A. Berkovich

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.

The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: Regulation of steroid biosynthesis

The rate-limiting step in the biosynthesis of steroids is the transport of the substrate cholesterol from the outer to the inner mitochondrial membrane, where cholesterol is metabolized to pregnenolone. This transport is markedly stimulated by the action of hormones, such as adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) for adrenocortical and testicular Leydig cells, respectively. Recently, it was demonstrated that the peripheral-type or mitochondrial benzodiazepine receptor, abundant in steroidogenic tissues, is involved in the regulation of steroid biosynthesis. In search for an endogenous ligand for mitochondrial benzodiazepine receptors, regulating steroidogenesis, the effects of Diazepam Binding Inhibitor (DBI) were studied. The model systems used were the Y-1 adrenocortical and the MA-10 Leydig cell lines, previously shown to be valid steroidogenic models. Both cell lines contain significant levels of immunoreactive DBI. Purified DBI from rat brain, at high nanomolar concentrations, increased formation of pregnenolone, when added to mitochondrial preparations of both cell types; but at concentrations of DBI above 1 microM, a decrease in the stimulation was observed. Flunitrazepam, a benzodiazepine which binds to mitochondrial benzodiazepine receptors, with high nanomolar affinity, inhibited the stimulatory action of DBI on the formation of mitochondrial pregnenolone, indicating that DBI exerts its stimulatory effects through an action on mitochondrial benzodiazepine receptors. In order to determine the biologically active amino acid sequence in the DBI molecule, various fragments of DBI were synthesized and tested; also, peptides structurally unrelated to DBI were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

DBI (Diazepam Binding Inhibitor): The Precursor of a Family of Endogenous Modulators of GABAA Receptor Function. History, Perspectives, and Clinical Implications*

Biochemical, electrophysiological, and lately, molecular biological techniques have shown that GABAA receptors are heterogeneous supramolecular complexes and can be divided into at least three major subgroups: GABAA1, GABAA2, and GABAA3. They differ mainly in the structural and functional properties of the allosteric modulatory center associated with each one of them. This paper will review the present state of research based on the evidence that DBI (diazepam binding inhibitor) and its natural processing products can selectively modulate GABAergic transmission at different GABAA receptor subtypes. Furthermore, the possibility that the DBI family of peptides represents a novel and meaningful neurochemical correlate for neuropsychiatric pathology, sustained by an alteration of GABAergic transmission, will be discussed.

A study of diazepam binding inhibitor (DBI) processing products in human cerebrospinal fluid and in postmortem human brain.

Diazepam binding inhibitor (DBI) is a neuropeptide of 11 kDa molecular size and is unevenly distributed in human and rat brain. It appears to function as a negative allosteric modulator of GABAA receptors. In the present paper, using antibodies directed against several synthetic peptides, which correspond to selective regions of human DBI (DBI 51-70, DBI 37-50, DBI 81-101), it is shown that DBI is processed into at least 6 peptide fragments in both postmortem human brain and in cerebrospinal fluid (CSF). One of these fragments was identified as the synthetic DBI 51-70 fragment (an eikosaneuropeptide, ENP) by combined chromatographic procedures. Immunoblotting analysis of the other fragments, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), revealed an apparent molecular size, ranging from 3-4 kDa for four of them and a larger molecular form of 8 kDa. On the basis of the immunological properties, a tentative amino acid sequence was deduced.