Dala²met⁵amide these losses in affinity have been confirmed by both ³H-labeled peptide saturation studies and by studying the peptide competition against the [³H]naloxone binding. Both analyses indicate a 7-fold loss in affinity accompanying the modification of this group by NEM. All of the opiate agonists, including the enkephalins, β-endorphin, oxymorphone, normorphine, and etorphine, exhibit losses in affinity. The magnitude of these losses is not constant, but a property of the particular agonist, and is found with even the mixed agonist-antagonist pentazocine. Therefore these losses are proposed to be a general property of opiate agonists in this system; pure opiate antagonists along with the mixed antagonist-agonist cyclazocine do not show this loss. Modification of this second site is accompanied by (a) an increased sensitivity to the selective effects of cations (rank order Na⁺ > Li⁺ > K⁺ = choline) to decrease preferentially the agonist affinity and (b) a loss in sensitivity of the affinity of Dala²met⁵amide to GTP. The second NEM-sensitive site is therefore distal to the ligand binding domain and is proposed to be involved selectively in the formation of high-affinity agonist-receptor complexes.