抄録
Neuropeptides, nocistatin (NST) and nociceptin/orpahnin FQ (N/OFQ), are produced from the same precursor, prepronociceptin/orphanin FQ (ppN/OFQ). Although all opioid precursors contain several bioactive peptides with similar functions, NST and N/OFQ have opposite central functions including pain transmission, learning and memory. Protein processing has been measured biochemically in the lysate of destroyed cells and a cell-free system. To investigate the regulation of NST and N/OFQ production from ppN/OFQ in living cells and in vivo, we developed a novel intra-molecular BRET system between a luciferase and fluorescent protein. This BRET system demonstrated high efficiency of BRET and the real-time and continuous BRET assay, and the change in BRET signals quantified the processing of the protein. This BRET had the desired profile for monitoring the protein processing of NST and N/OFQ in intact cells, and this BRET system showed that proprotein convertases, PC 1 and furin, were involved in the production of these peptides. Since our novel intra-molecular BRET system is particularly useful for studies on protein processing in living cells, it can be applicable to the elucidation of regulation of biological functions of NST and N/OFQ in vivo. [Jpn J Physiol 54 Suppl:S43 (2004)]
