Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit.
125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.
Howard
A D
AD
de La Baume
S
S
Gioannini
T L
TL
Hiller
J M
JM
Simon
E J
EJ
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002024:Bufo marinus; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004723:Endorphins / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D001615:beta-Endorphin
D004723:Endorphins; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D011957:Receptors, Opioid; D001615:beta-Endorphin