Integrity of tritiated orphanin FQ/Nociceptin: Implications for establishing a reliable binding assay

Denise Irene Quigley, Seksiri Arttamangkul, Richard Gordon Allen, David Kilgore Grandy

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

In the course of establishing a reliable and reproducible binding assay for the orphanin FQ/nociceptin (OFQ/N) ligand-receptor system we used reversed phase-high-performance liquid chromatography (HPLC) (RP-HPLC) to monitor the integrity of [3H]OFQ/N obtained from three different manufacturers. This means of analysis revealed that the stability of [3H]OFQ/N during storage varied considerably depending on the manufacturer. Furthermore, the integrity of [3H]OFQ/N was significantly compromised in the presence of COS-7 cell membranes. Interestingly, if the peptide was added to COS-7 membranes after they had been exposed to low pH it remained intact, suggesting that the peptide's breakdown during binding is, in part, enzymatically mediated. Although a variety of protease inhibitors were tested, none proved completely effective at protecting the tritiated peptide. The intention of the studies presented here was to evaluate OFQ/N binding components, namely the available [3H]OFQ/N ligands, in an effort to standardize the binding conditions for this receptor ligand system. Consequently, this study underscores the importance of monitoring the integrity of the trace ligand being used in a given binding assay. Copyright (C) 2000 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)1111-1118
Number of pages8
JournalPeptides
Volume21
Issue number7
DOIs
StatePublished - Jul 1 2000

Keywords

  • Nociceptin
  • OFQ receptor
  • Orphanin FQ
  • Peptide degradation
  • Radioligand binding

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Endocrinology
  • Cellular and Molecular Neuroscience

Fingerprint Dive into the research topics of 'Integrity of tritiated orphanin FQ/Nociceptin: Implications for establishing a reliable binding assay'. Together they form a unique fingerprint.

  • Cite this