LONDRAVILLE, RL; KINTER, M; University of Akron; Lerner Research Institute, Cleveland Clinic Foundation: Immunopurification of anti-leptin reactive peptide from brain of green sunfish, Lepomis cyanellus

We have isolated and sequenced peptides from fish brain that putatively align with mammalian leptins. Leptin is a small (16 kDa) protein hormone that influences fat storage, metabolic rate, onset of reproduction, immune function and bone density. Leptin existence and potential function is largely uncharacterized in non-mammals. We used an anti-mouse leptin antibody (that reacts to leptin-like peptide in fish) on an immobilized support to immunoprecipitate a putative fish leptin from green sunfish brain homogenate. This column eluted proteins that resolved into three major bands by SDS-PAGE. All bands were in the size range of mammalian leptin and one was recognized by the anti-leptin antibody (immunoblot). Each of the three bands were subjected to in-gel trypsin digestion followed by LC-tandem MS and Maldi-ToF analyses to generate sequence for over 25 individual peptides. Some of these peptides were identified by homology to highly conserved proteins from fishes (40S ribosomal protein and fatty acid-binding proteins). Three peptides putatively align with mammalian leptin (consensus sequence). We have generated primers based on these peptides, and are attempting RT-PCR amplification of the corresponding message from fish brain.