Immunoaffinity‐purified opiate receptor specifically binds the δ‐class opiate receptor ligand, cis‐(+)‐3‐methylfentanylisothiocyanate, SUPERFIT

Abstract

Using an antibody generated against the opiate receptor on NG108‐15 cells, we recently purified the putative receptor from this hybrid cell line. We herein report that the purified receptor complex specifically binds tritiated cis‐(+)‐3‐methylfentanylisothiocyanate (SUPERFIT), with the predominant binding associated with a 58 kDa polypeptide chain. Consistent with these findings is the in situ labeling of a 58 kDa protein with [3H]SUPERFIT on NG108‐15 cells.

Anti-Opioid Receptor Antibody Recognition of a Binding Site on Brain and Leukocyte Opioid Receptors

“…By comparison, immunocyte opioid receptors purified by affinity chromatography and subsequently analyzed by PAGE have a similar molecular weight to what has previously been reported for 8-class [4,5] and p-class [3,21 ] opioid receptors of neuroendocrine tissue. In addition, the molecular weight of the immunoaffinity-purified immunocyte receptor is consistent with the results of previous in situ labeling experiments of the 8-class opioid receptor on cells of the immune system [18], Moreover, the present study using purified mouse brain recep tors would indicate a similar structural profile to immune cell receptors.…”

“…Previous results also suggest that the 8-and u-class opioid receptors have similar if not identical molecular weights [3,5,21], Thus, the present studies were carried out in order to further address whether the binding sites are in close enough proximity to modulate ligand-binding capabilities of one another. Studies of brain opioid receptor structure using the site-directed p-(BIT) and 8-(SUPERFIT) class-specific reagents in in situ labeling techniques revealed that BIT (10-20 uA fig.…”

“…It is unclear whether these sites exist on one parent molecule or are com pletely separate entities. However, recent functional assays [2] and biochemical analysis [3][4][5] would suggest that the 8-and u-class opioid binding sites may be physically associated. Speci fically, opioid ligand labeling studies revealed that the li-and 8-class opioid receptor binding sites migrated at an identical molecular weight (58,000 daltons).…”

“…The antibodies were produced using the molecular recognition theory in which peptides (termed com plementary peptides) that are specified by the strand comple mentary to the strand coding for a ligand (in this case, y-endorphin) are used as immunogens. Antibody produced in this manner was shown to compele for binding with naloxone and |5-endorphin to NG108-15 cells and block l25I-|5-endorphin binding to NG108-15 cells [5,7], Likewise, a monoclonal an tibody (MAB) which recognizes the 8-class opioid receptor on NG108-15 cells has been produced using the complementary peptide to [Met]-enkephalin as an immunogen. The MAB could block binding of |;!5I-|i-endorphin to NG108-15 cells.…”

From the Neuroendocrinology of Lymphocytes toward a Molecular Basis of the Network Theory

“…Likewise, it was determined upon purification and SDS-polyacrylamide gel electro phoresis that the leukocyte opioid receptor was com posed of four polypeptide chains with Mr of 70,000, 56,000, 46,000 and 31,000 daltons [22], In order to fur ther investigate the physicochemical properties of the receptor, we addressed the functionality of the receptor from NG108-15 cells and leukocytes by determining if purified material could specifically bind an opiate ligand and which chain (or chains) was (were) responsible for its recognition. In studies using cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT), a delta-class-specific opiate receptor ligand, immunoaffinity-purified receptor from NG108-15 and leukocytes specifically recognized SUPERFIT and the 56-to 58-dalton polypeptide chain was associated with all of ihe binding [23].…”