Characterization of antisera to the naloxone-insensitive receptor for β- endorphin on U937 cells generated by using the complementary peptide strategy

N. A. Shahabi, K. L. Bost, T. C. Madhok, Burt Sharp

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Abstract

Antisera to the naloxone-insensitive receptor for β-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-β- endorphin was synthesized as predicted by reading the β-endorphin antisense mRNA 3' to 5'. By using enzyme-linked immunosorbent assay, rabbit antisera specific for the peptide complementary to β-endorphin (C'-peptide) were characterized. With the exception of C'-peptide, preabsorption of the antisera with human-β-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 β-[125I]endorphin. Immunoglobulin to C'-peptide (10-800 μg/tube) inhibited the binding of β-[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 β-[125I]endorphin to U937 cell membrane in the presence of β- 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 β-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 β-endorphin from the naloxone-insensitive receptor on U937 cells. The efficacy of the antisera with intact U937 cells suggests that the β-endorphin receptor is on the cell surface.

Original languageEnglish (US)
Pages (from-to)876-883
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume263
Issue number2
StatePublished - Jan 1 1992

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Endorphins
U937 Cells
Immune Sera
C-Peptide
Peptides
Immunoglobulins
Enzyme-Linked Immunosorbent Assay
Cell Membrane
naloxone receptor
Antibodies
Opioid Receptors
Naloxone
Sodium Dodecyl Sulfate
Immunoglobulin M
Sucrose
Reading
Polyacrylamide Gel Electrophoresis
Western Blotting
Rabbits

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

@article{136104742f8843d7b44e57de256f9734,
title = "Characterization of antisera to the naloxone-insensitive receptor for β- endorphin on U937 cells generated by using the complementary peptide strategy",
abstract = "Antisera to the naloxone-insensitive receptor for β-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-β- endorphin was synthesized as predicted by reading the β-endorphin antisense mRNA 3' to 5'. By using enzyme-linked immunosorbent assay, rabbit antisera specific for the peptide complementary to β-endorphin (C'-peptide) were characterized. With the exception of C'-peptide, preabsorption of the antisera with human-β-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 β-[125I]endorphin. Immunoglobulin to C'-peptide (10-800 μg/tube) inhibited the binding of β-[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 β-[125I]endorphin to U937 cell membrane in the presence of β- 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 β-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 β-endorphin from the naloxone-insensitive receptor on U937 cells. The efficacy of the antisera with intact U937 cells suggests that the β-endorphin receptor is on the cell surface.",
author = "Shahabi, {N. A.} and Bost, {K. L.} and Madhok, {T. C.} and Burt Sharp",
year = "1992",
month = "1",
day = "1",
language = "English (US)",
volume = "263",
pages = "876--883",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
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T1 - Characterization of antisera to the naloxone-insensitive receptor for β- endorphin on U937 cells generated by using the complementary peptide strategy

AU - Shahabi, N. A.

AU - Bost, K. L.

AU - Madhok, T. C.

AU - Sharp, Burt

PY - 1992/1/1

Y1 - 1992/1/1

N2 - Antisera to the naloxone-insensitive receptor for β-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-β- endorphin was synthesized as predicted by reading the β-endorphin antisense mRNA 3' to 5'. By using enzyme-linked immunosorbent assay, rabbit antisera specific for the peptide complementary to β-endorphin (C'-peptide) were characterized. With the exception of C'-peptide, preabsorption of the antisera with human-β-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 β-[125I]endorphin. Immunoglobulin to C'-peptide (10-800 μg/tube) inhibited the binding of β-[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 β-[125I]endorphin to U937 cell membrane in the presence of β- 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 β-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 β-endorphin from the naloxone-insensitive receptor on U937 cells. The efficacy of the antisera with intact U937 cells suggests that the β-endorphin receptor is on the cell surface.

AB - Antisera to the naloxone-insensitive receptor for β-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-β- endorphin was synthesized as predicted by reading the β-endorphin antisense mRNA 3' to 5'. By using enzyme-linked immunosorbent assay, rabbit antisera specific for the peptide complementary to β-endorphin (C'-peptide) were characterized. With the exception of C'-peptide, preabsorption of the antisera with human-β-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 β-[125I]endorphin. Immunoglobulin to C'-peptide (10-800 μg/tube) inhibited the binding of β-[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 β-[125I]endorphin to U937 cell membrane in the presence of β- 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 β-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 β-endorphin from the naloxone-insensitive receptor on U937 cells. The efficacy of the antisera with intact U937 cells suggests that the β-endorphin receptor is on the cell surface.

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M3 - Article

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