Irreversible labelling of δ-opioid receptors in rat brain and neuroblastoma cells by [H]azido-DTLET: Characterization of subunits and autoradiographic visualization of the covalent binding |
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Authors: | Jean-Marie Zajac William Rostne Bernard P Roques |
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Institution: | Jean-Marie Zajac, William Rostène,Bernard P Roques, |
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Abstract: | 3H]Az-DTLET (Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr), a photoaffinity probe for δ 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 (
) 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°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 δ-opioid receptors. |
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