The complete amino acid sequence of brevilysin H6 (H6), a zinc-protease isolated from Gloydius halys brevicaudus venom, was determined by a manual Edman degradation method. H6 has an amino-terminal pyroglutamic acid and consists of a total of 419 residues. An N-linked sugar chain is attached at Asn-181. The molecule is composed of three domains (metalloprotease, disintegrin-like and cysteine-rich domains), as commonly found in other high molecular mass metalloproteases from snake venoms. In the absence of calcium ions, H6 is autocatalytically degraded with a half-life of 47 min to give 29 and 45 kDa fragments, which correspond to residues 208-419 and 99-419 of H6, respectively. Thus, the autoproteolysis seemed to start from the cleavage of either the Leu(98)-Leu(99) or Asp(207)-Ile(208) bond. Calcium ions suppressed both the formation of the 45 kDa fragment and the rate of autoproteolysis. Calcium ions also contributed to the stability of H6 against pH, heating, urea and cysteine. More than twenty-five peptide bonds adjacent to hydrophobic residues in the metalloprotease domain were progressively cleaved during the autoproteolysis.

Two platelet aggregation inhibitors, ussuristatin 1 (US-1) and 2 (US-2), were newly isolated from the venom of Chinese viper (Agkistrodon ussuriensis) by SP-Toyopearl 650M column chromatography and reverse-phase HPLC. The Mrs of these polypeptides were estimated to be about 8,000 by SDS-PAGE. Analytical gel filtration revealed that US-2 exists as a dimer. Both polypeptides comprised 71 amino acids, whose sequences showed high similarities to those of other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-Gly-Asp (KGD). US-1 strongly suppressed platelet aggregation induced by ADP, collagen, thrombin, and epinephrine with IC50 = 17-33 nM. US-2 also inhibited the platelet aggregation, but the IC50s were about ten times higher. US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin, while US-2 did not inhibit the cell adhesion to fibronectin. This indicates that the KGD-bearing disintegrin is a specific inhibitor for the fibrinogen receptor.

Genes for Bowman-Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.Key words: cDNA cloning; Bowman-Birk inhibitors; Glycine soja; isoinhibitors; pseudogene Proteinase inhibitors are found in various plants and have been studied in the Leguminae, Gramineae, and Solanaceae.1,2) Especially, proteinase inhibitors of the Bowman-Birk family (BBIs) and the Kunitz family were discovered first in soybean seeds and have been well studied. [3][4][5] A number of functions have been proposed for BBIs, including the regulation of endogenous proteinases during germination, the storage of sulfur amino acids during dormancy, and the protection of the plant from insects and microorganisms, [6][7][8] while recent interest has focused on its medicinal utility as an antitumor agent, for instance.

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Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SPToyopearl 650M, Sephacryl S-200SF, and DEAEToyopearl 650S columns. Reverse-phase HPLCˆnally gave pure inhibitors. All of the inhibitors inhibited trypsin with dissociation constants of 3.2-6.2×10 -9 M. Some of the inhibitors inhibited chymotrypsin and elastase as well. Two inhibitors (VIIb and VIII) with a molecular weight of 20,000 were classiˆed as a soybean Kunitz inhibitor family. Others (I-VIIa) had a molecular weight of about 8,000, and were stable to heat and extreme pH, suggesting that these belonged to the Bowman-Birk inhibitor family. Partial amino acid sequences of four inhibitors were also analyzed. The complete sequence of inhibitor IV was ascertained from the nucleotide sequences of cDNA clones encoding isoinhibitors homologous to soybean C-II.