title: Multiple interconvertible affinity states for the delta opioid agonist-receptor complex.
abstract: Previously we demonstrated that rates of dissociation of [3H-D-Ala2-D-Leu5]enkephalin [( 3H]DADL) from bovine hippocampal synaptic plasma membranes (SPMs) varied depending upon association time, suggesting a multistep binding process. To characterize different kinetic intermediates, we examined the effects of guanine nucleotide on dissociation rate. Control off-rates were compared to those obtained when guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) (50 microM) was added either coincident with the radioligand at association or with 1 microM unlabeled DADL which initiated dissociation. delta site selectivity of [3H]DADL was ensured by addition of 20 nM unlabeled [D-Ala2-Me-Phe4-Gly-Ol5]enkephalin which suppressed mu site cross-reactivity in this preparation. Addition of Gpp(NH)p at the onset of dissociation increased the off-rate to a much greater extent after steady state binding was reached (60 min) compared to that following an association time of only 7 or 20 min. A slowly formed high affinity state appeared to be rapidly converted to a lower affinity state under these conditions. When Gpp(NH)p was present throughout the association period, the slowly dissociating state was no longer observed. Also, the off-rate following a 7-min association is linear and much faster than control, suggesting that Gpp(NH)p may affect an initial intermediate state as well as the high affinity complex. Pretreatment of the membranes with N-ethylmaleimide (NEM) eliminated the association time-dependent dissociation rates, apparently preventing time-dependent formation of a high affinity state. This state is thought to be possibly a ligand-receptor complex interacting with a GTP binding protein. However, the rate of dissociation from NEM-treated membranes was accelerated by addition of Gpp(NH)p and the effect was not association time-dependent. NEM treatment resulted in an increased potency for Gpp(NH)p inhibition of [3H]DADL steady state binding. These results suggest the occurrence of at least three steps in the association of DADL to bovine hippocampal synaptic membranes.
mesh_terms: D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives* / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine; D005033:Ethylmaleimide / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D009020:Morphine / Q000378:metabolism; D009271:Naltrexone / Q000378:metabolism; D011957:Receptors, Opioid / Q000378:metabolism*; D017465:Receptors, Opioid, delta; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*
chemical_list: D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D006165:Guanylyl Imidodiphosphate; D004743:Enkephalin, Leucine; D009271:Naltrexone; D016308:Enkephalin, Leucine-2-Alanine; D009020:Morphine; D005033:Ethylmaleimide