pH Selectivity of N‐Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

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Abstract
N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial SH groups at receptor binding sites. At physiological pH, NEM decreased GTP and Mn regulation but increased Na effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 .mu.M NEM in buffers ranging from pH 4.5-8.0. Lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4-6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 .mu.M) of NEM.