Characterization of antisera to the naloxone-insensitive receptor for beta-endorphin on U937 cells generated by using...

Antisera to the naloxone-insensitive receptor for beta-endorphin expressed on the U937 cell line were generated by using the complementary peptide strategy. A nanopeptide complementary to a C-terminal fragment of human beta-endorphin was synthesized as predicted by reading the beta-endorphin antisense mRNA 3' to 5'. By using enzyme-linked immunosorbent assay, rabbit antisera specific for the peptide complementary to beta-endorphin (C'-peptide) were characterized. With the exception of C'-peptide, preabsorption of the antisera with human-beta-endorphin1-31 or 10 unrelated peptides of 5 to 21 amino acids failed to reduce the enzyme-linked immunosorbent assay titer. Sucrose gradient separation was also used to show that the antisera failed to recognize beta-[125I]endorphin. Immunoglobulin to C'-peptide (10-800 micrograms/tube) inhibited the binding of beta-[125I]endorphin (1-2 nM) to intact U937 cells in a dose-dependent manner, whereas control immunoglobulin was ineffective. Moreover, immunoglobulin to C'-peptide failed to reduce [3H]naloxone binding to rat brain membrane. After binding and cross-linking of beta-[125I]endorphin to U937 cell membrane in the presence of beta-endorphin (2.5 x 10(-5) M), control immunoglobulin or anti-C'-peptide immunoglobulin, sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that anti-C'-peptide immunoglobulin inhibited binding to 44 and 59 kDa bands. Nonspecific antibody was completely ineffective, whereas beta-endorphin completely inhibited binding to the 44 kDa and partially to the 59 kDa band. Western blot analysis of U937 cell membrane showed bands at 64, 58 and 56 kDa. In summary, antibodies selective for C'-peptide displaced beta-endorphin from the naloxone-insensitive receptor on U937 cells.(ABSTRACT TRUNCATED AT 250 WORDS)