[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.