Affinity labeling of delta-opiate receptors using [D-Ala2,Leu5,Cys6]enkephalin. Covalent attachment via thiol-disulfide exchange.
[D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is a synthetic enkephalin analog which contains a sulfhydryl group. DALCE binds with high affinity to delta-receptors, with moderate affinity to mu-receptors, and with negligible affinity to kappa-receptors. Pretreatment of rat brain membranes with DALCE resulted in concentration-dependent loss of delta-binding sites. Using 2 nM [3H][D-Pen2,D-Pen5]enkephalin (where Pen represents penicillamine) to label delta-sites, 50% loss of sites occurred at about 3 microM DALCE. Loss of sites was not reversed by subsequent incubation in buffer containing 250 mM NaCl and 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), conditions which cause dissociation of opiate agonists. By contrast, the enkephalin analogs [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, [D-Pen2,D-Pen5]enkephalin, and [D-Ala2,D-Leu5,Lys6]enkephalin were readily dissociated by NaCl and Gpp(NH)p, producing negligible loss at 3 microM. This suggests that DALCE binds covalently to the receptors. Pretreatment of membranes with the reducing agents dithiothreitol and beta-mercaptoethanol had no effect on opiate binding. Thus, loss of sites required both specific recognition by opiate receptors and a thiol group. The irreversible effect of DALCE was completely selective for delta-receptors. Pretreatment with DALCE had no effect on binding of ligands to mu- or kappa-receptors. The effect of DALCE on delta-binding was: 1) markedly attenuated by inclusion of dithiothreitol in the preincubation buffer, 2) partially reversed by subsequent incubation with dithiothreitol, 3) slightly enhanced when converted to the disulfide-linked dimer, and 4) prevented by blocking the DALCE sulfhydryl group with N-ethylmaleimide or iodoacetamide. These results indicate that DALCE binds covalently to delta-receptors by forming a disulfide bond with a sulfhydryl group in the binding site. The mechanism may involve a thiol-disulfide exchange reaction.
Bowen
W D
WD
Section of Biochemistry, Brown University, Providence, Rhode Island 02912.
Hellewell
S B
SB
Kelemen
M
M
Huey
R
R
Stewart
D
D
D000818:Animals; D001921:Brain / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine / Q000031:analogs & derivatives*; D007425:Intracellular Membranes / Q000378:metabolism; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000187:drug effects / Q000378:metabolism*; D017465:Receptors, Opioid, delta; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*
D004220:Disulfides; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D013438:Sulfhydryl Compounds; C053821:enkephalin-Leu, Ala(2)-Cys(6)-; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; D004229:Dithiothreitol; D007460:Iodoacetamide