Interaction of Opiates with Opioid Binding Sites in the Bovine Adrenal Medulla: I. Interaction with δ and μ Sites

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Abstract
The interaction of opiates with the .delta. and .mu. opioid binding sites in the bovine adrenal medulla were examined. [3H][D-Ala2, D-Leu5]-enkephalin ([3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze .delta. site interactions, whereas either [3H]DADLE in the presence of saturating concentrations of [D-Ser2, Leu5]-enkephalin-Thr6 (DSLET) or [3H][D-Ala2, Me-Phe4, Gly5-ol]enkephalin ([3H]DAGO) was used for the determination of .mu. sites. Both binding sites were found to interact stereoselectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, .alpha.-chymotrypsin, pepsin), N-ethylmaleimide, and A2-phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with 1 class of binding sites, following simple 2nd-order biomolecular kinetics. Competition for binding by opiates and opioid peptides confirmed the .delta. and .mu. selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these 2 binding sites. In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalents ions modified the equilibirum dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.