Photoaffinity labeling of opioid delta receptors with an iodinated azido-ligand: [125I][D-Thr2,pN3Phe4,Leu5]enkephalyl-Thr6.
The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) was iodinated and purified by high performance liquid chromatography. Monoiodo-azido-DTLET displayed a high affinity (KD = 15 nM) and is selective (Kl mu/Kl delta = 9.8) for rat brain delta opioid receptors (for comparison, the corresponding values for tritiated azido-DTLET are KD = 1.66 nM and Kl mu/Kl delta = 27). On rat brain sections, the anatomical distribution of [125I]azido-DTLET binding sites revealed by autoradiography corresponds to that of delta receptors. On rat brain membrane homogenates and NG108-15 hybrid cells, UV irradiation of the receptor-ligand complex results in the irreversible binding to membrane proteins of 14% of the bound radioactivity Gel electrophoresis of [125I]azido-DTLET-labeled proteins followed by autoradiography shows a different pattern in rat brain and NG108-15 cells. In rat brain, labeling of two of these proteins, with molecular weights of 44,000 and 34,000, was inhibited by 30 nmol/liter of nonradioactive DTLET, a delta-selective ligand but not by the same concentration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-selective ligand. In NG108-15 cells, this 44-kDa protein was not visualized; the main band was at 33 kDa and disappeared in the presence of levorphanol.
Bochet
P
P
Laboratoire de Physiologie Nerveuse, CNRS, Gif-sur-Yvette, France.
Icard-Liepkalns
C
C
Pasquini
F
F
Garbay-Jaureguiberry
C
C
Beaudet
A
A
Roques
B
B
Rossier
J
J
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D009842:Oligopeptides; D010777:Photochemistry; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism; D017465:Receptors, Opioid, delta
D000345:Affinity Labels; D001386:Azides; D004745:Enkephalins; D008024:Ligands; D009842:Oligopeptides; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C037653:deltakephalin; C044210:tyrosyl-threonyl-glycyl-(4-azidophenylalanyl)-leucyl-threonine