Researcher Database

Takao Ojima
Faculty of Fisheries Sciences Marine Life Science Marine Biotechnology and Microbiology
Professor

Researcher Profile and Settings

Affiliation

  • Faculty of Fisheries Sciences Marine Life Science Marine Biotechnology and Microbiology

Job Title

  • Professor

URL

J-Global ID

Research Interests

  • 遺伝子工学   酵素化学   タンパク質化学   

Research Areas

  • Life sciences / Marine/Aquatic life sciences / Marine Biotechnology

Academic & Professional Experience

  • 1983/04 - Today Hokkaido University Faculty of Fisheries Sciences Professor

Education

  • 1979/04 - 1983/03  Graduate school of Hokkaido University  Graduate School of Fisheries  Department of Fisheries Chemistry
  • 1975/04 - 1979/03  Hokkaido University  Faculty of Fisheries  Department of Fisheries Chemistry

Association Memberships

  • 北海道海洋生物科学研究会   日本生物物理学会   マリンバイオテクノロジー学会   日本水産学会   日本農芸化学会   日本生化学会   日本応用糖質科学会   

Research Activities

Published Papers

  • Takao Ojima, Mohammad M. Rahman, Yuya Kumagai, Ryuji Nishiyama, Joemark Narsico, Akira Inoue
    Methods in Enzymology 605 457 - 497 1557-7988 2018/01/01 [Refereed][Invited]
     
    Seaweed polysaccharides have been widely used as viscosifier, gelling agents, and stabilizer in the various application fields, e.g., food, pharmaceutical, nutraceutical, and chemical industries. Applications of seaweed polysaccharides are further expanding to versatile directions, e.g., biofuels, bioactive compounds, and functional materials for medical and basic researches. Production of functional oligo- and monosaccharides by the use of specific enzymes is also expected to improve the value of seaweed polysaccharides. The enzymes that depolymerize seaweed polysaccharides are distributed largely among seaweed-associating organisms like marine invertebrates and bacteria. Among them, herbivorous marine gastropods such as abalone and sea hare are the most prominent producers of polysaccharide-degrading enzymes. To date, various kinds of polysaccharide-degrading enzymes have been isolated from the digestive fluid and hepatopancreas of these animals. Among them, alginate lyase, β-1,3-glucanase, mannanase, and cellulase are the major constituents of their digestive fluid. In this chapter, the authors describe the general methods for the preparation and activity assay of the gastropod polysaccharide-degrading enzymes and provide basic knowledge for their primary structures.
  • Crystal structure of laminarinase from Flavobacterium sp. UMI-01 reveals distinct thermostability and substrate specificity from other GH16 family members.
    H-M. Qin, T. Miyakawa, A. Inoue, A. Nakamura, T. Ojima, M. Tanokura
    Sci. Rep. 7 (11425) 1 - 9 2017 [Refereed][Not invited]
  • Continuous saccharification of laminarin by immobilized laminarinase ULam111 followed by ethanol fermentation with a marine-derived yeast.
    D. Mitsuya, M. Yamamoto, M. Okai, A. Inoue, T. Suzuki, T. Ojima, N. Urano
    Adv. in Microbiol. 7 387 - 403 2017 [Refereed][Not invited]
  • Identification of 2-keto-3-deoxy-D-gluconate kinase and 2-keto-3-deoxy-D-phosphogluconate aldolase in an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01.
    R. Nishiyama, A. Inoue, T. Ojima
    Mar. Drugs 15 (37) 1 - 17 2017 [Refereed][Not invited]
  • Structure and polymannuronate specificity of a eukaryotic member of the polysaccharide lyase family 14.
    H-M. Qin, T. Miyakawa, A. Inoue, R. Nishiyama, A. Nakamura, A. Asano, Y. Sawano, T. Ojima, M. Tanokura
    J. Biol. Chem. 292 2182 - 2190 2017 [Refereed][Not invited]
  • Akira Inoue, Ryuji Nishiyama, Takao Ojima
    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS 19 355 - 362 2211-9264 2016/11 [Refereed][Not invited]
     
    We recently reported the isolation of Flavobacterium sp. UMI-01 as a novel alginate-assimilating bacterium from decayed brown algae. In our previous study, FlAlyA was purified and characterized as an endolytic alginate lyase, producing alginate oligosaccharides (AOSs) from its polymer. Additionally, crude extracts from strain UMI-01 were found to be capable of producing of 4-deoxy-L-erythro-5-hexoseulose uronic acids (DEHs) through degradation of AOSs. Here, we report the enzymatic characteristics of four alginate lyases, i.e., FlAlyA, FlAlyB, FlAlyC, and FlAlex, found in the genomic sequence of UMI-01 using recombinant proteins. Our results demonstrated that the distinct roles of these enzymes facilitated complete degradation of alginate to DEH in strain UMI-01. First, FlAlyA attacked the alginate polymer in an endolytic manner, producing AOS. Recombinant FlAlyA could completely degraded alginate into AOS in vitro, and its activity was 20-52-fold higher than those of commercially available enzymes. Next, DEH was generated as a final degradation product through the functions of FlAlyB, FlAlyC, and FlAlex, which exhibited distinct substrate preferences; FlAlyB, FlAlyC, and FlAlex were identified as polyM-, polyMG-, and polyG-specific enzymes, respectively. Thus, our results confirmed that the combination of these four enzymes resulted in the most efficient degradation of alginate to DEH in vitro. (C) 2016 Elsevier B.V. All rights reserved.
  • Akira Inoue, Moe Anraku, Satoshi Nakagawa, Takao Ojima
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 (30) 15551 - 15563 0021-9258 2016/07 [Refereed][Not invited]
     
    Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of epsilon-Proteobacteria Nitratiruptor sp.SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli. Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 degrees C and around 6, respectively. Ahigh concentration of NaCl (0.8-1.4 M) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 degrees C for 30 min. Heat stability was decreased in the presence of 5 mM DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.
  • Yuka Tanino, Takashi Hashimoto, Takao Ojima, Masashi Mizuno
    JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 59 (1) 25 - 30 0912-0009 2016/07 [Refereed][Not invited]
     
    Fucoidan is a sulfated polysaccharide from brown sea algae. In the present study, it was demonstrated that oral administration of F-fucoidan from Saccharina japonica possessed anti-allergic effects using the passive cutaneous anaphylaxis reaction, but not by intraperitoneal administration. The inhibitory mechanism was dependent on galectin-9, which belongs to a soluble lectin family that recognizes p-galactoside and prevents IgE binding to mast cells. The anti-allergy properties of F-fucoidan were cancelled by an intravenous dose of anti-galectin-9 antibody or lactose, which bind competitively with galectin-9 before the passive cutaneous anaphylaxis reaction. F-fucoidan increased the expression level of galectin-9 mRNA in intestinal epithelial cells and serum galectin-9 levels. Oral treatment with F-fucoidan suppressed allergic symptoms through the induction of galectin-9. This is the first report that F-fucoidan can induce the secretion of galectin-9.
  • Akira Inoue, Aya Satoh, Mio Morishita, Yuko Tokunaga, Takuya Miyakawa, Masaru Tanokura, Takao Ojima
    ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS 16 282 - 291 2211-9264 2016/06 [Refereed][Not invited]
     
    Brown algae produce alginate that has various ratios and diverse sequences of two uronic acids, beta-D-mannuronic acid and alpha-L-guluronic acid, compared with those of alginate produced by bacteria. This diversity of alginate in brown algae is caused by mannuronan C5-epimerases (MC5Es), which catalyze the conversion of beta-D-mannuronic acid to alpha-L-guluronic acid. Although several bacterial MC5E enzymes have been well characterized, to date, there exists no information on the biochemical properties of eukaryotic MC5E. In this study, MC5E expression was detected in a brown alga Saccharina japonica sporophyte by immunoblot analysis. We also searched for MC5E mRNA from S. japonica by RT-PCR and revealed eight partial amino acid sequences, SjC5-I to -VIII. We focused on the highest frequency clone, SjC5-VI, and elucidated its full-length cDNA and putative gene structure. The translated SjC5-VI protein consists of 499 amino acids, with the N-terminal 21 amino acids predicted as a secretion signal sequence. Functional recombinant SjC5-VI (rSjC5-VI) was successfully expressed as a secreted protein using an insect-cell expression system, and we determined the optimal temperature, pH, and NaCl concentrations to be 35 degrees C, 7.0-8.2, and 300 mM, respectively, using the Ca2+-induced gel-formation assay. In addition, Ca2+ enhanced gelation by 1.7-fold following rSjC5-VI activity. Furthermore, H-1-NMR spectroscopy of rSjC5-VI-treated polyM revealed alternate epimerization of beta-D-mannuronic acid to alpha-L-guluronic acid. To the best of our knowledge, this is the first report on the characterization of MC5E activity in eukaryotes. (C) 2016 Elsevier B.V. All rights reserved.
  • Shogo Mochizuki, Ryuji Nishiyama, Akira Inoue, Takao Ojima
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 (52) 30962 - 30974 0021-9258 2015/12 [Refereed][Not invited]
     
    Abalone feeds on brown seaweeds and digests seaweeds' alginate with alginate lyases (EC 4.2.2.3). However, it has been unclear whether the end product of alginate lyases (i.e. unsaturated monouronate-derived 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)) is assimilated by abalone itself, because DEH cannot be metabolized via the Embden-Meyerhof pathway of animals. Under these circumstances, we recently noticed the occurrence of an NADPH-dependent reductase, which reduced DEH to 2-keto-3-deoxy-D-gluconate, in hepatopancreas extract of the pacific abalone Haliotis discus hannai. In the present study, we characterized this enzyme to some extent. The DEH reductase, named HdRed in the present study, could be purified from the acetone-dried powder of hepatopancreas by ammonium sulfate fractionation followed by conventional column chromatographies. HdRed showed a single band of similar to 40 kDa on SDS-PAGE and reduced DEH to 2-keto-3-deoxy-D-gluconate with an optimal temperature and pH at around 50 degrees C and 7.0, respectively. HdRed exhibited no appreciable activity toward 28 authentic compounds, including aldehyde, aldose, ketose, alpha-keto-acid, uronic acid, deoxy sugar, sugar alcohol, carboxylic acid, ketone, and ester. The amino acid sequence of 371 residues of HdRed deduced from the cDNA showed 18-60% identities to those of aldo-keto reductase (AKR) superfamily enzymes, such as human aldose reductase, halophilic bacterium reductase, and sea hare norsolorinic acid (a polyketide derivative) reductase-like protein. Catalytic residues and cofactor binding residues known in AKR superfamily enzymes were fairly well conserved in HdRed. Phylogenetic analysis for HdRed and AKR superfamily enzymes indicated that HdRed is an AKR belonging to a novel family.
  • Satoshi Kimura, Kei Nakayama, Masahisa Wada, Ung-Jin Kim, Kaoru Azumi, Takao Ojima, Akino Nozawa, Shin-Ichi Kitamura, Euichi Hirose
    DISEASES OF AQUATIC ORGANISMS 116 (2) 143 - 148 0177-5103 2015/10 [Refereed][Not invited]
     
    Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.
  • Akira Inoue, Chieco Mashino, Toshiki Uji, Naotsune Saga, Koji Mikami, Takao Ojima
    Current biotechnology 4 (3) 240 - 248 2015/08 [Refereed][Not invited]
     
    BACKGROUND: Alginate lyases belonging to polysaccharide lyase family-7 (PL-7) are the most well studied on their structures and functions among whole alginate lyases. However, all characterized PL-7 alginate lyases are from prokaryotic bacteria cells. Here we report the first identification of eukaryotic PL-7 alginate lyase from marine red alga Pyropia yezoensis. METHODS: The cDNA encoding an alginate lyase PyAly was cloned and was used for the construction of recombinant PyAly (rPyAly) expression system in Escherichia coli. Purified rPyAly was assayed to identify its enzymatic properties. Its expression pattern in P. yessoensis was also investigated. RESULTS: PyAly is likely a secreted protein consisting of an N-terminal signal peptide of 25 residues and a catalytic domain of 216 residues. The amino-acid sequence of the catalytic domain showed 19-29% identities to those of bacterial characterized alginate lyases classified into family PL-7. Recombinant PyAly protein, rPyAly, which was produced with E. coli BL21(DE3) by cold-inducible expression system, drastically decreased the viscosity of alginate solution in the early stage of reaction. The most preferable substrate for rPyAly was the poly(M) of alginate with an optimal temperature and pH at 35oC and 8.0, respectively. After reaction, unsaturated tri- and tetra-saccharides were produced from poly(M) as major end products. These enzymatic properties indicated that PyAly is an endolytic alginate lyase belonging to PL-7. Moreover, we found that the PyAly gene is split into 4 exons with 3 introns. PyAly was also specifically expressed in the gametophytic haplopid stage. CONCLUSION: This study demonstrates that PyAly in marine red alga P. yezoensis is a novel PL-7 alginate lyase with an endolytic manner. PyAly is a gametophyte-specifically expressed protein and its structural gene is composed of four exons and three introns. Thus, PyAly is the first enzymatically characterized eukaryotic PL-7 alginate lyase.
  • Florian Lelchat, Stephane Cerantola, Christophe Brandily, Sylvia Colliec-Jouault, Anne-Claire Baudoux, Takao Ojima, Claire Boisset
    CARBOHYDRATE POLYMERS 124 347 - 356 0144-8617 2015/06 [Refereed][Not invited]
     
