Unexpectedly high levels of opioid peptides in rat brain membranes.
Several considerations led us to directly measure, using radioimmunoassay, the levels of dynorphin-A(1-8), cholecystokinin-8, and Met5-enkephalin-Arg6-Gly7-Leu8 (MERGL) in lysed-P2 membranes, membranes incubated 60 min at 25 degrees C in 50 mM TRIS-HCl at pH 7.4, and membranes preincubated 60 min at 25 degrees C in TRIS buffer containing 0.4 M NaCl. Lysed-P2 membranes contained neuropeptides at levels between 0.253 and 1.727 pmol/mg protein. Incubating membranes in buffer alone decreased membrane peptide levels by 40 to 60%. Preincubations in the presence of 0.4 M NaCl resulted in even greater reductions in the peptide content of membranes (between 58 and 90%), without changing [3H]etorphine binding. Detailed comparisons of the two binding sites labeled by [3H]naloxone revealed changes in binding parameters induced by preincubation with NaCl. Taken collectively, these studies demonstrate that lysed-P2 membranes contain sequestered peptides that are not bound to opiate receptors.
D000818:Animals; D001923:Brain Chemistry; D002462:Cell Membrane / Q000032:analysis; D004399:Dynorphins / Q000032:analysis*; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000032:analysis; D007028:Hypothalamic Hormones / Q000032:analysis*; D008297:Male; D010446:Peptide Fragments / Q000032:analysis*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000032:analysis; D012844:Sincalide / Q000032:analysis*
D007028:Hypothalamic Hormones; D010446:Peptide Fragments; D011957:Receptors, Opioid; D004744:Enkephalin, Methionine; D004399:Dynorphins; C026770:dynorphin (1-8); C033453:enkephalin-Met, Arg(6)-Gly(7)-Leu(8)-; D012844:Sincalide