NON-RADIOACTIVE DETECTION OF RECEPTORS IN BRAIN

THE OVERALL GOAL OF THIS PROJECT IS TO ESTABLISH THE TECHNOLOGY FOR CARRYING OUT RECEPTOR STUDIES IN THE BRAIN WITH NONRADIOACTIVE LIGANDS. THE SPECIFIC AIMS OF THE RESEARCH PROGRAM ARE TO DETERMINE OPTIMAL METHODS FOR: (1) COUPLING BIOTIN TO SYNTHETIC NEUROPEPTIDES WITHOUT CHANGING THEIR RECEPTOR AFFINITY; (2) IMMOBILIZING THE DERIVATIZED LIGAND, IF NECESSARY, ON THE BOUND RECEPTOR; AND (3) DETECTING THE BOUND LIGAND VIA ENZYME-LINKED, AVIDIN-BASED REAGENTS. THE PROGRAM WILL BE CARRIED OUT BY USING A NOVEL SITE-SPECIFIC BIOTINYLATION PROCEDURE TO SELECTIVELY LABEL AMINO ACIDS AT A NONCRITICAL REGION OF THE PEPTIDE LIGAND, WHICH WILL BE A BETA- ENDORPHIN IN THIS FEASIBILITY STUDY. RECEPTOR-BINDING STUDIES WILL THEN BE CARRIED OUT USING RAT BRAIN MEMBRANE PREPARATIONS OR HISTOLOGICAL SECTIONS. USINGAVIDIN-BIOTINYLATED HORSERADISH PEROXIDASE AS THE DETECTION REAGENT, BINDING WILL BE DETECTED SPECTROPHOTOMETRICALLY (MEMBRANE ASSAYS) OR BY LIGHT MICROSCOPY (HISTOCHEMICAL ASSAYS). BECAUSE OF THE FUNDAMENTAL AND CLINICAL SIGNIFICANCE OF RECEPTOR BIOLOGY, THERE IS A VERY LARGE USER BASE FOR RADIOLABELED RECEPTOR LIGANDS. HOWEVER, THE INTRODUCTION OFUSEFUL NONRADIOACTIVE LIGANDS WOULD PROVIDE SAFER, LESS EXPENSIVE, AND MORE STABLE REAGENTS FOR RECEPTOR STUDIES.