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 K_i 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 GABA_A receptor-mediated effects.