TY - JOUR
T1 - Integrity of tritiated orphanin FQ/Nociceptin
T2 - Implications for establishing a reliable binding assay
AU - Quigley, Denise Irene
AU - Arttamangkul, Seksiri
AU - Allen, Richard Gordon
AU - Grandy, David Kilgore
N1 - Funding Information:
The work was supported by a grant from NIDA (DA 10703, DKG and DA 11282, RGA).
PY - 2000/7
Y1 - 2000/7
N2 - 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.
AB - 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.
KW - Nociceptin
KW - OFQ receptor
KW - Orphanin FQ
KW - Peptide degradation
KW - Radioligand binding
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U2 - 10.1016/S0196-9781(00)00248-5
DO - 10.1016/S0196-9781(00)00248-5
M3 - Article
C2 - 10998545
AN - SCOPUS:0033816845
SN - 0196-9781
VL - 21
SP - 1111
EP - 1118
JO - Peptides
JF - Peptides
IS - 7
ER -