    We have studied the exopolysaccharide produced by Cobetia marina DSMZ 4741, a marine bacterium isolated from coastal seawater. This strain is able to produce a polysaccharide in presence of carbon sources as glucose, mannitol and alginate. The maximum production occurs in aerobic condition, during the end of the exponential phase. The polymer is a non-viscous, acidic heteropolysaccharide of 270 kDa constituted of a repeating unit of: -> 2)-beta-D-Ribf-(1 -> 4)-[7,8-O-(Pyr)]-alpha-D-KDOp-(2 -> This kind of chemical structure is generally related to K-antigen polysaccharide of pathogenic Escherichla coli strains. This is the first time this type of EPS is described from a marine bacterium. Moreover the polysaccharide exhibits a pyruvate substitution on its 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) residue never encountered before. The discovery of such an unexpected EPS with high biotechnological potential is a new incentive for a better exploration of bioactive marine resources. (c) 2015 Elsevier Ltd. All rights reserved.
  • Akira Inoue, Ryuji Nishiyama, Shogo Mochizuki, Takao Ojima
    MARINE DRUGS 13 (1) 493 - 508 1660-3397 2015/01 [Refereed][Not invited]
     
    In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-L-ery thro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-D-gluconate (KDG) by a specific reductase, and metabolized through the Entner-Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%-88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.
  • Akira Inoue, Kohei Takadono, Ryuji Nishiyama, Kenji Tajima, Takanori Kobayashi, Takao Ojima
    MARINE DRUGS 12 (8) 4693 - 4712 1660-3397 2014/08 [Refereed][Not invited]
     
    A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of similar to 30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 degrees C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I-Escherichia coli BL21(DE3) expression system and similar to eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.
  • Yuya Kumagai, Takuya Satoh, Akira Inoue, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 167 1 - 7 1096-4959 2014/01 [Refereed][Not invited]
     
    Endo-beta-1,3-glucanases (laminarinase, EC 3.2.1.6) from marine molluscs specifically degrades laminarin from brown algae producing laminaribiose and glucose, but hardly degrades laminaribiose. For the complete depolymerization of laminarin, other enzymes that can hydrolyze laminaribiose appear to be necessary. In the present study, we successfully isolated a laminaribiose-hydrolyzing enzyme from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatographies. This enzyme, AkLab, named after the scientific name of this animal and substrate specificity toward laminaribiose, shows an approximate molecular mass of 110 kDa on SDS-PAGE, and optimum pH and temperature at around pH 5.5 and 50 degrees C, respectively. AkLab rapidly hydrolyzes laminaribiose and p-nitrophenyl-beta-D-glucoside, and slowly cellobiose, gentiobiose and lactose, but not sucrose and maltose. AkLab shows high transglycosylation activity and can produce a series of laminarioligosaccharides larger than laminaritetraose from laminaribiose (a donor substrate) and laminaritriose (an acceptor substrate). This enzyme is suggested to be a member of glycosyl hydrolase family 1 by the analysis of partial amino-acid sequences. (C) 2013 Elsevier Inc. All rights reserved.
  • Mohammad M. Rahman, Akira Inoue, Takao Ojima
    FRONTIERS IN CHEMISTRY 2 1 - 13 2014 [Refereed][Not invited]
     
    The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95, 66, 45, and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40 degrees C, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55 degrees C for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic beta-1,4-glucanase (EC 3.2.1.4). A cDNA of 1013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.
  • Rendong Ren, Yosuke Azuma, Takao Ojima, Takashi Hashimoto, Masashi Mizuno, Yosuke Nishitani, Masaru Yoshida, Takeshi Azuma, Kazuki Kanazawa
    BRITISH JOURNAL OF NUTRITION 110 (5) 880 - 890 0007-1145 2013/09 [Refereed][Not invited]
     
    Laminaria japonica is traditionally eaten in Japan as a beneficial food for thrombosis. The alga contains two specific ingredients, a xanthophyll fucoxanthin (FX) and a polysaccharide, F-fucoidan (FD). The aim of the present study was to investigate whether FX or FD exhibited anti-thrombotic effects. For this purpose, three types of capsules, containing 1 mg FX, 400 mg fucoidan, and both, were prepared from the alga and administered to volunteers for 5 weeks. The dose of FD or FD + FX significantly shortened lysis time (LT) of the thrombus measured by a global thrombosis test in the blood, but FX did not. Examining the mechanism, dietary FD increased H2O2 and the secretion of prostacyclin (PGI(2)), a potent inhibitor of platelet aggregation, in the blood, although FD was under the detection limit in the blood, determining with its monoclonal antibody. Furthermore, in mouse experiments, dietary FD was totally excreted into the faeces and was not incorporated into the blood. We then employed a co-culture system of a Caco-2 cell monolayer with fresh human blood. The addition of FD to Caco-2 cells stimulated the expression of NADPH oxidase 1 (NOX1) and dual oxidase 2 (DUOX2) mRNA and secreted H2O2 onto the blood side accompanied by a significant increase in serum PGI2 production. These effects were invalidated by the combined addition of FD with its monoclonal antibody. The results suggested that dietary FD stimulated the expression of H2O2-producing enzymes in intestinal epithelial cells and released H2O2 into the blood, which played a signalling role to increase PGI(2) production and then shortened LT for thrombi.
  • Takuya Satoh, Akira Inoue, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 1 166 (1) 15 - 22 1096-4959 2013/09 [Refereed][Not invited]
     
    Previously, we isolated two alpha-amylase isozymes, HdAmy58 and HdAmy82, from the digestive fluid of the Pacific abalone Haliotis discus hannai (Kumagai et al., 2013, Comp. Biochem. Physiol., B 164, 80-88). These enzymes degraded starch producing maltooligosaccharides but not glucose. However, the digestive fluid itself could produce glucose from starch, indicating that the digestive fluid contains alpha-glucosidase-like enzymes together with the alpha-amylases. Thus, in the present study, we isolated this alpha-glucosidase-like enzyme from the digestive fluid and characterized it to some extent. Isolation of this enzyme was carried out by ammonium sulfate fractionation followed by conventional column chromatographies and FPLC. The purified enzyme showed an apparent molecular mass of 97 kDa on SDS-PAGE, and optimal temperature and pH of 45 degrees C and 3.8-5.5, respectively. This enzyme could degrade various sizes of maltooligosaccharides into glucose and released glucose from starch producing no appreciable intermediate oligosaccharides. We concluded that this enzyme is an alpha-glucosidase (EC 3.2.1.20) exolitically acting on polymer substrate and named HdAgl. HdAgl efficiently degraded maltose but hardly degraded p-nitrophenyl alpha - D-glucopyranoside (alpha-pNPG) and isomaltose. This enzyme was regarded as a maltase-like alpha-glucosidase that preferably degrades maltose but scarcely aryl glucosides. When starch was used as a substrate, HdAgl converted approximately 40% (w/w) of the starch to glucose. If an abalone alpha-amlylase HdAmy58 was added to the reaction mixture, the glucose yield increased to 60% (w/w). These results suggested that both HdAgl and HdAmy58 play important roles for the production of glucose from dietary starch in the digestive fluid. The amino-acid sequence of 887 residues for HdAgl was deduced by the cDNA method. This sequence showed 41-46% amino-acid identities to those of mammalian and avian alpha-glucosidases belonging to glycoside-hydrolase-family31. (C) 2013 Elsevier Inc. All rights reserved.
  • Hiroyuki Tanaka, Hiroki Takahashi, Takao Ojima
    FEBS LETTERS 16 587 (16) 2612 - 2616 0014-5793 2013/08 [Refereed][Not invited]
     
    Invertebrate troponin C typically contains Ca2+-specific binding sites, sites II and IV, in the N- and C-terminal domains, respectively. To investigate the roles of these sites for Ca2+-dependent regulation of muscle contraction, we generated lobster troponin C mutants, and analyzed their Ca2+-binding properties and regulatory effects on actomyosin-tropomyosin Mg-ATPase activity. The results suggest that Ca2+ binding to site IV is responsible for regulation at relatively low Ca2+ concentrations, while site II has an essential role in full activation at higher Ca2+ concentrations. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Takuya Ueda, Yuka Nakamura, Caleb M. Smith, Bryan A. Copits, Akira Inoue, Takao Ojima, Satoko Matsunaga, Geoffrey T. Swanson, Ryuichi Sakai
    GLYCOBIOLOGY 23 (4) 412 - 425 0959-6658 2013/04 [Refereed][Not invited]
     
    Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100 degrees C for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and 18 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, whereas matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry indicated broad ion clusters centered at 16,216 and 16,423, respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologs of animal prototype galectins and that multiple isolectins comprised the CchGs. One of the isolectins was expressed as a recombinant protein and exhibited physico-chemical and biological properties comparable with those of the natural lectins. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.
  • Yuya Kumagai, Takuya Satoh, Akira Inoue, Takao Ojima
    Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology 2 164 (2) 80 - 88 1096-4959 2013/02 [Refereed][Not invited]
     
    Two α-amylase (EC 3.2.1.1) isozymes, HdAmy58 and HdAmy82, with approximate molecular masses of 58. kDa and 82. kDa, respectively, were isolated from the digestive fluid of the Pacific abalone Haliotis discus hannai. Optimal temperatures and pHs for HdAmy58 and HdAmy82 were at 30 °C and 6.7, and 30 °C and 6.1, respectively. Both enzymes similarly degraded starch, glycogen, and maltooligosaccharides larger than maltotriose producing maltose and maltotriose as the major degradation products. However, the activity toward maltotetraose was appreciably higher in HdAmy82 than HdAmy58. cDNAs encoding HdAmy58 and HdAmy82 were cloned and the amino-acid sequences of 511 and 694 residues for HdAmy58 and HdAmy82, respectively, were deduced. The putative catalytic domains of HdAmy58 and HdAmy82 were located in the 17-511th and 19-500th amino-acid regions, respectively, and they showed approximately 50% amino-acid identity to each other. These sequences also showed 62-99% amino-acid identity to the catalytic domains of known α-amylases that belong to glycoside-hydrolase-family 13. The difference in the molecular masses between HdAmy58 and HdAmy82 was ascribed to the extension of approximately 190 residues in the C-terminus of HdAmy82. This extended region showed 41-63% amino-acid identity with the ancillary domains of several α-amylases previously reported. © 2012 Elsevier Inc.
  • Masaru Tanokura, Yusuke S. Kato, Fumiaki Yumoto, Takuya Miyakawa, Yumiko Miyauchi, Daijiro Takeshita, Yoriko Sawano, Takao Ojima, Iwao Ohtsuki
    Biological Chemistry 394 (1) 55 - 68 1431-6730 2013/01 [Refereed][Not invited]
     
    Troponin C (TnC) is the Ca 2 +-sensing subunit of troponin that triggers the contraction of striated muscles. In scallops, the striated muscles consume little ATP energy in sustaining strong contractile forces. The N-terminal domain of TnC works as the Ca 2 + sensor in vertebrates, whereas scallop TnC uses the C-terminal domain as the Ca 2 + sensor, suggesting that there are differences in the mechanism of the Ca 2 +-dependent regulation of muscles between invertebrates and vertebrates. Here, we report the crystal structure of Akazara scallop ( Chlamys nipponensis akazara ) adductor muscle TnC C-terminal domain (asTnC C ) complexed with a short troponin I fragment (asTnI S ) and Ca 2 +. The electron density of a Ca 2 + ion is observed in only one of the two EF-hands. The EF-hands of asTnC C can only be in the fully open conformation with the assistance of asTnI S. The number of hydrogen bonds between asTnC C and asTnI S is markedly lower than the number in the vertebrate counterparts. The Ca 2 + modulation on the binding between asTnC C and asTnI S is weaker, but structural change of the complex depending on Ca 2 + concentration was observed. Together, these findings provide a detailed description of the distinct molecular mechanism of contractile regulation in the scallop adductor muscle from that of vertebrates.
  • Takuya Miyakawa, Hiroto Shinomiya, Fumiaki Yumoto, Yumiko Miyauchi, Hiroyuki Tanaka, Takao Ojima, Yusuke S. Kato, Masaru Tanokura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 3-4 429 (3-4) 137 - 141 0006-291X 2012/12 [Refereed][Not invited]
     
    Plastins are Ca2+-regulated actin-bundling proteins, and essential for developing and stabilizing actin cytoskeletons. T-plastin is expressed in epithelial and mesenchymal cells of solid tissues, whereas L-plastin is expressed in mobile cells such as hemopoietic cell lineages and cancer cells. Using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry, we here demonstrate that the EF-hand motifs of both T- and L-plastin change their structures in response to Ca2+, but the sensitivity to Ca2+ is lower in T-plastin than in L-plastin. These results suggest that T-plastin is suitable for maintaining static cytoskeletons, whereas L-plastin is suitable for dynamic rearrangement of cytoskeletons. (C) 2012 Elsevier Inc. All rights reserved.
  • Mohammad Matiur Rahman, Ling Wang, Akira Inoue, Takao Ojima
    CARBOHYDRATE RESEARCH 360 69 - 77 0008-6215 2012/10 [Refereed][Not invited]
     
    Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate. (C) 2012 Elsevier Ltd. All rights reserved.
  • Mohammad Matiur Rahman, Ling Wang, Akira Inoue, Takao Ojima
    CARBOHYDRATE RESEARCH 360 69 - 77 0008-6215 2012/10 [Refereed][Not invited]
     
