Shigeyuki Terada

Purification and Characterization of Proteinase Inhibitors from Wild Soja (Glycine soja) Seeds

Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SP-Toyopearl 650M, Sephacryl S-200SF, and DEAE-Toyopearl 650S columns. Reverse-phase HPLC finally 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 classified 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.

Multigene family for Bowman-Birk type proteinase inhibitors of wild soja and soybean: the presence of two BBI-A genes and pseudogenes.

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.

[1] Analysis of ascorbic acid, dehydroascorbic acid, and transformation products by ion-pairing high-performance liquid chromatography with multiwavelength ultraviolet and electrochemical detection

Publisher Summary This chapter focuses on the analysis of ascorbic acid, dehydroascorbic acid, and transformation products using ion-pairing high-performance liquid chromatography with multiwavelength ultraviolet and electrochemical detection. Preparative separation of transformation products is carried out by DEAE-Sepharose column chromatography. Elution is carried out with 10 m M sodium phosphate buffer, pH 6.0. The eluates are examined by absorption spectrum and electrochemical detection–high-performance liquid chromatography (ECD–HPLC) analysis. L -ascorbic acid (AsA), erythro- L -ascorbic acid (EAsA), and the reductant R-345, possess an absorption maximum at 345 nm. Analysis by ECD–HPLC reveals three main ECD-active spots—AsA, EAsA, and a reductant possessing an absorption maximum at 290 nm. The latter reductant has a retention time a little less than that of AsA, and it is eluted in the void volume on a DEAE-Sepharose column, showing a less negative charge. Its absorption maximum at 290 nm in neutral aqueous solution shifts to 335 nm above pH 9.0.

Purification and characterization of a non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus venom

A non-hemorrhagic metalloprotease (protease L4) was purified from the venom of Chinese Mamushi (Agkistrodon halys brevicaudus) by gel filtration and anion-exchange chromatography. Protease L4 has the molecular weight of 22,000 and its optimum pH was 8.5. The protein was stable in the pH range of 5-9 and below 40 degrees C. The proteolytic activity was inhibited by metal-chelating agents and some metal ions. Calcium ion activated the activity dose-dependently, but had only a minor effect on the thermal and pH stability. L4 showed fibrinogenase activity, hydrolyzing only the A alpha chain of fibrinogen. The protease cleaved preferentially at the N-terminal of Leu and His residues of some peptides.

Purification, Amino Acid Sequence, and cDNA Cloning of Trypsin Inhibitors from Onion (Allium cepa L.) Bulbs

Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3×10-9 M, 2.3×10-7 M, and 3.1×10-7 M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.

Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum.

Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.

Structural requirement for the biological activity of serum thymic factor

Several polypeptides have been claimed for thymic factors (hormones) which mediate the differentiation of T lymphocyte precursors to more matured T cells [ l]. Bach et al. [2] have obtained a circulating thymic factor from pig serum and shown its thymus origin. This factor has been reported to exhibit various immunological activities including @-conversion in vitro and in vivo [3], ind~lction of suppressor T cells in NZB mice [4], normalization of the abnormally high level of autologous erythrocyte-binding cells in ATx mice [S]. The chemical structure of this factor, designated Facteur Thymique Sdrique (FTS), was elucidated as a nonapeptide, <GIu-Ala-Lys-SerGln-Gly-Gly-Ser-Asn-OH [6]. There exist few reports on the influence of the chemical structure of this factor on the biological activity [7,8]. Here, we describe the 8-conversion activity of synthetic FTS and its short chain analogs. The pentapeptide moiety (Lys-Ser-Gln-Gly-Gly) is shown to be a minimum essential part for the expression of full agonistic activity and the C-terminal part may have an enhancing role in the binding of this pentapeptide portion.

Accelerated evolution of small serum proteins (SSPs)-The PSP94 family proteins in a Japanese viper.

Five small serum proteins (SSPs) with molecular masses of 6.5-10 kDa were detected in Habu (Trimeresurus flavoviridis) serum; this included two novel proteins SSP-4 and SSP-5. The amino acid sequences of these proteins and of SSP-1, SSP-2, and SSP-3, which were reported previously, were determined on the basis of the nucleotide sequences of their cDNAs. Although these proteins exhibited only limited sequence identity to mammalian prostatic secretory protein of 94 amino acids (PSP94), the topological pattern of disulfide bonds in SSPs was identical to that of the mammalian proteins. SSP-3 and SSP-4 lacked approximately 30 residues at the C-terminal. Each of the full-length cDNAs encoded a mature protein of 62-90 residues and a highly conserved signal peptide. The evolutionary distances between SSPs estimated on the basis of the amino acid changes were significantly greater than those of the synonymous nucleotide substitutions; these finding, together with results from analyses of nonsynonymous to synonymous rates of change (dN/dS) suggest that snake SSPs have endured substantial accelerated adaptive protein evolution. Such accelerated positive selection in SSPs parallels other findings of similar molecular evolution in snake venom proteins and suggests that diversifying selection on both systems may be linked, and that snake SSP genes may have evolved by gene duplication and rapid diversification to facilitate the acquisition of various functions to block venom activity within venomous snakes.

Snake fetuin: isolation and structural analysis of new fetuin family proteins from the sera of venomous snakes.

Novel proteins were isolated from the sera of Chinese Mamushi (Gloydius blomhoffi brevicaudus) and Habu (Trimeresurus flavoviridis). The primary structures of these proteins were determined by protein sequencing, and the nucleotide sequences were established by cDNA cloning from the liver mRNAs. They belonged to the fetuin family having a double-headed cystatin-like domain and a His-rich domain, akin to HSF, an antihemorrhagic factor isolated from Habu serum. They showed no antihemorrhagic activity and were designated HSF-like proteins (HLPs). Mamushi serum contained two different HLPs termed HLP-A and HLP-B. Both HLP-A and Habu HLP had a unique 17-residue deletion in their His-rich domains. HLP-B comprised two glycosylated polypeptide chains and inhibited the precipitation of calcium phosphate as potently as does bovine fetuin. HLP-B was hence identified as a snake fetuin. The phylogenetic analysis of the fetuin family of proteins showed that antihemorrhagins and HLPs have evolved from this snake fetuin.

Primary structures of concanavalin a-like lectins from seeds of two species of Canavalia

The amino acid sequences of two lectins from the seeds of Canavalia lineata and C. virosa have been determined by the manual Edman degradation method. Both proteins were found to be highly homologous to concanavalin A, a lectin from C. ensiformis. All the residues suggested to participate in binding to carbohydrates and metal ions are completely conserved in the proteins.