Irreversible labelling of delta-opioid receptors in rat brain and neuroblastoma cells by [3H]azido-DTLET: characterization of subunits and autoradiographic visualization of the covalent binding.
[3H]Az-DTLET (Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr), a photoaffinity probe for delta opioid receptors binds to a single class of sites in rat brain membranes with a high affinity (KD = 1.66 nM). The selectivity index of Az-DTLET (KI delta/KI mu = 0.036) is better than that of its precursor DTLET (0.053). Rat brain or neuroblastoma glioma cells membranes were incubated with 10 nM [3H]Az-DTLET, washed and irradiated with U.V. After irradiation a fraction (20-30%) of specific binding was found to remain indissociable after 10 min at 60 degrees C and was considered as irreversible. This fraction increased as a function of the irradiation time. The radioactivity irreversibly bound to rat brain membranes, solubilized by sodium cholate, was associated with high molecular weight species (200,000 daltons). In denaturing conditions (SDS 2%), the [3H]Az-DTLET specific binding was associated with molecular components of 45-50 K and 90-100 K daltons. In contrast, when opioid receptors were prelabelled by [3H]Az-DTLET, solubilized by Na-cholate and irradiated, the radioactivity was only recovered with subunits of 45-50 K daltons. The autoradiographic localization of the irreversibly bound [3H]Az-DTLET in rat brain was identical to that of reversibly bound [3H]DTLET or [3H]Az-DTLET. These results suggest that [3H]Az-DTLET represents an adequate specific probe for studies on the structure, function and anatomical distribution at light and even electron microscopic level of delta-opioid receptors.
D000818:Animals; D001345:Autoradiography; D001386:Azides; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D003342:Corpus Striatum / Q000378:metabolism; D008297:Male; D008566:Membranes / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009842:Oligopeptides; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000378:metabolism*; D012995:Solubility; D014316:Tritium
D001386:Azides; D009842:Oligopeptides; D011957:Receptors, Opioid; D014316:Tritium; C044210:tyrosyl-threonyl-glycyl-(4-azidophenylalanyl)-leucyl-threonine