Comparison of [125I]beta-endorphin binding to rat brain and NG108-15 cells using a monoclonal antibody directed against the opioid receptor.
The properties of [125I]beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of [125I]beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM [125I]beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM [3H] [D-penicillamine2, D-penicillamine5] enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of [125I]beta h-endorphin to brain membranes, the antibody also displaced [125I]beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting [125I]beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit [125I]beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.
Bidlack
J M
JM
Department of Pharmacology, University of Rochester, School of Medicine and Dentistry, New York 14642.
O'Malley
W E
WE
Schulz
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001921:Brain / Q000378:metabolism*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine; D004745:Enkephalins / Q000378:metabolism / Q000494:pharmacology; D005910:Glioma; D007075:Immunoglobulin M / Q000494:pharmacology; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008297:Male; D008970:Molecular Weight; D009447:Neuroblastoma; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000378:metabolism*; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D014407:Tumor Cells, Cultured / Q000378:metabolism; D001615:beta-Endorphin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004745:Enkephalins; D007075:Immunoglobulin M; D007457:Iodine Radioisotopes; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004743:Enkephalin, Leucine; D001615:beta-Endorphin; D016308:Enkephalin, Leucine-2-Alanine; D020881:Enkephalin, D-Penicillamine (2,5)-