Anti-opioid receptor antibody recognition of a binding site on brain and leukocyte opioid receptors.
Opioid receptors reportedly exist on neuronal tissue of central and peripheral origin as well as on cells of the immune system. Previously, an opioid receptor has been purified from the neuroblastoma x glioma hybrid cell line, NG108-15 cells. In an effort to compare these results with opioid receptors isolated from primary neuronal tissue, we employed a methodology based on the molecular recognition theory to develop a monoclonal antibody which was used to isolate and biochemically characterize murine brain opioid receptors. We herein report the purification of an opioid receptor from mouse brain with a molecular weight of 65,000 daltons (range was 62-70 kD under reducing conditions) using a monoclonal antibody to an (the) opioid receptor. In situ labeling experiments with the delta-class selective opioid receptor affinity ligand, cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT) of brain membrane confirmed these observations. Moreover, SUPERFIT, when coupled to the binding site, could block the recognition of the receptor by the monoclonal antibody. However, the selective, mu-class opioid receptor affinity reagent, 2-(p-ethoxybenzyl)-1-N,N-diethylaminoethyl-5-isothiocyanatobenz imidazole was ineffective at masking the binding site from recognition by the monoclonal antibody. Likewise, opioid-like receptors were purified from murine leukocytes which migrated at a molecular weight of 58,000 daltons under nonreducing conditions and 70,000 daltons under reducing conditions. In addition, immunoaffinity-purified receptor is shown to specifically bind the delta-class-selective opioid ligand, cis-(+)-3-methylfentanylisothiocyanate as well as the endogenous opioid peptides, beta-endorphin and [Met]-enkephalin.(ABSTRACT TRUNCATED AT 250 WORDS)
Carr
D J
DJ
Department of Physiology and Biophysics, University of Alabama at Birmingham.
DeCosta
B R
BR
Kim
C H
CH
Jacobson
A E
AE
Bost
K L
KL
Rice
K C
KC
Blalock
J E
JE
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005283:Fentanyl / Q000031:analogs & derivatives; D007120:Immunochemistry; D007202:Indicators and Reagents; D017879:Isothiocyanates; D007962:Leukocytes / Q000378:metabolism*; D008566:Membranes; D051379:Mice; D008810:Mice, Inbred C57BL; D011957:Receptors, Opioid / Q000032:analysis* / Q000276:immunology; D013861:Thiocyanates; D014316:Tritium
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D017879:Isothiocyanates; D011957:Receptors, Opioid; D013861:Thiocyanates; D014316:Tritium; C046222:3-methylfentanyl isothiocyanate; C042097:2-(4-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimidazole; D005283:Fentanyl