Integrity of tritiated orphanin FQ/nociceptin: implications for establishing a reliable binding assay.
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 [(3)H]OFQ/N obtained from three different manufacturers. This means of analysis revealed that the stability of [(3)H]OFQ/N during storage varied considerably depending on the manufacturer. Furthermore, the integrity of [(3)H]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 [(3)H]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.
Quigley
D I
DI
Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, 97201, Portland, OR, USA.
Arttamangkul
S
S
Allen
R G
RG
Grandy
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary / Q000378:metabolism; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D018847:Opioid Peptides / Q000737:chemistry* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D013997:Time Factors; D000097629:Nociceptin
D003546:Cysteine Endopeptidases; D018076:DNA, Complementary; D008024:Ligands; D009097:Multienzyme Complexes; D018847:Opioid Peptides; D046988:Proteasome Endopeptidase Complex; D000097629:Nociceptin