The objective of this proposal is to develop sensitive, non-isotopic bioluminescent ligand receptor assays for oxytocin (OT) and arginine-vasopressin (AVP). Our laboratory has developed methods to covalently bind firefly luciferin to various biologically active molecules. Under the appropriate conditions, the luciferin-derivatized molecule retains it biologic function and also has luciferin activity (ie., the ability to produce photons of light in the presence of luciferase, magnesium and ATP). In effect, we are able to """"""""label"""""""" various molecules, such as proteins, peptides and steroids with a non-radioactive probe. The luciferin-derivatized molecule may then be used in bioluminescent ligand receptor assays, analogous to radio-ligand receptor assays. Because of the sensitivity of the luciferin-luciferase reaction (measureable light is produced with 10 atamoles of luciferin) these assays are 500-5000 fold more sensitive than existing assays using H-3-labeled compounds. This increased sensitivity is extremely important for the localization and characterization of specific neuropeptide receptor sites in discrete brain regions. OT and AVP neurons have been visualized in many brain regions however specific binding sites have been difficult to localize and characterize due the relatively low specific activity of the available radiolabeled ligands. Consequently, the increased sensitivity of luciferin-derivatized OT and AVP will be extremely important for the localization and characterization of specific receptor sites in discrete brain regions. This information will be very important in further elucidation of the physiological importance of OT and AVP in brain function. The second goal of this research will be to develop luciferin analogs which may be more readily bound to other molecules, enabling us to more widely utilize luciferin-derivatized compounds as an alternative to radiolabeled compounds.
