Affinity cross-linked delta-opioid receptor in NG108-15 cells is low molecular weight (25 kDa) and coupled to GTP-binding proteins.
The affinity cross-linking of the delta-opioid receptor in neuroblastoma x glioma NG108-15 cells was undertaken using (3-[125I]iodotyrosyl27)human-beta-endorphin ([125I]beta-endorphin) and disuccinimidyl suberate (DSS) or bis(sulfosuccinimidyl) suberate (BS3) in order to estimate molecular size. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, two radioactive bands were observed. Labeling of a major band of 29 kDa diminished in the presence of unlabeled selective delta-opioid agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), in a concentration-dependent manner, while labeling of a minor band of 58 kDa was hardly affected. The labeling intensity of the 29 kDa band decreased by addition of guanosine 5'-(3-o-thio)triphosphate (GTP gamma S) or by pretreatment of cells with pertussis toxin. These results, taking the molecular weight of covalently bound beta-endorphin (3.6 kDa) into consideration, suggest that the delta-opioid receptor in NG108-15 cell membrane is a 25 kDa protein which is coupled to pertussis toxin-sensitive guanosine triphosphate-binding proteins (G-proteins).
Harada
J
J
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Ueda
H
H
Iso
Y
Y
Satoh
M
M
D002462:Cell Membrane / Q000737:chemistry; D005591:Chemical Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009447:Neuroblastoma / Q000378:metabolism; D037342:Pertussis Toxin; D011957:Receptors, Opioid / Q000737:chemistry / Q000378:metabolism*; D013388:Succinimides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D001615:beta-Endorphin / Q000494:pharmacology*
D011957:Receptors, Opioid; D013388:Succinimides; D010566:Virulence Factors, Bordetella; D001615:beta-Endorphin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; C019358:disuccinimidyl suberate