    Herbivorous marine snails like Littorina species are known to possess alginate lyases in their digestive tracts. The Littorina enzymes have been identified as endolytic polymannuronate (poly(M)) lyases (EC 4.2.2.3); however, it is still unclear which polysaccharide-lyase family (PL) the Littorina enzymes belong to, since no complete primary structure of Littorina enzymes has been determined. Thus, in the present study, we analyzed the primary structure of LbAly28, a 28 kDa alginate lyase isozyme of Littorina brevicula, by the cDNA method. LbAly28 cDNAs were amplified by PCR followed by 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA. A cDNA covering entire coding region of LbAly28 consisted of 1129 bp and encoded an amino-acid sequence of 291 residues. The deduced amino-acid sequence comprised an initiation methionine, a putative signal peptide of 14 residues, a propeptide-like region of 16 residues, and a mature LbAly28 domain of 260 residues. The mature LbAly28 domain showed 43-53% amino-acid identities with other molluscan PL-14 enzymes. The catalytically important residues in PL-14 enzymes, which were identified in the Chlorella virus glucuronate-specific lyase vAL-1 and Aplysia poly(M) lyase AkAly30, were also conserved in LbAly28. Site-directed mutagenesis regarding these residues, that is, replacements of Lys94, Lys97, Thr121, Arg 123, Tyr135, and Tyr137 to Ala, decreased the activity of recombinant LbAly28 to various degrees. From these results we concluded that LbAly28 is a member of PL-14 alginate lyases. Besides the effects of above mutations, we noticed that the replacement of T121 by Ala changed the substrate preference of LbAly28. Namely, the activities toward sodium alginate and poly(MG)-block substrate increased and became comparable with the activity toward poly(M)block substrate. This suggests that the region including T121 of LbAly28 closely relates to the recognition of poly(MG) region of alginate. (C) 2012 Elsevier Ltd. All rights reserved.
  • Syuto Hasegawa, Kazuhiro Ura, Hiroyuki Tanaka, Takao Ojima, Yasuaki Takagi
    FISHERIES SCIENCE 78 (5) 1107 - 1115 0919-9268 2012/09 [Refereed][Not invited]
     
    We isolated a cellulase from the digestive organs of the short-spined sea urchin Strogylocentrotus intermedius using a combination of ion-exchange chromatography and gel filtration together with an assay for carboxymethylcellulase activity. The isolated cellulase was stained as a single band by Congo red. The molecular weight of the isolated cellulase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, was 59 kDa. The isolated cellulase exhibited hydrolytic activity toward carboxymethyl cellulose, with an optimum temperature and pH of 30 A degrees C and pH 8.0, respectively. The thermal stability of the enzyme was characterized by determining the temperature at which activity decreased by 50 % with treatment for 30 min at pH 7.0 and found to be 32 A degrees C. Cellulase activity remained at a high level at 5-20 A degrees C, which is the growth temperature of the short-spined sea urchin. These results confirm that the short-spined sea urchin should preferably be reared at a water temperature of < 20 A degrees C.
  • Ling Wang, Mohammad Matiur Rahman, Akira Inoue, Takao Ojima
    FISHERIES SCIENCE 78 (4) 889 - 896 0919-9268 2012/07 [Refereed][Not invited]
     
    Previously we isolated the major alginate lyase isozyme LbAly35 from a marine snail Littorina brevicula and showed that this enzyme was significantly heat stable in a broad pH range compared with other molluscan alginate lyases (Hata et al., Fish Sci 75:755-763, 2009). LbAly35 showed practically no similarity to other molluscan alginate lyases in the N-terminal amino-acid sequence of 20 residues and no cross-reactivity with anti-abalone alginate lyase antiserum. These led us to consider that the primary structure of LbAly35 is considerably deviated from other molluscan enzymes. Thus, in the present study, we first compared the thermal stability of LbAly35 with an abalone alginate lyase, HdAly, and found that the first order inactivation rate constants for LbAly35 at 40 and 45 A degrees C were 1/20 and 1/45 of those for HdAly, respectively. Then, we cloned cDNAs encoding LbAly35 and characterized its deduced amino-acid sequence comparing with those of other molluscan alginate lyases. The cDNAs were amplified by polymerase chain reaction (PCR) and 5'- and 3'-RACE PCRs from the L. brevicula hepatopancreas cDNA using degenerated primers synthesized on the basis of partial amino-acid sequences of LbAly35. The cDNA covering the entire translational region of LbAly35 comprised 1,093 bp and encoded an amino-acid sequence of 296 residues. The amino-acid sequence consisted of an initiation methionine, a putative signal peptide for secretion (22 residues), a propeptide-like region (10 residues), and a mature LbAly35 domain of 263 residues. Although the N-terminal region of LbAly35 was significantly deviated from those of other molluscan alginate lyases, the catalytic domain of LbAly35 showed similar to 45 % identity to other molluscan enzymes which had been classified under polysaccharide-lyase-family-14 (PL-14). In addition, the amino-acid residues crucially important for the catalytic actions of PL-14 enzymes were also conserved in LbAly35. Accordingly, LbAly35 was regarded as a member of PL-14 as other molluscan alginate lyases despite the significant deviation of its N-terminal region.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 1-3 162 (1-3) 24 - 33 1096-4959 2012/05 [Refereed][Not invited]
     
    A beta-D-mannosidase (EC 3.2.1.25) with a molecular mass of approximately 100 kDa was purified from the digestive fluid of a marine gastropod Aplysia kurodai by ammonium sulfate fractionation followed by column chromatographies on TOYOPEARL Butyl-650 M, TOYOPEARL DEAE-650 M, and Superdex 200 10/300 GL This enzyme, named AkMnsd in the present study, showed optimal activities at pH 4.5 and 40 degrees C and was stable at the acidic pH range from 2.0 to 6.7 and the temperature below 38 degrees C. The Km and Vmax values for AkMnsd determined at pH 6.0 and 30 degrees C with p-nitrophenyl beta-D-mannopyranoside were 0.10 mM and 3.75 mu mol/min/mg, respectively. AkMnsd degraded various polymer mannans as well as mannooligosaccharides liberating mannose as a major degradation product. Linear mannan from green alga Codium fragile was completely depolymerized by AkMnsd in the presence of AkMan, an endolytic beta-mannanase, which we previously isolated from the same animal (Zahura et al., Comp. Biochem. Physiol. B 157, 137-148 (2010)). A cDNA encoding AkMnsd was amplified from the Aplysia hepatopancreas cDNA by the PCR using degenerated primers designed on the basis of N-terminal and internal amino-acid sequences of AkMnsd. The cloned AkMnsd cDNA consisted of 2985 bp and encoded an amino-acid sequence of 931 residues with the calculated molecular mass of 101,970 Da. The deduced sequence of AkMnsd showed 20-43% amino-acid identity to those of glycoside-hydrolase-family 2 (GHF2) beta-mannosidases. The catalytically important amino-acid residues determined in GHF2 enzymes were completely conserved in AkMnsd. Thus, AkMnsd is regarded as a new member of GHF2 mannosidase from marine gastropod. (C) 2012 Elsevier Inc. All rights reserved.
  • Kazuhiro Ura, Syuto Hasegawa, Eri Tanaka, Takahiro Gotoh, Takao Ojima, Yasuaki Takagi
    ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY 295 (1) 73 - 77 1932-8486 2012/01 [Refereed][Not invited]
     
    Subtilase, a major protease in the short-spined sea urchin (Strongylocentrotus intermedius), was isolated and used as antigen for the subsequent production of a specific polyclonal antibody. Immunoreactive cells were observed by immunohistochemical analysis in granules in the anterior and posterior stomach and the anterior intestine. These granules, which were most numerous in the anterior stomach, also stained intensely with methylene blue-Azure II. However, granules in cells of the esophagus, posterior intestine, and rectum were not stained by this antibody. We conclude that subtilase mainly localizes in the stomach and anterior intestine of the sea urchin. Anat Rec, 2012. (C) 2011 Wiley Periodicals, Inc.
  • Mitsunori Yanagisawa, Takao Ojima, Kiyohiko Nakasaki
    JOURNAL OF MATERIAL CYCLES AND WASTE MANAGEMENT 13 (4) 321 - 326 1438-4957 2011/12 [Refereed][Not invited]
     
    The mid-gut gland of the scallop contains saccharification enzymes such as cellulase and amylase, and these enzymes have been disposed of together with the mid-gut gland after the removal of the adductor muscle, an edible part of the scallop. We used a drip from the mid-gut gland of the scallop, obtained by squeezing the gland, as an inexpensive enzyme mixture and tried to produce bioethanol from the glucans present in sea lettuce by the method of simultaneous saccharification and fermentation (SSF) with the use of baker's yeast. The ethanol concentration attained was as high as 7.2 g/L, which corresponded to similar to 37% of the conversion of glucans in sea lettuce in the solid-state SSF. Furthermore, we ascertained that the drip and sea lettuce contain nutrients that are indispensable for maintaining the yeast activity, and, thus, the SSF did not require any additional nutrients, such as yeast extract or peptone, the use of which increases the cost of fermentation to a high level.
  • Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    BIOCHIMIE 93 (10) 1720 - 1730 0300-9084 2011/10 [Refereed][Not invited]
     
    Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5'- and 3'-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed similar to 40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg: however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1. (C) 2011 Elsevier Masson SAS. All rights reserved.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 159 (4) 227 - 235 1096-4959 2011/08 [Refereed][Not invited]
     
    Previously we isolated an endo-beta-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1392 bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96 Da. Comparison of the primary structure of AkMan with other beta-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 beta-1,4-mannanases indicated that AkMan together with other molluscan beta-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21 (DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells. (C) 2011 Elsevier Inc. All rights reserved.
  • Akira Inoue, Chieco Mashino, Teina Kodama, Takao Ojima
    MARINE BIOTECHNOLOGY 13 (2) 256 - 263 1436-2228 2011/04 [Refereed][Not invited]
     
    Functional recombinant abalone alginate lyase (rHdAly) and beta-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30A degrees C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35A degrees C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (< 1 x 10(3) protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 x 10(6) protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 mu m, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.
  • Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 157 (4) 317 - 325 1096-4959 2010/12 [Refereed][Not invited]
     
    Two alginate lyase isozymes, AkAly28 and AkAly33, with approximate molecular masses of 28 and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare, Aplysia kurodai. Both of AkAly28 and AkAly33 were regarded as the endolytic polymannuronate (poly(M)) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate producing unsaturated tri- and disaccharides and rapidly decreased the viscosity of sodium alginate solution in the initial phase of degradation. Optimal pH and temperature of the two enzymes were similarly observed at pH 6.7 and 40 degrees C, respectively. Temperature that caused a half inactivation of the two enzymes during 20-min incubation was also similar to each other, i.e., 38 degrees C. However, NaCl requirement and activity toward oligosaccharide substrates of the two enzymes were significantly different from each other. Namely, AkAly28 showed practically no activity in the absence of NaCl and the maximal activity at NaCl concentrations higher than 0.2 M, whereas AkAly33 showed similar to 20% of maximal activity despite the absence of NaCl and the maximal activity at around 0.1 M NaCl. AkAly28 hardly degraded oligosaccharides smaller than tetrasaccharide, while AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and 2-keto-3-deoxy-gluconaldehyde (an open chain form of unsaturated monosaccharide). Analysis of the N-terminal and internal amino-acid sequences of AkAly28 and AkAly33 indicated that both of the two enzymes belong to polysaccharide lyase family 14. (C) 2010 Elsevier Inc. All rights reserved.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 157 (1) 137 - 143 1096-4959 2010/09 [Refereed][Not invited]
     
    A mannan-degrading enzyme was isolated from the digestive fluid of the common sea hare Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatography. The purified enzyme, named AkMan in the present paper, showed a single band with an approximate molecular mass of 40,000 Da on SDS-PAGE and preferably degraded a linear beta-1,4-mannan from green algae Codium fragile producing tri-and disaccharides. The optimal temperature of AkMan was 55 degrees C at pH 7.0 and temperature that caused 50% inactivation of AkMan during a 20-min incubation was 52 degrees C. AkMan retained high activity at pH 4.0-7.5 and was not inactivated in such acidic pH range by the incubation at 40 degrees C for 20 min. AkMan could degrade glucomannan from konjak root and galactomannan (tara gum and guar gum) as well as the linear beta-1,4-mannan, while not carboxymethyl cellulose, agarose, dextran and xylan. These results indicate that AkMan is a typical endo-beta-1,4-mannanase (EC 3.2.1.78) splitting internal beta-1,4-mannosyl linkages of mannan. The N-terminal and internal amino-acid sequences of AkMan shared similar to 55% amino-acid identity to the corresponding sequences of an abalone beta-1,4-mannanase, HdMan, which belongs to glycosyl hydrolase family 5 (GHF5). Thus, AkMan was also regarded as a member of GHF5 beta-1,4-mannanases. (C) 2010 Elsevier Inc. All rights reserved.
  • Chikako Nagasato, Akira Inoue, Masashi Mizuno, Kazuki Kanazawa, Takao Ojima, Kazuo Okuda, Taizo Motomura
    PLANTA 232 (2) 287 - 298 0032-0935 2010/07 [Refereed][Not invited]
     
    During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.
  • Fumihiko Okumura, Hiroyuki Kameda, Takao Ojima, Shigetsugu Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 395 (3) 352 - 355 0006-291X 2010/05 [Refereed][Not invited]
     
    We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. (C) 2010 Elsevier Inc. All rights reserved.
  • Yuya Kumagai, Takao Ojima
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 155 (2) 138 - 144 1096-4959 2010/02 [Refereed][Not invited]
     
    Two types of beta-1,3-glucanases, AkLam36 and AkLam33 with the molecular masses of 36 kDa and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare Aplysia kurodai. AkLam36 was regarded as an endolytic enzyme (EC 32.1.6) degrading laminarin and laminarioligosaccharides to laminaritriose, laminaribiose, and glucose, while AkLam33 was regarded as an exolytic enzyme (EC 32.1.58) directly producing glucose from polymer laminarin. AkLam36 showed higher activity toward beta-1,3-glucans with a few beta-1,6-linked glucose branches such as Laminaria digitata laminarin (LLam) than highly branched beta-1,3-glucans such as Eisenia bicyclis laminarin (ELam). AkLam33 showed moderate activity toward both Ram and LLam and high activity toward smaller substrates such as laminaritetraose and laminaritriose. Although both enzymes did not degrade laminaribiose as a sole substrate, they were capable of degrading it via transglycosylation reaction with laminaritriose. The N-terminal amino-acid sequences of AkLam36 and AkLam33 indicated that both enzymes belong to the glycosyl hydrolase family 16 like other molluscan beta-1,3-glucanases. (C) 2009 Elsevier Inc. All rights reserved.

