Isolation and identification of a peptide from rat brain which inhibits [3H]TCP binding

1. A stereospecific radioreceptor binding assay for the phencyclidine analogue [3H]TCP was utilized to screen for inhibitors of binding in extracts of rat brain. 2. Extracts were prepared from rat cortex and hippocampus by methods employing aqueous acid or acidified methanol. Samples were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The most active fraction was further purified by high performance-size exclusion chromatography. 3. Size exclusion chromatography revealed two zones of activity, corresponding to mol. wts of 4000-8000 Da and 1000-2000 Da. Final purification of the lower molecular weight material was achieved by RP-HPLC. 4. Two well-separated peaks were shown to be homogeneous. Their amino acid sequences were determined by automated Edman degradation and data base searching identified these two peaks as the undecapeptide Substance P and its oxidized counterpart (Substance P sulfoxide). 5. Comparative HPLC of synthetic Substance P, or its sulfoxide, as well as spectral analysis confirmed the identity of the isolated peptides. 6. Synthetic Substance P inhibits specific [3H]TCP binding in the radioreceptor assay.