Selective protection of stereospecific enkephalin and opiate binding against inactivation by N-ethylmaleimide: evidence for two classes of opiate receptors.

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Abstract
Stereospecific binding of 3H-labeled [D-Ala2,D-Leu5]enkephalin is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide. This inactivation, like that of opiate binding, exhibits pseudo 1st-order kinetics with a half-inactivation time of 10-12 min. The presence of opiates or enkephalins during incubation with N-ethylmaleimide provides good protection. Quantitative studies of protection demonstrate that naltrexone and morphine are 20 and 8 times, respectively, more effective in protecting the binding of [3H]naltrexone than that of [3H]enkephalin. [D-Ala2,Leu]enkephalin and [D-Ala2,Met]enkephalin are more effective (7 and 30 times, respectively) for the protection of 3H-labeled [D-Ala2,D-Leu5]enkephalin binding. These results provide strong evidence for the existence of 2 classes of opiate receptor in rat brain.