Books etc

  • Marine Enzymes for Biocatalysis
    OJIMA Takao (Joint workPolysaccharide-degrading enzymes from herbivorous marine invertebrates)
    Woodhead Publishing Ltd. 2013/10
  • マリンバイオテクノロジーの新潮流
    (Joint workバイオ触媒)
    シーエムシー出版 2011
  • 「水圏生化学」
    恒星社厚生閣 2008
  • 魚貝類筋肉タンパク質-その構造と機能
    恒星社厚生閣 1999
  • 演習で学ぶ生化学
    三共出版 1999
  • 水産動物筋肉タンパク質の比較生化学
    恒星社厚生閣 1989
  • Comparative biochemistry of muscular proteins from aquatic animals.
    1989

MISC

  • OJIMA TAKAO  日本水産学会誌 = Bulletin of the Japanese Society of Scientific Fisheries  78-  (5)  2012/09  [Not refereed][Not invited]
  • 熊谷 裕也, 尾島 孝男  化学と生物  50-  (6)  396  -398  2012/06/01  [Not refereed][Not invited]
  • Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima  BIOCHIMIE  93-  (10)  1720  -1730  2011/10  [Not refereed][Not invited]
     
    Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5'- and 3'-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed similar to 40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg: however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1. (C) 2011 Elsevier Masson SAS. All rights reserved.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY  159-  (4)  227  -235  2011/08  [Not refereed][Not invited]
     
    Previously we isolated an endo-beta-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1392 bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96 Da. Comparison of the primary structure of AkMan with other beta-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 beta-1,4-mannanases indicated that AkMan together with other molluscan beta-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21 (DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells. (C) 2011 Elsevier Inc. All rights reserved.
  • Akira Inoue, Chieco Mashino, Teina Kodama, Takao Ojima  MARINE BIOTECHNOLOGY  13-  (2)  256  -263  2011/04  [Not refereed][Not invited]
     
    Functional recombinant abalone alginate lyase (rHdAly) and beta-1,4-endoglucanase (rHdEG66) were expressed as secreted proteins with baculoviral expression systems. The specific activity of each recombinant enzyme, 2,490 and 18.2 U/mg for rHdAly and rHdEG66, respectively, was comparable to its native form at 30A degrees C. Purified rHdAly and rHdEG66 showed the highest specific activity both at 35A degrees C and optimum pH 8.7 and 5.9, respectively. These properties were also comparable to those of the native enzymes. Protoplast isolation was attempted from Laminaria japonica using both rHdAly and rHdEG66. When L. japonica blades were incubated in artificial seawater containing rHdAly and rHdEG66, very low numbers of protoplasts (< 1 x 10(3) protoplasts/g fresh weight) resulted. However, using blades pretreated with proteinase K, the protoplast was increased up to 5 x 10(6) protoplasts/g fresh weight. Since the average diameter of isolated protoplasts was 11.6 mu m, these cells were mostly derived from the epidermal layer rather than the cortical layer. Our results suggest that at least three enzymes, alginate lyase, cellulase, and protease, are essential for effective protoplast isolation from L. japonica. The protoplast isolation method in this study is more useful than earlier methods because it preferentially yielded protoplasts of the epidermal layer, which are known to be able to be regenerated.
  • Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY  157-  (4)  317  -325  2010/12  [Not refereed][Not invited]
     
    Two alginate lyase isozymes, AkAly28 and AkAly33, with approximate molecular masses of 28 and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare, Aplysia kurodai. Both of AkAly28 and AkAly33 were regarded as the endolytic polymannuronate (poly(M)) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate producing unsaturated tri- and disaccharides and rapidly decreased the viscosity of sodium alginate solution in the initial phase of degradation. Optimal pH and temperature of the two enzymes were similarly observed at pH 6.7 and 40 degrees C, respectively. Temperature that caused a half inactivation of the two enzymes during 20-min incubation was also similar to each other, i.e., 38 degrees C. However, NaCl requirement and activity toward oligosaccharide substrates of the two enzymes were significantly different from each other. Namely, AkAly28 showed practically no activity in the absence of NaCl and the maximal activity at NaCl concentrations higher than 0.2 M, whereas AkAly33 showed similar to 20% of maximal activity despite the absence of NaCl and the maximal activity at around 0.1 M NaCl. AkAly28 hardly degraded oligosaccharides smaller than tetrasaccharide, while AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and 2-keto-3-deoxy-gluconaldehyde (an open chain form of unsaturated monosaccharide). Analysis of the N-terminal and internal amino-acid sequences of AkAly28 and AkAly33 indicated that both of the two enzymes belong to polysaccharide lyase family 14. (C) 2010 Elsevier Inc. All rights reserved.
  • Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY  157-  (1)  137  -143  2010/09  [Not refereed][Not invited]
     
    A mannan-degrading enzyme was isolated from the digestive fluid of the common sea hare Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatography. The purified enzyme, named AkMan in the present paper, showed a single band with an approximate molecular mass of 40,000 Da on SDS-PAGE and preferably degraded a linear beta-1,4-mannan from green algae Codium fragile producing tri-and disaccharides. The optimal temperature of AkMan was 55 degrees C at pH 7.0 and temperature that caused 50% inactivation of AkMan during a 20-min incubation was 52 degrees C. AkMan retained high activity at pH 4.0-7.5 and was not inactivated in such acidic pH range by the incubation at 40 degrees C for 20 min. AkMan could degrade glucomannan from konjak root and galactomannan (tara gum and guar gum) as well as the linear beta-1,4-mannan, while not carboxymethyl cellulose, agarose, dextran and xylan. These results indicate that AkMan is a typical endo-beta-1,4-mannanase (EC 3.2.1.78) splitting internal beta-1,4-mannosyl linkages of mannan. The N-terminal and internal amino-acid sequences of AkMan shared similar to 55% amino-acid identity to the corresponding sequences of an abalone beta-1,4-mannanase, HdMan, which belongs to glycosyl hydrolase family 5 (GHF5). Thus, AkMan was also regarded as a member of GHF5 beta-1,4-mannanases. (C) 2010 Elsevier Inc. All rights reserved.
  • RAHMAN Mohammad Matiur  日本水産学会誌 = Bulletin of the Japanese Society of Scientific Fisheries  76-  (4)  2010/07  [Not refereed][Not invited]
  • Chikako Nagasato, Akira Inoue, Masashi Mizuno, Kazuki Kanazawa, Takao Ojima, Kazuo Okuda, Taizo Motomura  PLANTA  232-  (2)  287  -298  2010/07  [Not refereed][Not invited]
     
    During cytokinesis in brown algal cells, Golgi-derived vesicles (GVs) and flat cisternae (FCs) are involved in building the new cell partition membrane. In this study, we followed the membrane fusion process in Silvetia babingtonii zygotes using electron microscopy together with rapid freezing and freeze substitution. After mitosis, many FCs were formed around endoplasmic reticulum clusters and these then spread toward the future cytokinetic plane. Actin depolymerization using latrunculin B prevented the appearance of the FCs. Fusion of GVs to FCs resulted in structures that were thicker and more elongated (EFCs; expanded flat cisternae). Some complicated membranous structures (MN; membranous network) were formed by interconnection of EFCs and following the arrival of additional GVs. The MN grew into membranous sacs (MSs) as gaps between the MNs disappeared. The MSs were observed in patches along the cytokinetic plane. Neighboring MSs were united to form the new cell partition membrane. An immunocytochemical analysis indicated that fucoidan was synthesized in Golgi bodies and transported by vesicles to the future cytokinetic plane, where the vesicles fused with the FCs. Alginate was not detected until the MS phase. Incubation of sections with cellulase-gold showed that the cellulose content of the new cross wall was not comparable to that of the parent cell wall.
  • Fumihiko Okumura, Hiroyuki Kameda, Takao Ojima, Shigetsugu Hatakeyama  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  395-  (3)  352  -355  2010/05  [Not refereed][Not invited]
     
    We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. (C) 2010 Elsevier Inc. All rights reserved.
  • Yuya Kumagai, Takao Ojima  Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology  155-  (2)  138  -144  2010/02  [Not refereed][Not invited]
     
    Two types of β-1,3-glucanases, AkLam36 and AkLam33 with the molecular masses of 36 kDa and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare Aplysia kurodai. AkLam36 was regarded as an endolytic enzyme (EC 3.2.1.6) degrading laminarin and laminarioligosaccharides to laminaritriose, laminaribiose, and glucose, while AkLam33 was regarded as an exolytic enzyme (EC 3.2.1.58) directly producing glucose from polymer laminarin. AkLam36 showed higher activity toward β-1,3-glucans with a few β-1,6-linked glucose branches such as Laminaria digitata laminarin (LLam) than highly branched β-1,3-glucans such as Eisenia bicyclis laminarin (ELam). AkLam33 showed moderate activity toward both ELam and LLam and high activity toward smaller substrates such as laminaritetraose and laminaritriose. Although both enzymes did not degrade laminaribiose as a sole substrate, they were capable of degrading it via transglycosylation reaction with laminaritriose. The N-terminal amino-acid sequences of AkLam36 and AkLam33 indicated that both enzymes belong to the glycosyl hydrolase family 16 like other molluscan β-1,3-glucanases. © 2009 Elsevier Inc. All rights reserved.
  • KOBAYASHI Junya, SUZUKI Takashi, BANDO Tadanori, OJIMA Takao, KOBAYASHI Takanori  Research reports Hakodate Technical college  44-  25  -28  2010  [Not refereed][Not invited]
     
    The authors produced a sub-critical reactor with 0.5L of capacity, 32MPa of resistant pressure and 400℃ of heat-resistant. We used the equipment for degradation reactions to monosaccharides in several kinds of unused seaweeds. It was found that the degradability was low at 200℃, however, degradations to monosaccharides were promoted in sea lettuces more than other seaweeds. Conspicuous catalytic effects were not seen in a sulfated ZrO2 catalyst, which is a solid superacid. Celluloses and starches were also examined at 200℃ so as to compare degradation reactions. Small and significant catalyst's effects were recognized in celluloses and starches, respectively on improvement of degradability for glucose.
  • Douchi Matsuba, Takako Terui, Jin O-Uchi, Hiroyuki Tanaka, Takao Ojima, Iwao Ohtsuki, Shin'ichi Ishiwata, Satoshi Kurihara, Norio Fukuda  JOURNAL OF GENERAL PHYSIOLOGY  133-  (6)  571  -581  2009/06  [Not refereed][Not invited]
     
    Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during beta-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca2+ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca2+ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca2+ sensitivity, but also abolished the Ca2+-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca2+ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca2+ sensitivity, and PKA further decreased Ca2+ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.
  • Mami Hata, Yuya Kumagai, Mohammad Matiur Rahman, Satoru Chiba, Hiroyuki Tanaka, Akira Inoue, Takao Ojima  FISHERIES SCIENCE  75-  (3)  755  -763  2009/05  [Not refereed][Not invited]
     
    Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via beta-elimination, producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino acid sequences of alginate lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate lyases. The alginate lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate lyases with molecular masses of approximately 28, 34, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate lyases with molecular masses of 35, 32, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher-order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino acid sequences, while the L. brevicula enzymes were not.
  • Masashi Mizuno, Yosuke Nishitani, Takeshi Tanoue, Yoshie Matoba, Taka Ojima, Takashi Hashimoto, Kazuki Kanazawa  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  73-  (2)  335  -338  2009/02  [Not refereed][Not invited]
     
    The establishment of a simple technique to determine the concentration of fucoidan wag developed by using a monoclonal antibody against fucoidan. This antibody reacted with fucoidans purified from Laminaria japonica Areschoug (Makombu in Japanese) and Kjellmaniella gyrate Miyabe (Gagome), but not with polysaccharides from Undaria pinnatifida Suringar (Wakame). Neither laminarin nor algenic acid, which are constituents in Laminaria japonica, were recognized by the prepared antibody. Application of the enzymed-linked immunosorbent assay (ELISA) inhibition assay increased the specificity of fucoidan in measuring the fucoidan contents. On the basis of these results, it was ascertained that the ELISA inhibition assay of using the anti-fucoidan monoclonal antibody was rapid, accurate, and sensitive in measuring the content of fucoidan. In addition, the localization of fucoidan in Laminaria japonica was investigated. This is the first report of fucoidan being restricted to the outer cortical layer.
  • KUMAGAI Yuya, OJIMA Takao  Comparative Biochemistry and Physiology  154-  (1)  113  -120  2009  [Not refereed][Not invited]
  • Sayo Yamamoto, Takehiko Sahara, Daisuke Sato, Kosei Kawasaki, Satoru Ohgiya, Akira Inoue, Takao Ojima  ENZYME AND MICROBIAL TECHNOLOGY  43-  (6)  396  -402  2008/11  [Not refereed][Not invited]
     
