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.
Highlights
The coupling of receptor to guanine nucleotide binding protein (G-protein) hasbeen central to many signal transduc
In several tion of [3H-~-Ala2-~-Leu6]enkephal(i[‘nHIDADL) studies, changes in receptor affinity have been attributed to a from bovine hippocampal synaptic plasma membranes multistep association process which results in the formation (SPMs) varied depending upon association time, sug- of an intermediate complex consisting of agonist, receptor, gesting a multistep binding process
It has been demonstrated that opiates inhibit different kinetic intermediates, we examined the effects of guanine nucleotide on dissociation rate
Summary
DADL (plus 20 nM unlabeled DAMGE) was incubated with 200-400 pg ofprotein at 25 "C in a finavlolume of 1ml for 1h before collection of bound ligand on glass fiber filters In these experiments, nonspecific binding routinely amounted to 15-30%. Length of association was readily demonstrated for DADL in the presence of unlabeled DAMGE (Fig. 1).Previously [8], we indicated that a minimum of two steps areinvolved in the association of opioid agonists, but the results which follow suggest that a three-step model is more appropriate. Inclusion of Gpp(NH)p atthe onset of dissociation from equilibrium caused aconcentration-dependent increase in the rate of dissociation with the maximal effect seen at 25 p~ (data not shown) This acceleration of dissociation was most pronounced after 60-min association (Fig. 1)but was much less apparent when preceded by a shortened association period (Fig. 2B).
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