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Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturationinducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin.gonadotropin ͉ gonad-stimulating substance ͉ insulin-like growth factor/relaxin superfamily ͉ 1-methyladenine ͉ in situ hybridization

Venomous mammals are rare, and their venoms have not been characterized. We have purified and characterized the blarina toxin (BLTX), a lethal mammalian venom with a tissue kallikrein-like activity from the submaxillary and sublingual glands of the shorttailed shrew Blarina brevicauda. Mice administered BLTX i.p. developed irregular respiration, paralysis, and convulsions before dying. Based on the amino acid sequence of purified protein, we cloned the BLTX cDNA. It consists of a prosequence and an active form of 253 aa with a typical catalytic triad of serine proteases, with a high identity with tissue kallikreins. BLTX is an N-linked microheterogeneous glycoprotein with a unique insertion of 10 residues, L 106 TFFYKTFLG 115 . BLTX converted kininogens to kinins, which may be one of the toxic pathogens, and had dilatory effects on the blood vessel walls. The acute toxicity and proteolytic activity of BLTX were strongly inhibited by aprotinin, a kallikrein inhibitor, suggesting that its toxicity is due to a kallikrein-like activity of the venom.

A homolog of the serinehhreonine protein kinase (~3 4~~~' ) , encoded by the cdcZf gene of the fission yeast (Schizosaccharomyces pombe), is a catalytic subunit of maturation-promoting factor and a key regulator of the cell cycle. We have raised a monoclonal antibody against the most conserved amino acid sequence, the PSTAIR sequence (EGVPSTAIREISLLKE) of ~3 4~~~' This antibody recognizes 31 -34 kDa proteins by immunoblotting in all species examined so far. The proteins recognized by the anti-PSTAIR antibody are probably either ~3 4~~~' itself or proteins highly homologous to ~3 4~~~' in the given species, since, in all species studies to date, they are all precipitated with p13suc1, the fission yeast S U C~+ gene product, which binds to ~3 4~~~' with high specificity. The anti-PSTAIR imrnunoprecipitate had no histone H1 kinase activity and did not contain cyclin 6, suggesting that the PSTAIR region is masked when ~34'~'' forms a complex with cyclin B as an active kinase. lmmunoblotting with the anti-PSTAIR antibody demonstrated that the fastest-migrating form of ~3 4~~~' homologues becomes abundant, when oocytes mature or the cell enters M phase. The possible significance of this observation is discussed in relation to the phosphorylation and activity state of p34cdc2 The observed broad cross-reactivity of the anti-PSTAIR antibody against ~34'~'' homologues in various species should permit us to examine the role of ~34'~'' homologues in the regulation of the cell cycle in a variety of organisms.

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Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 muM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.

A cDNA encoding a nuclear 17K K,20L L-dihydroxy-4-pregnen-3-one (17K K,20L L-DP, spermiation-inducing hormone in fish) receptor (DPR) was, for the first time, isolated from an eel testis cDNA library. The amino acid sequence of DPR shows high homology with those of human and chicken progesterone receptors. The affinity of the bacterial recombinant DPR ligand binding domain protein for 17K K,20L L-DP is higher than that of progesterone. In transfection experiments using COS7 cells, the DPR showed progestin-dependent activation of transcription. 17K K,20L L-DP was the most effective activator of transcription. These results indicate that the cDNA encodes a functional eel DPR, and show that 17K K,20L L-DP has a nuclear receptormediated action in eel testes.z 2000 Federation of European Biochemical Societies.

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