title: Relationship between opioid-receptor occupancy and stimulation of low-Km GTPase in brain membranes.
abstract: Treatment of rat brain membranes with the irreversible opioid ligand cis-3-methylfentanylisothiocyanate (Superfit) was used to reduce gradually the number of available binding sites for the delta-selective agonist [3H][D-Ser2,Leu5]enkephalin-Thr6 ([ 3H]DSLET). Subsequently, the correlation between ligand binding and low-Km GTPase was investigated. Alkylation with 10 microM and 25 microM Superfit inactivated 66% and 71% of high-affinity (KD, 1 nM) binding sites without decreasing the affinity of the remaining sites and the stimulation of low-Km GTPase by DSLET. Following exposure of the membranes to 50 microM and 75 microM Superfit, ligand binding was confined to the low-affinity (KD, 20 nM) sites. In these membranes, the delta-agonists DSLET and [D-Pen2,D-Pen5]enkephalin still stimulated low-Km GTPase, and these effects were blocked by ICI 174864 (N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH; AIB, alpha-aminoisobutyric acid), a delta-selective antagonist. A similar relationship between low-affinity ligand binding and GTPase stimulation was observed following alkylation of the delta-opioid receptor with the non-selective irreversible antagonist beta-chlornaltrexamine in the presence of protective concentrations of DSLET. The results reveal spare receptor sites in the coupling of the delta-opioid receptor to low-Km GTPase in brain and identify low-affinity ligand binding as a functional component in the process.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109.
authors.forename: M J
authors.initials: MJ
authors.lastname: Clark
authors.forename: G L
authors.initials: GL
authors.lastname: Nordby
authors.forename: F
authors.initials: F
authors.lastname: Medzihradsky
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D020881:Enkephalin, D-Penicillamine (2,5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives*; D004745:Enkephalins / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology; D009271:Naltrexone / Q000031:analogs & derivatives / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology
chemical_list: D004745:Enkephalins; D009842:Oligopeptides; D011957:Receptors, Opioid; D004743:Enkephalin, Leucine; D009271:Naltrexone; C017120:chlornaltrexamine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D006160:Guanosine Triphosphate; D020881:Enkephalin, D-Penicillamine (2,5)-; D012964:Sodium; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D008274:Magnesium