Isolation and characterization of a rat brain triakontatetraneuropeptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro 5-4864 recognition site.
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 octadecaneuropeptide, 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 intracerebroventricularly 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 microM. TTN also enhances picrotoxinin inhibition of gamma-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.
Slobodyansky
E
E
FIDIA-Georgetown Institute for the Neurosciences, Georgetown University School of Medicine, Washington, D.C. 20007.
Guidotti
A
A
Wambebe
C
C
Berkovich
A
A
Costa
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex; D001253:Astrocytes / Q000378:metabolism; D001570:Benzodiazepinones / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002531:Cerebellum / Q000378:metabolism; D003220:Conflict, Psychological / Q000187:drug effects; D003292:Convulsants / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D026261:Diazepam Binding Inhibitor; D004597:Electroshock; D008297:Male; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D009830:Olfactory Bulb / Q000378:metabolism; D010446:Peptide Fragments; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism
D000936:Antigen-Antibody Complex; D001570:Benzodiazepinones; D003292:Convulsants; D026261:Diazepam Binding Inhibitor; D009479:Neuropeptides; D010446:Peptide Fragments; C060941:triakontatetraneuropeptide; C028513:4'-chlorodiazepam