Molecular Pharmacology, Vol 20, 255-262, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Two Different Modifications of the Neuroblastoma x Glioma Hybrid Opiate Receptors Induced by N-Ethylmaleimide

¹ Department of Physiological Chemistry and Pharmacology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110

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, -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.

Note:
ACKNOWLEDGMENTS The authors wish to thank R. J. Lefkowitz and A. De Lean, Duke University, for providing their computer program. Their assistance, along with that of A. Alexander, Management Information Service, Hoffmann-La Roche, Inc., in implementing this program is also gratefully acknowledged.

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