    Alginate lyase is an enzyme that degrades alginate chains via beta-elimination and has been used for the production of alginate oligosaccharides and protoplasts from brown algae. Previously, we deduced the amino-acid sequence of an abalone alginate lyase, HdAly, from its cDNA sequence and, through multiple amino-acid sequence alignment, found that several basic amino-acid residues were highly conserved among the polysaccharide-lyase family 14 (PL-14) enzymes including HdAly. In the present study, we assessed the functional importance of the conserved basic amino-acid residues in HdAly by using site-directed mutants that were produced with a cold-inducible yeast expression system consisting of an expression vector pLTex321sV5H and a host Saccharomyces cerevisiae BY4743. At first, we prepared wildtype HdAly with the yeast expression system and confirmed that this recombinant possesses nearly the same properties as native HdAly with respect to specific activity (1300 U/mg), optimal pH (7.8), optimal temperature (35 C), and protoplast-producing ability from the brown alga Laminaria japonica. Then, we prepared a series of site-directed mutants by replacing the conserved basic amino-acid residues of HdAly with Ala and determined the alginate-degrading activity of these mutants. As a result, we found that the replacement of Lys95 caused complete inactivation of HdAly and those of Arg92, Arg110, and Arg119 caused 65% or more inactivation. These results indicate that the region spanning Arg92 to Arg119 is closely related to the catalytic activity of HdAly. (C) 2008 Elsevier Inc. All rights reserved.
  • Akira Inoue, Mayu Kagaya, Takao Ojima  JOURNAL OF APPLIED PHYCOLOGY  20-  (5)  633  -640  2008/10  [Not refereed][Not invited]
     
    Laminaria japonica protoplasts were released with high yields using the abalone alginate lyase HdAly in combination with a cellulase and chelating agents. Addition of EDTA at concentrations higher than 10 mM to Laminaria thalli which had been preincubated with HdAly and Cellulase Onozuka, dramatically improved the yield of protoplasts. EDTA was far more effective than EGTA, indicating that chelating divalent metal ions such as Mg2+ and Sr2+ in addition to Ca2+ is a key factor for high-yield production of Laminaria protoplasts. Protoplasts had a mean diameter of 27 mu m, suggesting that most protoplasts were derived from cortical cells rather than epidermal layer cells. Recombinant HdAly (rHdAly) was produced from a cDNA clone in the Sf9 insect cell expression system. rHdAly had substantially the same enzymatic properties and protoplast-producing ability as did native HdAly. The optimal conditions for high yield production of protoplasts from Laminaria using native and recombinant HdAlys were investigated.
  • Yuya Kumagai, Akira Inoue, Hiroyuki Tanaka, Takao Ojima  FISHERIES SCIENCE  74-  (5)  1127  -1136  2008/10  [Not refereed][Not invited]
     
    The mid-gut gland of scallop Patinopecten yessoensis has been discarded in scallop processing factories as a fishery waste and various attempts have been made to turn the waste into valuable resources. In the present study, we tried to use mid-gut gland drips from scallop as a source of beta-1,3-glucanase. The mid-gut gland drips were collected in a local fishery factory in Yubetsu-cho, Hokkaido Prefecture. beta-1,3-Glucanase was purified from the mid-gut gland drips by ammonium sulfate fractionation followed by successive chromatography on Toyopearl Phenyl-650M and Toyopearl DEAE-650M. The scallop beta-1,3-glucanase, named PyLam38 in the present study, showed a molecular mass of approximately 38 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and hydrolyzed laminarin, a beta-1,3-glucan from Laminaria sp., producing laminaribiose and glucose with an optimal pH and temperature of 6.0 and 45 degrees C, respectively. PyLam38 exhibited high transglycosylation activity toward various accepter substrates such as monosaccharides, alcohols and xylooligosaccharides. Thus, PyLam38 was found to be useful for the production of various novel heterooligosaccharides consisting of laminarioligosaccharides and various accepters.
  • Hiroyuki Tanaka, Hisoka Suzuki, Iwao Ohtsuki, Takao Ojima  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS  1784-  (7-8)  1037  -1042  2008/07  [Not refereed][Not invited]
     
    Molluscan troponin regulates muscle contraction through a novel Ca2+-dependent activating mechanism associated with Ca2+-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin 1. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin 1, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca2+-dependent activation of the Mg-ATPase activity of rabbit-actomyosin-scallop-tropomyosin. In addition, the actin-tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca2+ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin-tropomyosin filaments and is essential for regulation. (C) 2008 Elsevier B.V. All rights reserved.
  • Fumiaki Yumoto, Hiroyuki Tanaka, Koji Nagata, Yumiko Miyauchi, Takuya Miyakawa, Takao Ojima, Masaru Tanokura  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  369-  (1)  109  -114  2008/04  [Not refereed][Not invited]
     
    Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca2+ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca2+-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC Globe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca2+-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca2+ -binding properties of the complex of TnC Globe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca2+-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the Globe of scallop TnC. We therefore assumed that the effect of Ca2+-binding must lead to a change in the interaction mode between the Globe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca2+ signal to TnI in Tit ternary complex. (c) 2007 Elsevier Inc. All rights reserved.
  • Yukiko Nishida, Ken-ichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima  BIOCHIMIE  89-  (8)  1002  -1011  2007/08  [Not refereed][Not invited]
     
    Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54 kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases. (c) 2007 Elsevier Masson SAS. All rights reserved.
  • Fumiaki Yumoto, Koji Nagata, Yumiko Miyauchi, Takao Ojima, Hiroyuki Tanaka, Kiyoyoshi Nishita, Iwao Ohtsuki, Masaru Tanokura  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS  63-  535  -537  2007/06  [Not refereed][Not invited]
     
    Troponin C (TnC) is the Ca2+-binding component of troponin and triggers muscle contraction. TnC of the invertebrate Akazara scallop can bind only one Ca2+ at the C-terminal EF-hand motif. Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. The crystals diffracted X-rays to 1.80 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.1, b = 42.2, c = 60.0 angstrom. The asymmetric unit was assumed to contain one molecular complex of the Akazara scallop TnC C-lobe and TnI fragment, with a Matthews coefficient of 1.83 angstrom(3) Da(-1) and a solvent content of 33.0%.
  • Atsushi Nakamura, Fumi Sasaki, Kazuhiko Watanabe, Takao Ojima, Dong-Hyun Ahn, Hiroki Saeki  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY  54-  (25)  9529  -9534  2006/12  [Not refereed][Not invited]
     
    The effect of the Maillard reaction on the allergenicity of squid tropomyosin (TM) was investigated. When TM was reacted with ribose (TM-ribose), its human-specific IgE-binding ability decreased markedly and R-chymotryptic digestibility of TM was also altered at the early stage of the Maillard reaction. On the other hand, the modification of the lysine residues in TM using 2,4,6-trinitrobenzenesulfonic acid had no effect on the allergenicity and R-chymotryptic digestibility of TM. Therefore, the structural change in TM induced by the Maillard reaction would cause the reduction of the allergenicity, rather than the block of lysine residues. Although peptic digestion diminished the specific IgE-binding ability of TM, the reduction of the allergenicity by the Maillard reaction remained after peptic digestion. These results suggest that hypersensitive reaction of TM-ribose in the human body might be lower than that of native TM.
  • Shuuji Ootsuka, Naotsune Saga, Ken-ichi Suzuki, Akira Inoue, Takao Ojima  JOURNAL OF BIOTECHNOLOGY  125-  (2)  269  -280  2006/09  [Not refereed][Not invited]
     
    An endo-beta-1,4-mannanase was isolated from digestive fluid of Pacific abalone, Haliotis discus hannai, by successive chromatographies on TOYPEARL CM-650 M, hydroxyapatite, and TOYOPEARL HW50F The abalone mannanase, named HdMan in the present paper, showed a molecular mass of approximately 39,000 Da on SDS-PAGE, and exhibited high hydrolyic activity on both galactomannan from locust bean gum and glucomannan from konjac at an optimal pH and temperature of 7.5 and 45 degrees C, respectively. HdMan could degrade either beta-1,4-mannan or beta-1,4-mannooligosaccharides to mannotriose and mannobiose similarly to beta-1,4-mannanases from Pomacea, Littorina, and Mytilus. In addition, HdMan could disperse the fronds of a red alga Porphyra yezoensis into cell masses consisting of 10-20 cells that are available for cell engineering of this alga. cDNAs encoding HdMan were amplified by polymerase chain reaction from an abalone-hepatopancreas cDNA library. From the nucleotide sequences of the cDNAs, the sequence of 1232 bp in total was determined and the amino-acid sequence of 377 residues was deduced from the translational region of 1134 bp locating at nucleotide positions 15-1148. The N-terminal region of 17 residues except for the initiation Met, was regarded as the signal peptide of HdMan because it was absent in the HdMan protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Accordingly, mature HdMan was considered to consist of 359 residues with the calculated molecular mass of 39,627.2 Da. HdMan is classified into glycoside hydrolase family 5 (GHF5) on the basis of sequence homology to GHF5 enzymes. (c) 2006 Elsevier B.V. All rights reserved.
  • Harumasa Suzuki, Ken-ichi Suzuki, Akira Inoue, Takao Ojima  CARBOHYDRATE RESEARCH  341-  (11)  1809  -1819  2006/08  [Not refereed][Not invited]
     
    We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [Carbohydr. Res. 2003, 338, 2841-2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42 degrees C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62-67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate Iyase belonging to PL14. (c) 2006 Elsevier Ltd. All rights reserved.
  • OOTSUKA Shuuji, FUKUDA Satoru, SHIBATA Daisuke, SAGA Naotsune, OJIMA Takao  水産増殖  54-  (1)  83  -88  2006/03/20  [Not refereed][Not invited]
  • Preparation of an enzyme degrading cell-wall polysaccharides from digestive fluid of abalone
    Suisan Zoushoku  54-  83  -88  2006  [Not refereed][Not invited]
  • F Yumoto, QW Lu, S Morimoto, H Tanaka, N Kono, K Nagata, T Qjima, F Takahashi-Yanaga, Y Miwa, T Sasaguri, K Nishita, M Tanokura, Ohtsuki, I  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  338-  (3)  1519  -1526  2005/12  [Not refereed][Not invited]
     
    bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+ sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations nuclear magnetic resonance spectroscopy revealed that this Mutation does not extensively affect the Structure of the whole cTnI molecule, but induces all unexpectedly subtle change in the structure of a region around the Mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tin binding region of cTnI. The present study also Suggests that both HCM and RCM involving cTnI Mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM. (c) 2005 Elsevier Inc. All rights reserved.
  • Tanaka H, Ohtsuki I, Ojima T  Biophysics  45-  (1)  2005/10/19  [Not refereed][Not invited]
  • Suzuki H, Tanaka H, Ohtsuki I, Ojima T  Biophysics  45-  (1)  2005/10/19  [Not refereed][Not invited]
  • A Nakamura, K Watanabe, T Ojima, DH Ahn, H Saeki  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY  53-  (19)  7559  -7564  2005/09  [Not refereed][Not invited]
     
    Scallop tropomyosin (TM), the major allergen of shellfish, was prepared from adductor muscles and reacted with four reducing sugars to investigate the effect of the Maillard reaction on the allergenicity of TM. The lgE-binding ability of TM increased significantly with the progress of the reaction with glucose, ribose, and maltose, but not with maltotriose. The allergenicity was enhanced at the early stage of the Maillard reaction, and the trend of the effect depended on the type of reducing sugar used. 2,4,6-Trinitrobenzenesulfonic acid treatment of the lysine residues in TM showed that the protein surface charge resulting from the Maillard reaction had no effect on the enhancement of the allergenicity. Thus, the change in the allergenicity would be closely related to the structural change caused by the Maillard reaction.
  • H Tanaka, Y Takeya, T Doi, F Yumoto, M Tanokura, Ohtsuki, I, K Nishita, T Ojima  FEBS JOURNAL  272-  (17)  4475  -4486  2005/09  [Not refereed][Not invited]
     
    Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca2+-dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI(232-292)), which contains the inhibitory region (residues 104-115 of rabbit TnI) and the regulatory TnC-binding site (residues 116-131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca2+. These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI(130-252), which contains the structural TnC-binding site (residues 1-30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca2+-dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI(1-116)) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca2+-dependent activation.
  • T Doi, A Satoh, H Tanaka, A Inoue, F Yumoto, M Tanokura, Obtsuki, I, K Nishita, T Ojima  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS  436-  (1)  83  -90  2005/04  [Not refereed][Not invited]
     
    Ca2+-binding sites I and II in the N-terminal lobe of Molluscan troponin C (TnC) have lost the ability to bind Ca2+ due to substitutions of the amino acid residues responsible for Ca2+ liganding. To evaluate the functional importance of the Ca2+-deficient N-terminal lobe in the Ca2+-regulatory function of Molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using Ca-45(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca2+/mol and 1 mol Ca2+/Mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca2+ binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6 M urea, if Ca2+ was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin 1, they showed different Ca2+-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca2+ sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca2+ sensitivity. Our findings indicate that the N-terminal lobe of Molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca2+. (c) 2005 Elsevier Inc. All rights reserved.
  • Nara M, Yumoto F, Nagata K, Tanokura M, Kagi H, Ojima T, Nishita K  Biophysics  44-  (1)  2004/11/10  [Not refereed][Not invited]
  • KISHIMURA HIDEKI, OJIMA TAKAO, HAYASHI KENJI, NISHITA KIYOYOSHI  Bulletin of the Japanese Society of Scientific Fisheries  70-  (5)  770  -771  2004/09/15  [Not refereed][Not invited]
  • A Inoue, T Ojima, K Nishita  JOURNAL OF BIOCHEMISTRY  136-  (1)  107  -114  2004/07  [Not refereed][Not invited]
     
