title: Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation.
abstract: The mobility of purified mu opioid binding protein in SDS-polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration-dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, mu greater than delta much greater than kappa. For mu receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.
authors.affiliation: Department of Psychiatry, New York University Medical Center, NY 10016.
authors.forename: T L
authors.initials: TL
authors.lastname: Gioannini
authors.forename: Y F
authors.initials: YF
authors.lastname: Liu
authors.forename: Y H
authors.initials: YH
authors.lastname: Park
authors.forename: J M
authors.initials: JM
authors.lastname: Hiller
authors.forename: E J
authors.initials: EJ
authors.lastname: Simon
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides / Q000032:analysis* / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004723:Endorphins / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism; D006168:Guinea Pigs; D008024:Ligands; D008970:Molecular Weight; D009271:Naltrexone / Q000378:metabolism; D011957:Receptors, Opioid / Q000032:analysis* / Q000378:metabolism
chemical_list: D004220:Disulfides; D004723:Endorphins; D004745:Enkephalins; D008024:Ligands; D011957:Receptors, Opioid; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009271:Naltrexone; D004229:Dithiothreitol