Identification of the binding subunit of the sigma-type opiate receptor by photoaffinity labeling with 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2-methyl[4,6-3H]phenyl)guanidine

摘要: Abstract The sigma-type opiate receptor is a distinct binding site in the brain that may mediate some of psychotomimetic effects caused by benzomorphan opiates and phencyclidine humans. We have developed synthetic, highly selective ligand for this receptor, 1,3-di-o-tolylguanidine (DTG). To identify protein(s) sigma we now synthesized radiolabeled azide derivative DTG, 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2-methyl[4,6-3H]phenyl)-guanidine ([3H]N3DTG). In guinea pig membrane assays conducted dark, [3H]N3DTG bound reversibly, selectively, with high affinity (Kd = 10 nM) to receptors. The drug specificity profile reversible [3H]-N3DTG was identical [3H]DTG 3H-labeled (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine indicating ligand. Guinea membranes were photoaffinity-labeled [3H]N3DTG. NaDodSO4/PAGE detergent-solubilized extract identified single 29-kDa radioactive band. Sepharose Cl-6B gel chromatography photolabeled solubilized nondenaturing detergent sodium cholate showed complex Stoke's radius 4.6 nm (Mr, 150,000) represent intact complex. material polypeptide be subunit receptor. specific photoaffinity described here should useful tool purifying characterizing