    Akazara scallop striated muscle tropomyosin mutants without a fused amino acid (nf-Tm), and with Ala- (A-Tm) or Asp-Ala- (DA-Tm) fused at the N-terminus were expressed in Escherichia coli cells. Among them, nf-Tm alone has an initial methionine. The native Akazara scallop tropomyosin and DA-Tm showed similar a-helix contents and intrinsic viscosity, but nf-Tm and A-Tm exhibited lower values than those of the native tropomyosin. According to the relative viscosity, all the expressed tropomyosins appear to have lost head-to-tail polymerization ability. Though nf-Tm has extremely low actin-binding ability, the ability was almost completely recovered with a two amino acid fusion but incompletely with a one amino acid fusion. On the other hand, an amino acid fusion, irrespective of the number, seemed to inhibit the Mg-ATPase activity of actomyosin. However, the bacterially expressed tropomyosins together with Akazara scallop troponin do not confer the full Ca2+-regulation ability of Mg-ATPase activity of actomyosin. These results support that N-terminal blocking probably by an acetyl group of Akazara scallop tropomyosin plays an important role not only in head-to-tail polymerization and actin-binding, as known for vertebrate tropomyosin, but also in maintaining the secondary or higher structure and Ca2+-regulation together with troponin.
  • M Nara, F Yumoto, K Nagata, M Tanokura, H Kagi, T Ojima, K Nishita  BIOPOLYMERS  74-  (1-2)  77  -81  2004/05  [Not refereed][Not invited]
     
    Troponin C (TnC) is the Ca2+-binding regulatory protein of the troponin complex in muscle tissue, Vertebrate fast skeletal muscle TnCs bind four Ca2+, while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca2+ at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca2+. Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg2+ bound in a mutant Akazaro scallop TnC (E142Q) in D 20 solution. The result showed that the side-chain COO- groups of Asp 131 and Asp 133 in the Ca2+-binding site of E142Q bind to Mg2+ in the pseudo-bridging mode. Mg2+ titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the bond at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO- groups. As a result, the binding constants of them for Mg2+ were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO- group of Glu 142 of the wild type has no relation to the Mg2+ ligation. The effect of Mg2+ binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg2+ as well as Ca2+. (C) 2004 Wiley Periodicals, Inc.
  • H Tanaka, Y Maezawa, T Ojima, K Nishita  FISHERIES SCIENCE  70-  (1)  198  -200  2004/02  [Not refereed][Not invited]
  • H Tanaka, Y Maezawa, T Ojima, K Nishita  FISHERIES SCIENCE  70-  (1)  198  -200  2004/02  [Not refereed][Not invited]
  • Structural characteristics of a cellulase from the Pacific Abalone Haliotis discus hannai.
    Marine Biotechnology  6-  S502-S505  2004  [Not refereed][Not invited]
  • KISHIMURA HIDEKI, OJIMA TAKAO, HAYASHI KENJI, NISHITA KIYOYOSHI  Bulletin of the Japanese Society of Scientific Fisheries  69-  (6)  968  -974  2003/11/15  [Not refereed][Not invited]
     
    Site-directed mutagenesis study of phospholipase A_2 (PLA_2) from the pyloric ceca of starfish Asterina pectinifera was used to probe the relationship between polar-group specificity and structure of the pancreatic loop region. The sequence of the cDNA encoding the starfish PLA_2 was exchanged by the oligonucleotide-directed dual amber-long and accurate polymerase chain reaction method to insert Lys residue between Cys 62 and Gly63 of the wild-type PLA_2 (WT PLA_2) expressed by Escherichia coli. The modified cDNA was inserted into the expression plasmid pET-16b, and PLA2 mutant was expressed in E. coli BL21 (DE3) by induction with isopropyl-β-D (-)-thiogalactopyranoside. The PLA_2 mutant produced as inclusion bodies was dissociated with urea and 2-mercaptoethanol and renatured by dialyzing against Tris-HCl buffer. Renatured PLA_2 mutant showed essentially the same properties as the WT PLA_2 with respect to positional specificity of substrate, optimum pH and Ca^<2+> requirement. However, the PLA_2 mutant hydrolyzed phosphatidylethanolamine (PE) more effectively than the WT PLA_2. Therefore, it was suggested that the structure of the pancreatic loop region may be associated with polar-group specificity of PE.
  • E Shimizu, T Ojima, K Nishita  CARBOHYDRATE RESEARCH  338-  (24)  2841  -2852  2003/11  [Not refereed][Not invited]
     
    An alginate lyase, termed HdAly in the present paper, was isolated from the hepatopancreas of abalone, Haliotis discus hannai, by ammonium sulfate fractionation, followed by TOYOPEARL CM-650M column chromatography. Enzymatic properties of HdAly were similar to those of previously reported Haliotis and Turbo poly(M) lyases, e.g., it preferentially degraded a poly(beta-D-mannuronate)-rich substrate with an optimal pH and temperature at pH 8.0 and 45 degreesC, respectively. In order to determine the primary structure of abalone lyase that is still poorly understood, cDNAs for HdAly were cloned by PCR from the abalone hepatopancreas cDNA library and sequenced. From the nucleotide sequences of the cDNAs, the sequence of 909 bp in total was determined, and the amino acid sequence of 273 residues was deduced from the translational region of 822 bp locating at nucleotide positions 27-848. The N-terminal region of 16 residues, except for the initiation Met in the deduced sequence, was regarded as the signal peptide since it was absent in the HdAly protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretary proteins. This suggests that HdAly is initially produced as a precursor possessing the signal peptide in hepatopancreatic cells and then secreted into digestive tract as the mature form. Thus, the mature HdAly was regarded to consist of 256 residues with the calculated molecular mass of 28895.5 Da. The amino acid sequence of HdAly showed 85 and 28% identity to those of Turbo cornutus alginate lyase SP2 and the C-terminal region of Chlorella virus lyase-like protein CL2, respectively, while it showed no significant identity to those of any bacterial alginate lyases. In order to provide the basis for the structure-function studies and various applications of the abalone lyase, a bacterial expression system was constructed by means of the HdAly-cDNA and pET-3a expression plasmid. Although the active recombinant HdAly was hardly produced at a cultivation temperature 37 degreesC in Escherichia coli BL21 (DE3), a small amount of soluble and active enzyme could be produced when the temperature was lowered to 19 degreesC. (C) 2003 Elsevier Ltd. All rights reserved.
  • Nara M, Yumoto F, Nagata K, Tanokura M, Kagi H, Ojima T, Nishita K  Biophysics  43-  (1)  2003/08/25  [Not refereed][Not invited]
  • OJIMA TAKAO  Bulletin of the Japanese Society of Scientific Fisheries  69-  (3)  326  -329  2003/05/15  [Not refereed][Not invited]
  • 尾島 孝男, 鈴木 賢一, 清水 映里  バイオインダストリ-  20-  (3)  21  -27  2003/03  [Not refereed][Not invited]
  • K Suzuki, T Ojima, K Nishita  EUROPEAN JOURNAL OF BIOCHEMISTRY  270-  (4)  771  -778  2003/02  [Not refereed][Not invited]
     
    A cellulase [endo-beta-1,4-d-glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 degreesC and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56-1840 gave an amino-acid sequence of 594 residues including the initiation methionine. The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C-terminal region of 453 residues in HdEG66, i.e. approximately the C-terminal three quarters of the protein, showed 42-44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca . While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region. By genomic PCR using specific primers to the 3'-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself.
  • 岸村栄毅, 尾島孝男, 林賢治, 西田清義  日本水産学会誌  69-  (6)  968  -974  2003  [Not refereed][Not invited]
  • NMR structural study of troponin C C-terminal domain complexed with troponin I fragment from Akazara scallop
    Advances of Experimental Medical Biology  538-  195  -201  2003  [Not refereed][Not invited]
  • 「海産無脊椎動物からの有用酵素の探索」
    バイオインダストリー  20-  (3)  21  -27  2003  [Not refereed][Not invited]
  • かまぼこ-その科学と技術<山澤正勝・関伸夫・福田裕編>
    恒星社厚生閣  2003  [Not refereed][Not invited]
  • Nippon Suisan Gakkaishi  69-  968  -974  2003  [Not refereed][Not invited]
  • Ojima Takao, Higuchi Tomoyuki, Nishita Kiyoyoshi  Fisheries Science  68-  (0)  1507  -1510  2002/11  [Not refereed][Not invited]
  • Y Iwamni, T Ojima, A Inoue, K Nishita  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY  133-  (2)  257  -267  2002/10  [Not refereed][Not invited]
     
    The nucleotide sequence of the cDNA encoding myosin heavy chain of chum salmon Oncorhynchus keta fast skeletal muscle was determined. The sequence consists of 5994 bp, including 5814 bp of translated region deducing an amino acid sequence of 1937 residues. The deduced sequence showed 79% homology to that of rabbit fast skeletal myosin and 84-87% homology to those of fast skeletal myosins from walleye pollack, white croaker and carp. The putative binding-sites for ATP, actin and regulatory light-chains in the subfragment-1 region of the salmon myosin showed high homology with the fish myosins (78-100% homology). However, the Loop-1 and Loop-2 showed considerably low homology (31-60%). On the other hand, the deduced sequences of subfragment-2 (533 residues) and light meromyosin (564 residues) showed 88-93% homology to the corresponding regions of the fish myosins. It becomes obvious that several specific residues of the rabbit LMM are substituted to Gly in the salmon LMM as well as the other fish LMMs. This may be involved in the structural instability of the fish myosin tail region. (C) 2002 Elsevier Science Inc, All.rights reserved.
  • T Higuchi, T Ojima, K Nishita  FISHERIES SCIENCE  68-  (5)  1145  -1150  2002/10  [Not refereed][Not invited]
     
    Heat-induced structural changes and aggregation properties of walleye pollack myosin, light meromyosin (LMM) and heavy meromyosin (HMM) were investigated. According to the circular dichroism (CD) measurement, the (x-helix content of the pollack myosin and LMM were estimated to be 72% and 90% at 5degreesC but decreased to 22% and 21% by increasing the temperature to 60degreesC with two transitions at 35degreesC and 50degreesC, respectively. In contrast, that of HMM decreased gradually from 37% to 33% by increasing the temperature from 5degreesC to 40degreesC, and decreased steeply to 20% above 50degreesC. These results indicate that the decrease in the (X-helix content in the myosin molecule upon heating was attributable mainly to the decrease in the (X-helix content in the LMM region. In contrast, 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence and light scattering intensity of both,myosin and HMM were remarkably increased above 25degreesC and 35degreesC, respectively, while those of LMM showed only a slight change even above 60degreesC. Although LMM alone formed no aggregates detectable by the light scattering measurement, it formed coprecipitates with myosin but not with HMM upon heating at 40degreesC for 10 min. These facts suggest that LMM bind to the LMM region of the myosin. Further, it was found that myosin gel formed in a test tube by the same heating conditions was significantly weakened by coexistence of LMM. These results suggest that the association of the LMM region of myosin molecules is essential for the heat-induced gelation of myosin.
  • Structure and function of starfish phospholipase A2
    Fisheries Science  68, supplement II/,1623-1624-  2002  [Not refereed][Not invited]
  • Interaction of walleye pollack light meromyosin with tail region of the pollack myosin by heat-treatment
    Fisheries Science  68, supplement II/,1625-1626-  2002  [Not refereed][Not invited]
  • Unfolding manner of walleye pollack light meromyosin upon heating
    Fisheries Science  68, supplement II/,1507-1510-  2002  [Not refereed][Not invited]
  • F Yumoto, M Nara, H Kagi, W Iwasaki, T Ojima, K Nishita, K Nagata, M Tanokura  EUROPEAN JOURNAL OF BIOCHEMISTRY  268-  (23)  6284  -6290  2001/12  [Not refereed][Not invited]
     
    FTIR spectroscopy has been applied to study the coordination structures of Mg2+ and Ca2+ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2+ binding site. The region of the COO- antisymmetric stretch provides information about the coordination modes of COO- groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm(-1) in the apo state, whereas additional bands were observed at 1543 and 1601 cm(-1) in the Ca2+-bound and Mg2+-bound states, respectively. The intensity of the band at 1567 cm(-1) in the Mg2+-bound state was identical to that in the apo state. Therefore, the sidechain COO- group of Glu142 at the 12th position in the Ca2+-binding site coordinates to Ca2+ in the bidentate mode but does not interact with Mg2+ directly. A slight upshift of COO- antisymmetric stretch due to Asp side-chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO- groups of Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the coordination structure of Mg2+ was different from that of Ca2+ in the Ca2+-binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2+-bound and Ca2+-bound states of Akazara scallop TnC, as spectral changes upon either Ca2+ or Mg2+ binding were very small in the infrared amide-I' region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2+ binding were larger than that upon Mg2+ binding. Moreover, gel-filtration experiments indicated that the molecular sizes of TnC had the order apo TnC > Mg2+-bound TnC > Ca2+-bound TnC. These results suggest that the tertiary structures are different in the Ca2+- and Mg2+-bound states. The present study may provide direct evidence that the side-chain COO- groups in the Ca2+-binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC.
  • H Kishimura, T Ojima, K Hayashi, K Nishita  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY  128-  (3)  565  -573  2001/03  [Not refereed][Not invited]
     
    Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta -D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris-HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE-cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca2+ requirement. (C) 2001 Elsevier Science Inc. All rights reserved.
  • Comparative Biochemistry and Physiology  129B-  787  2001  [Not refereed][Not invited]
  • 『かまぼこの足形成』<関伸夫、伊藤慶明>
    恒星社厚生閣  2001  [Not refereed][Not invited]
  • T Ojima, N Koizumi, K Ueyama, A Inoue, K Nishita  JOURNAL OF BIOCHEMISTRY  128-  (5)  803  -809  2000/11  [Not refereed][Not invited]
     
    Scallop troponin C (TnC) binds only one Ca2+/mol and the single Ca2+-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, Ii. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca2+-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca2+-regulation was investigated using a site-directed mutant with an inactivated site TV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and wild-type TnC (TnC(C)). Equilibrium dialysis using Ca-45(2+) demonstrated that TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca2+/mol, but that TnC-ZEQ and TnC(N) bind virtually no Ca2+. The UV difference spectra of TnC(W) and TnC(C) showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca2+-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addition, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl2, unlike TnC-ZEQ and TnC(N). These results indicate that Ca2+ binds only to site IV and that Ca2+-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N) were shown to form soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and Tnc, conferred Ca2+-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca2+-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca2+-sensitivity. Thus, the major functions of scallop TnC, such as Ca2+-binding and interaction with TnI, are located in the C-terminal domain, however, the full Ca2+-regulatory function requires the presence of the N-terminal domain.
  • T Ojima, N Koizumi, K Ueyama, A Inoue, K Nishita  JOURNAL OF BIOCHEMISTRY  128-  (5)  803  -809  2000/11  [Not refereed][Not invited]
     
    Scallop troponin C (TnC) binds only one Ca2+/mol and the single Ca2+-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, Ii. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca2+-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca2+-regulation was investigated using a site-directed mutant with an inactivated site TV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnC(N) and TnC(C)), and wild-type TnC (TnC(C)). Equilibrium dialysis using Ca-45(2+) demonstrated that TnC(W) and TnC(C) bind 0.6-0.8 mol of Ca2+/mol, but that TnC-ZEQ and TnC(N) bind virtually no Ca2+. The UV difference spectra of TnC(W) and TnC(C) showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca2+-binding, while TnC-ZEQ and TnC(N) did not show these bands. In addition, TnC(W) and TnC(C) showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl2, unlike TnC-ZEQ and TnC(N). These results indicate that Ca2+ binds only to site IV and that Ca2+-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnC(W), TnC-ZEQ, and TnC(C), but not TnC(N) were shown to form soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnC(W), TnC-ZEQ, or TnC(C), but not TnC(N). These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and Tnc, conferred Ca2+-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnC(N)-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca2+-sensitivity, while the TnC(C)-TnT-TnI complex conferred weak Ca2+-sensitivity. Thus, the major functions of scallop TnC, such as Ca2+-binding and interaction with TnI, are located in the C-terminal domain, however, the full Ca2+-regulatory function requires the presence of the N-terminal domain.
  • Yumoto F, Nara M, Kagi H, Nagata K, Iwasaki W, Ojima T, Nishita K, Tanokura M  Biophysics  40-  (1)  2000/08/05  [Not refereed][Not invited]
  • KISHIMURA H  Comp. Biochem. Physiol.  126-  (4)  579  -586  2000/08  [Not refereed][Not invited]
     
    Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A2 (PLA2), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA2 protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA2 protein, suggesting the presence of multiple forms in the starfish PLA2. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA2. In phylogenetic tree, the starfish PLA2 should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA2, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA2 from porcine pancreas.
  • M Togashi, M Kakinuma, Y Hirayama, H Fukushima, S Watabe, T Ojima, K Nishita  FISHERIES SCIENCE  66-  (2)  349  -357  2000/04  [Not refereed][Not invited]
     
    The amino acid sequences of myosin rod containing subfragment-2 (S2) and light meromyosin (LMM) were determined by cDNA cloning for walleye pollack fast skeletal myosin heavy chain. While S2 and LMM were composed of 442 and 656 amino acid residues, a total of 1937 amino acid residues accounted for the whole myosin heavy chain molecule with previously determined sequence for the subfragment-1 heavy chain region of this fish. Both regions for S2 and LMM showed a seven-residue repeat pattern characteristic to fibrous proteins with a coiled-coil structure of two alpha-helices, displaying a, b, c, d, e, f, and g where positions a and d were frequently occupied by hydrophobic amino acids and c and g often contained charged residues. The occurrence of a 28-residue unit with repetitive sequence was also strongly suggested, when one and three skip residues were adopted into S2 and LMM, respectively. Thus, walleye pollack S2 and LMM consisted of 17 and 24 zones with a 28-residue repeat rearrangement. There were several amino acid substitutions which might account for a low thermal stability of walleye pollack myosin heavy chain in comparison with the sequences of higher vertebrate counterparts. However, it seemed difficult to interpret such low stability only from the comparison in the 28-residue repeat arrangement at the primary structure.
  • M Togashi, M Kakinuma, Y Hirayama, H Fukushima, S Watabe, T Ojima, K Nishita  FISHERIES SCIENCE  66-  (2)  349  -357  2000/04  [Not refereed][Not invited]
     
    The amino acid sequences of myosin rod containing subfragment-2 (S2) and light meromyosin (LMM) were determined by cDNA cloning for walleye pollack fast skeletal myosin heavy chain. While S2 and LMM were composed of 442 and 656 amino acid residues, a total of 1937 amino acid residues accounted for the whole myosin heavy chain molecule with previously determined sequence for the subfragment-1 heavy chain region of this fish. Both regions for S2 and LMM showed a seven-residue repeat pattern characteristic to fibrous proteins with a coiled-coil structure of two alpha-helices, displaying a, b, c, d, e, f, and g where positions a and d were frequently occupied by hydrophobic amino acids and c and g often contained charged residues. The occurrence of a 28-residue unit with repetitive sequence was also strongly suggested, when one and three skip residues were adopted into S2 and LMM, respectively. Thus, walleye pollack S2 and LMM consisted of 17 and 24 zones with a 28-residue repeat rearrangement. There were several amino acid substitutions which might account for a low thermal stability of walleye pollack myosin heavy chain in comparison with the sequences of higher vertebrate counterparts. However, it seemed difficult to interpret such low stability only from the comparison in the 28-residue repeat arrangement at the primary structure.
  • H Kishimura, T Ojima, H Tanaka, K Hayashi, K Nishita  FISHERIES SCIENCE  66-  (1)  104  -109  2000/02  [Not refereed][Not invited]
     
    The complete amino acid sequence of phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera was determined by automated Edman degradation. The A. pectinifera PLA(2) (APLA(2)) consists of 137 amino acids with an unblocked N-terminus and its molecular weight is calculated to be 15 300.1. The enzyme contains 14 cysteine (Cys) residues at the corresponding positions of the same residues which have been shown to be involved in intramolecular disulfide bonds in mammalian pancreatic PLA(2). The region involving an active site and a Ca2+-binding loop shows fairly high sequence homology (75%) between the APLA(2) and porcine pancreatic PLA(2). The APLA(2) conserved the amino acid sequence of the loop portion of the porcine pancreatic PLA(2) except for the deletion of two amino acids. These features indicate that the APLA(2) can be classified into the group 1 type PLA(2). In contrast, the homology between the APLA(2) and porcine pancreatic PLA(2) was calculated to be 47% in the whole region. Further, the insertion of sixteen residues and the deletion of three residues were required in the sequence of the APLA(2) to align the corresponding region to the beta-wing of porcine pancreatic PLA(2). These differences in amino acid sequence of the APLA(2) may account for its specific properties such as the higher activity and the characteristic substrate specificity.
  • H Kishimura, T Ojima, H Tanaka, K Hayashi, K Nishita  FISHERIES SCIENCE  66-  (1)  104  -109  2000/02  [Not refereed][Not invited]
     
    The complete amino acid sequence of phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera was determined by automated Edman degradation. The A. pectinifera PLA(2) (APLA(2)) consists of 137 amino acids with an unblocked N-terminus and its molecular weight is calculated to be 15 300.1. The enzyme contains 14 cysteine (Cys) residues at the corresponding positions of the same residues which have been shown to be involved in intramolecular disulfide bonds in mammalian pancreatic PLA(2). The region involving an active site and a Ca2+-binding loop shows fairly high sequence homology (75%) between the APLA(2) and porcine pancreatic PLA(2). The APLA(2) conserved the amino acid sequence of the loop portion of the porcine pancreatic PLA(2) except for the deletion of two amino acids. These features indicate that the APLA(2) can be classified into the group 1 type PLA(2). In contrast, the homology between the APLA(2) and porcine pancreatic PLA(2) was calculated to be 47% in the whole region. Further, the insertion of sixteen residues and the deletion of three residues were required in the sequence of the APLA(2) to align the corresponding region to the beta-wing of porcine pancreatic PLA(2). These differences in amino acid sequence of the APLA(2) may account for its specific properties such as the higher activity and the characteristic substrate specificity.
  • Comparative Biochemistry and Physiology  126B-  579  2000  [Not refereed][Not invited]
  • A Inoue, T Ojima, K Nishita  FISHERIES SCIENCE  65-  (5)  772  -776  1999/10  [Not refereed][Not invited]
     
    cDNA clones encoding tropomyosin of akazara scallop Chlamys plipponensis akazara striated adductor muscle were isolated and sequenced. The largest cDNA obtained is composed of 1,995 bp including an open reading frame of 852 'bp at nucleotide positions 27-878, which deduced amino acid sequence of 284 residues with a calculated molecular weight of 32,540. According to database searches on NBRF-PIR 49.0 and GenBank, the amino acid sequence showed higher homology of 73% and 700ibto bloodfluke planorb tropomyosin and mussel anterior byssus retractor muscle tropomyosin, respectively, and considerably high homology of 53% and 51% to rabbit alpha- and beta-tropomyosins, respectively. The sequences corresponding to the critical region for actin-binding (residues 1-9) and troponin-T-binding region (near residues 150-180) of rabbit alpha-tropomyosin are conserved also in akazara scallop tropomyosin. As the nine residues at both of the N-terminus and C-terminus are generally regarded to form a head-to-tail interaction region, the residues at the N-terminus of akazara scallop tropomyosin show high sequence homology to those of various muscle tropomyosins. However, those at the C-terminus show lower sequence homology than those of vertebrate tropomyosin. Thus, head-to-tail interaction of akazara scallop tropomyosin may be different from rabbit tropomyosin on account of low homology of the C-terminal sequence.
  • A Inoue, T Ojima, K Nishita  FISHERIES SCIENCE  65-  (5)  772  -776  1999/10  [Not refereed][Not invited]
     
    cDNA clones encoding tropomyosin of akazara scallop Chlamys plipponensis akazara striated adductor muscle were isolated and sequenced. The largest cDNA obtained is composed of 1,995 bp including an open reading frame of 852 'bp at nucleotide positions 27-878, which deduced amino acid sequence of 284 residues with a calculated molecular weight of 32,540. According to database searches on NBRF-PIR 49.0 and GenBank, the amino acid sequence showed higher homology of 73% and 700ibto bloodfluke planorb tropomyosin and mussel anterior byssus retractor muscle tropomyosin, respectively, and considerably high homology of 53% and 51% to rabbit alpha- and beta-tropomyosins, respectively. The sequences corresponding to the critical region for actin-binding (residues 1-9) and troponin-T-binding region (near residues 150-180) of rabbit alpha-tropomyosin are conserved also in akazara scallop tropomyosin. As the nine residues at both of the N-terminus and C-terminus are generally regarded to form a head-to-tail interaction region, the residues at the N-terminus of akazara scallop tropomyosin show high sequence homology to those of various muscle tropomyosins. However, those at the C-terminus show lower sequence homology than those of vertebrate tropomyosin. Thus, head-to-tail interaction of akazara scallop tropomyosin may be different from rabbit tropomyosin on account of low homology of the C-terminal sequence.
  • Yumoto F, Nagata K, Nara M, Iwasaki W, Ojima T, Nishita K, Adachi K, Konno A, Nemoto N, Tanokura M  Biophysics  39-  (1)  1999/09/02  [Not refereed][Not invited]
  • T Ojima, T Higuchi, K Nishita  FISHERIES SCIENCE  65-  (3)  459  -465  1999/04  [Not refereed][Not invited]
     
