Binding kinetics of delta opioid receptors differ for microsomal and synaptic sites.
Earlier, we demonstrated that agonist binding to synaptic plasma membranes involves a multi-step association process. In this study, high affinity binding kinetics of an agonist, [3H]D-Ala2-D-Leu5-enkephalin (DADLE), to delta sites on bovine hippocampal microsomal and synaptic plasma membranes (SPM) were compared. delta site selectivity of DADLE was ensured by suppressing undesirable mu site binding with 20 nM unlabeled D-Ala2-MePhe4-Glyol5-enkephalin. The kinetics of receptor binding to microsomal delta sites are generally more rapid than those of SPMs. Furthermore, the association time-dependent rate of dissociation, which is readily observed with SPMs, was not detected for microsomal binding sites. Although the apparent KD of DADLE did not differ significantly from that in SPMs, kinetic analysis indicated that little or no formation of the high affinity, slowly dissociating, complex occurred with microsomes. The absence of this complex, shown previously in SPMs to be most sensitive to guanine nucleotides, appeared to account for the attenuated effect of guanyl 5'-yl-imidodiphosphate [Gpp(NH)p] on dissociation from microsomes. Nevertheless, the presence in microsomes of inhibitory guanine nucleotide binding proteins was demonstrated by specific 32P-labeling by pertussis toxin of bands at 39 and 41 kDa, attributable to the alpha subunit of Go and Gi, respectively. The action of 100 mM Na+ to increase the off-rate is similar for both preparations. In contrast, addition of Mn2+ reduced the rates of association and dissociation for both subcellular fractions. The off-rate in the presence of Mn2+ is similar for SPMs and microsomes, displaying association time-dependent rates of dissociation for both. To determine whether Mn2+ promotes coupling in microsomes, the effect of Gpp(NH)p was examined. After a 60-min association, Gpp(NH)p did not affect microsomal kinetics but increased the off-rate from SPMs. The actions of both Na+ and Mn2+ appear to be mediated at early steps in the association process.
Spain
J W
JW
Department of Biochemistry, St. Louis University School of Medicine, Missouri 63104.
Petta
D E
DE
Coscia
C J
CJ
D000818:Animals; D002417:Cattle; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine; D019204:GTP-Binding Proteins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006624:Hippocampus / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism*; D011957:Receptors, Opioid / Q000378:metabolism*; D017465:Receptors, Opioid, delta; D012964:Sodium / Q000494:pharmacology; D013569:Synapses / Q000378:metabolism*
D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; D012964:Sodium; D019204:GTP-Binding Proteins; D008274:Magnesium