We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH(2) sequence, SIKPSAYLPLRF-NH(2) (RFamide peptide), from the quail brain. This quail RFamide peptide was shown to decrease gonadotropin release from the cultured anterior pituitary and to be located at least in the quail hypothalamo-hypophysial system. We therefore designated this RFamide peptide gonadotropin inhibitory hormone (GnIH). In the present study we characterized the GnIH cDNA from the quail brain by a combination of 3' and 5' rapid amplification of cDNA ends ('RACE'). The deduced GnIH precursor consisted of 173 amino acid residues, encoding one GnIH and two putative gene-related peptide (GnIH-RP-1 and GnIH-RP-2) sequences that included -LPXRF (X=L or Q) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the gene encoding GnIH in the diencephalon including the hypothalamus. Furthermore, mass spectrometric analyses detected the mass numbers for matured GnIH and GnIH-RP-2, revealing that both peptides are produced from the precursor in the diencephalon as an endogenous ligand. Taken together, these results lead to the conclusion that GnIH is a hypothalamic factor responsible for the negative regulation of gonadotropin secretion. Furthermore, the presence of a novel RFamide peptide family containing a C-terminal -LPXRF-NH(2) sequence has been revealed.

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Four peptidyl inhibitors of the small-conductance Ca2+-activated K' channels (SK,,) have been isolated from the venom of the Chinese scorpion Buthus martensi. These peptides were identified by screening C,, HPLC fractions of the crude venom by means of mass analysis by matrix-assisted-laser-desorption/ ionization time-of-flight mass spectrometry, and toxicological tests in mice. Edman degradation analysis of the purified peptides showed sequences of 28-31 amino acids including 6 cysteine residues. Three of the sequences were similar to the PO1 peptides from Androctonus scorpions, showing 76% sequence similarity for the most closely related, named BmPO1, and 46% for the other two, named BmP02 and BmP03. Like the PO1 peptides, these molecules showed a low toxic activity in mice after intracerebroventricular injection, and competed (K,,, > 1 pM) with iodinated apamin for binding to its receptor site from rat brain, which has been proved to be the SK,, channels. The fourth toxin was structurally related to the POS/leiurotoxin I toxin family, with 90% similarity, and was named BmPO5. This toxin exhibited a high toxic activity with lethal effects in mice. Due to its small representation in the venom [less than 0.01% (by mass)], its biological properties have been assessed on the synthetic analogue of BmPOS, which was assembled on a solid phase by means of Fmoc methodology. The synthetic peptide was physicochemically identical to the natural peptide, as shown by comparison of their molecular masses and amino acid compositions, and by their coelution after coinjection on capillary electrophoresis. These results confirmed the primary structure of BmPO5 including an amidated C-terminus. Similarly to natural BmPO5, synthetic BmPO5 produced toxic and lethal effects after intracerebroventricular injection in mice (LD,,, = 37 ng), and was able to compete with iodinated apamin for binding to its receptor in rat brain

Three novel toxins belonging to the scorpion K+ channel-inhibitor family were purified to homogeneity from the venom of the Chinese scorpion Buthus martensi. They have been identified according to their molecular mass (3800-4300 Da) and their neurotoxicity in mice and characterized as 37-amino acid peptides. One of them shows 81-87% sequence identity with members of the kaliotoxin group (named BmKTX), whereas the other two, named BmTX1 and BmTX2, show 65-70% identity with toxins of the charybdotoxin group. Their chemical synthesis by the Fmoc methodology allowed us to show that BmKTX, unlike BmTX1 and BmTX2, possesses an amidated C-terminal extremity. Toxicity assays in vivo established that they are lethal neurotoxic agents in mice (LD50s of 40-95 ng per mouse). Those toxins proved to be potent inhibitors of the voltage-gated K+ channels, as they were able to compete with [125I]kaliotoxin for its binding to rat brain synaptosomes (IC50s of 0.05-1 nM) and to block the cloned voltage-gated K+ channel Kv1.3 from rat brain, expressed in Xenopus oocytes (IC50s of 0.6-1.6 nM). BmTX1 and BmTX2 were also shown to compete with [125I]charybdotoxin for its binding to the high-conductance Ca2+-activated K+ channels present on bovine aorta sarcolemmal membranes (IC50s of 0.3-0.6 nM). These new sequences show multipoint mutations when compared to the other related scorpion K+ channel toxins and should prove to be useful probes for studying the diverse family of K+ channels.