Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5‐4864 Recognition Site

Abstract: This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17‐50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octa‐decaneuropeptide, DBI 33‐50) antibodies coupled to CNBr‐Sepharose 4B followed by two reverse‐phase HPLC steps. The amino acid sequence of the purified peptide is: Thr‐Gln‐Pro‐Thr‐Asp‐Glu‐Glu‐Met‐Leu‐Phe‐Ile‐Tyr‐Ser‐His‐Phe‐Lys‐Gln‐Ala‐Thr‐Val‐Gly‐Asp‐Val‐Asn‐Thr‐Asp‐Arg‐Pro‐Gly‐Leu‐Leu‐Asp‐Leu‐Lys. Synthetic TTN injected intra‐cerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific “peripheral” benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the “central” BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5‐4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 μM. TTN also enhances picrotoxinin inhibition of γ‐aminobutyric acid (GABA)‐stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at “Ro 5‐4864 preferring” BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor‐mediated effects.