    Reversibility of unfolding of alpha-helix in walleye pollack light meromyosin (LMM) by heat-treatment was investigated by circular dichroism spectrometry and alpha-chymotryptic digestion. By increasing the temperature from 5 to 50 degrees C, alpha-helix content of the LMM was decreased from 88% to 15% with a major and a minor transition at around 28 degrees C and 40 degrees C, respectively. On the other hand, alpha-helix content of the LMM was increased from 15% to 68% by decreasing the temperature from 50 degrees C to 5 degrees C, while it was not fully recovered to the original value, suggesting that some alpha-helical portions were irreversibly unfolded by heating. Therefore, the LMM was heated at 50 degrees C for 5 min, cooled to 0 degrees C, and then digested with alpha-chymotrypsin to assign the reversible and irreversible unfolding regions. As a result, the 42-kDa fragment and some smaller peptides which were hardly formed by the digestion of unheated LMM were newly formed. The 42-kDa fragment was identified as an N-terminal 2/3 region of the LMM by the amino acid sequencing of both N- and C-terminal regions, while most of the smaller peptides were derived from the C-terminal 1/3 region of the LMM. The 42-kDa fragment showed 90% alpha-helix content and its unfolding upon heating was almost reversible unlike the original LMM. From these results, we propose that the N-terminal 2/3 and C-terminal 1/3 regions of the pollack LMM are reversibly and irreversibly unfolding regions, respectively, by the heat-treatment.
  • Fisheries Science  65-  (3)  459  -465  1999  [Not refereed][Not invited]
  • T Ojima, N Kawashima, A Inoue, A Amauchi, M Togashi, S Watabe, K Nishita  FISHERIES SCIENCE  64-  (5)  812  -819  1998/10  [Not refereed][Not invited]
     
    Primary structure of heavy meromyosin region of walleye pollack Theragra chalcogramma myosin heavy chain was determined by cDNA cloning. By using one PCR product and five cDNA clones isolated from a lambda gt11-cDNA library for the pollack dorsal muscle, a nucleotide sequence of 3,923 bp comprising 60 bp of 5'-untranslational region and 3,863 bp of coding region was determined. The deduced sequence of 1,287 amino acids showed considerably high homology to the corresponding regions of carp myosin (83%) and chicken and rabbit myosins (both 79%). The sequences of the regions for the putative ATP-binding, actin-binding, and regulatory light chain-binding were well conserved among the pollack, carp, chicken, and rabbit myosins (83-100% homology). On the other hand, relatively low sequence homologies were seen in the essential light chain-binding site (52-78%), junctions between 20-kDa and 50-kDa domains (27-33%) and 25-kDa and 50-kDa domains (53-57%) of subfragment-1.
  • T Ojima, N Kawashima, A Inoue, A Amauchi, M Togashi, S Watabe, K Nishita  FISHERIES SCIENCE  64-  (5)  812  -819  1998/10  [Not refereed][Not invited]
     
    Primary structure of heavy meromyosin region of walleye pollack Theragra chalcogramma myosin heavy chain was determined by cDNA cloning. By using one PCR product and five cDNA clones isolated from a lambda gt11-cDNA library for the pollack dorsal muscle, a nucleotide sequence of 3,923 bp comprising 60 bp of 5'-untranslational region and 3,863 bp of coding region was determined. The deduced sequence of 1,287 amino acids showed considerably high homology to the corresponding regions of carp myosin (83%) and chicken and rabbit myosins (both 79%). The sequences of the regions for the putative ATP-binding, actin-binding, and regulatory light chain-binding were well conserved among the pollack, carp, chicken, and rabbit myosins (83-100% homology). On the other hand, relatively low sequence homologies were seen in the essential light chain-binding site (52-78%), junctions between 20-kDa and 50-kDa domains (27-33%) and 25-kDa and 50-kDa domains (53-57%) of subfragment-1.
  • Yumoto F, Iwasaki W, Nagata K, Ojima T, Nishita K, Tanokura M  Biophysics  38-  (2)  1998/09/07  [Not refereed][Not invited]
  • H Tanaka, T Ojima, K Nishita  JOURNAL OF BIOCHEMISTRY  124-  (2)  304  -310  1998/08  [Not refereed][Not invited]
     
    The complete amino acid sequence of Akazara scallop, Chlamys nipponensis akazara, troponin-I was determined by automated Edman degradation. It is composed of 292 amino acid residues with a blocked N-terminus. The nl,is calculated to be 34,678, about 14,000 larger than that of vertebrate skeletal troponin-I but significantly smaller than the 52,000 that had been estimated by SDS-polyacrylamide gel electrophoresis. The homologous sequence to vertebrate and arthropoda troponin-Is is found in the C-terminal region. In particular, the sequence of the regions essential for binding to actin and troponin-C is highly conserved. On the other hand, Akazara scallop troponin-I has 100-133 extra residues at the N-terminus compared with vertebrate troponin-I. This extra region is rich in Glu and Arg and has a unique sequence, that shows in part a high sequence homology with the tropomyosin-binding site of troponin-T and caldesmon.
  • A Inoue, T Ojima, K Nishita  FISHERIES SCIENCE  64-  (3)  459  -463  1998/06  [Not refereed][Not invited]
     
    A cDNA clone encoding troponin-rr of ezo-giant scallop Patinopecten yessoensis striated adductor muscle has been isolated and sequenced. The cDNA is composed of 1,939 bp with a translational region of 975 bp at the nucleotide positions 111-1,085. It deduced 325 amino acid residues with a molecular weight of 38,438. Ezo-giant scallop troponin-T showed high homology with akazara scallop troponin-rr (92%). However, it possesses 11 amino acid residues more than akazara scallop troponin-T and substitutions of 26 residues. According to sequence comparison with rabbit troponin-T, the residues 39-126 and 180-233 of ezo-giant scallop troponin-T appears to be tropomyosin-interacting regions and the residues 206-210 to be troponin-I-interacting region. Moreover, ezo-giant scallop troponin-rr was assumed to have close phylogenetic relations to troponin-Ts of akazara scallop, nematode Caenorhabditis elegans, and fruit fly Drosophila melanogaster.
  • A Inoue, T Ojima, K Nishita  FISHERIES SCIENCE  64-  (3)  459  -463  1998/06  [Not refereed][Not invited]
     
    A cDNA clone encoding troponin-rr of ezo-giant scallop Patinopecten yessoensis striated adductor muscle has been isolated and sequenced. The cDNA is composed of 1,939 bp with a translational region of 975 bp at the nucleotide positions 111-1,085. It deduced 325 amino acid residues with a molecular weight of 38,438. Ezo-giant scallop troponin-T showed high homology with akazara scallop troponin-rr (92%). However, it possesses 11 amino acid residues more than akazara scallop troponin-T and substitutions of 26 residues. According to sequence comparison with rabbit troponin-T, the residues 39-126 and 180-233 of ezo-giant scallop troponin-T appears to be tropomyosin-interacting regions and the residues 206-210 to be troponin-I-interacting region. Moreover, ezo-giant scallop troponin-rr was assumed to have close phylogenetic relations to troponin-Ts of akazara scallop, nematode Caenorhabditis elegans, and fruit fly Drosophila melanogaster.
  • 尾島 孝男  日本水産学会誌  64-  (3)  1998/05/15  [Not refereed][Not invited]
  • A Inoue, T Ojima, K Nishita  FISHERIES SCIENCE  64-  (1)  164  -165  1998/02  [Not refereed][Not invited]
  • A Inoue, T Ojima, K Nishita  FISHERIES SCIENCE  64-  (1)  164  -165  1998/02  [Not refereed][Not invited]
  • Journal of Biochemistry  124-  304  1998  [Not refereed][Not invited]
  • T Ojima, S Yoshikawa, K Nishita  FISHERIES SCIENCE  63-  (5)  811  -815  1997/10  [Not refereed][Not invited]
     
    Actomyosin was extracted from myofibrils obtained from walleye pollack frozen surimi with 0.5 M KCl, 1 M sorbitol, and 1 mM Mg-ATP. Myosin was separated from F-actin by ultracentrifugation in the presence of MgCl2 and ATP. The myosin consisted of heavy chains of Mr 200,000 and light chains of Mr 25,000, 18,000, and 17,000. Ca-ATPase activity of the myosin decreased approximately 20% per day at 0 degrees C in 0.5 M KCl (pH 7.0) containing 1 M sorbitol. Ca-ATPase specific activity was approximately 8 times lower but EDTA-ATPase specific activity was several times higher than those of rabbit myosin at 0.05-0.5 M KCl, although the respective activities of both myosins showed similar dependences on KCI and pH. The Mg-ATPase activity of pollack myosin was increased approximately 100 times by the addition of an equal weight of rabbit F-actin and the activity was approximately 5 times higher than that of rabbit myosin. On the other hand, the actomyosin showed a significant increase in Mg-ATPase activity upon incubating at 20-30 degrees C, probably due to some irreversible conformational changes of myosin. The pollack myosin formed mini-filaments of about 0.5 mu m length and the filaments tended to aggregate with each other.
  • Y Nishimura, T Ojima, K Nishita  FISHERIES SCIENCE  63-  (5)  802  -806  1997/10  [Not refereed][Not invited]
     
    Tropomyosins were isolated from striated and smooth adductor muscles of akazara scallop Chlamys nipponensis akazara and ezo-giant scallop Patinopecten yessoensis and smooth adductor muscle of surf clam Spisula sacharinensis. These bivalve tropomyosins inhibited Mg-ATPase activity of rabbit reconstituted actomyosin to 15% of original activity, i.e., approximately 1.4 times stronger than rabbit and carp tropomyosins did at a weight ratio of 2:1 for F-actin: tropomyosin. On the other hand, the relative viscosities in the absence of KCl were 3.5-6 times higher than those of rabbit and carp tropomyosins. The intrinsic viscosities were, however, comparable to that of rabbit tropomyosin, i.e., 0.5 dl/g for bivalve tropomyosins and 0.4 dl/g for rabbit tropomyosin. These viscosities suggest that the bivalve tropomyosins tend to form a highly polymerized form compared with rabbit tropomyosin. Circular dichroism spectra and amino acid compositions of the bivalve tropomyosins are similar to one another and also to rabbit and carp tropomyosins. C-terminal three amino acids of bivalve tropomyosins were sequenced as -Ala-Gly-Tyr (scallops) and -Gly-Tyr-Thr (surf clam), which are quite different from -Thr-Ser-Ile and Thr-Ser-Leu of rabbit alpha- and beta-tropomyosin, respectively.
  • T Ojima, S Yoshikawa, K Nishita  FISHERIES SCIENCE  63-  (5)  811  -815  1997/10  [Not refereed][Not invited]
     
    Actomyosin was extracted from myofibrils obtained from walleye pollack frozen surimi with 0.5 M KCl, 1 M sorbitol, and 1 mM Mg-ATP. Myosin was separated from F-actin by ultracentrifugation in the presence of MgCl2 and ATP. The myosin consisted of heavy chains of Mr 200,000 and light chains of Mr 25,000, 18,000, and 17,000. Ca-ATPase activity of the myosin decreased approximately 20% per day at 0 degrees C in 0.5 M KCl (pH 7.0) containing 1 M sorbitol. Ca-ATPase specific activity was approximately 8 times lower but EDTA-ATPase specific activity was several times higher than those of rabbit myosin at 0.05-0.5 M KCl, although the respective activities of both myosins showed similar dependences on KCI and pH. The Mg-ATPase activity of pollack myosin was increased approximately 100 times by the addition of an equal weight of rabbit F-actin and the activity was approximately 5 times higher than that of rabbit myosin. On the other hand, the actomyosin showed a significant increase in Mg-ATPase activity upon incubating at 20-30 degrees C, probably due to some irreversible conformational changes of myosin. The pollack myosin formed mini-filaments of about 0.5 mu m length and the filaments tended to aggregate with each other.
  • K Nishita, T Ojima, A Takahashi, A Inoue  JOURNAL OF BIOCHEMISTRY  121-  (3)  419  -424  1997/03  [Not refereed][Not invited]
     
    Troponin which can confer Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity has been prepared from the smooth adductor muscle of Ezo-giant scallop (Patinopecten yessoensis). The troponin comprises 40-, 20-, and 19-kDa components, In order to characterize the components, they were separated from each other by CM-Toyopearl column chromatography in the presence of BM urea, Consequently, the 20-kDa component was identified as troponin C, based on the Ca2+-binding ability, The amount of Ca2+ bound to the troponin C was estimated to be 0.75 mol/mol at 10(-4) M Ca2+ by the equilibrium dialysis method, The 19-kDa component was identified as troponin I on the basis of not only its inhibitory effect on rabbit actomyosin Mg-ATPase activity along with the smooth adductor tropomyosin, but also the releasing effect of the smooth adductor troponin C on the inhibition, On the other hand, the 40-kDa component was regarded as troponin T on the basis that it bound to F-actin-tropomyosin filament and was indispensable for conferring Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity, along with troponin C and troponin I, The above assignments were confirmed by both amino acid analysis and immunoblotting using rabbit antisera raised against counterparts of scallop striated muscle troponin.
  • K Nishita, T Ojima, A Takahashi, A Inoue  JOURNAL OF BIOCHEMISTRY  121-  (3)  419  -424  1997/03  [Not refereed][Not invited]
     
    Troponin which can confer Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity has been prepared from the smooth adductor muscle of Ezo-giant scallop (Patinopecten yessoensis). The troponin comprises 40-, 20-, and 19-kDa components, In order to characterize the components, they were separated from each other by CM-Toyopearl column chromatography in the presence of BM urea, Consequently, the 20-kDa component was identified as troponin C, based on the Ca2+-binding ability, The amount of Ca2+ bound to the troponin C was estimated to be 0.75 mol/mol at 10(-4) M Ca2+ by the equilibrium dialysis method, The 19-kDa component was identified as troponin I on the basis of not only its inhibitory effect on rabbit actomyosin Mg-ATPase activity along with the smooth adductor tropomyosin, but also the releasing effect of the smooth adductor troponin C on the inhibition, On the other hand, the 40-kDa component was regarded as troponin T on the basis that it bound to F-actin-tropomyosin filament and was indispensable for conferring Ca2+-sensitivity upon rabbit actomyosin Mg-ATPase activity, along with troponin C and troponin I, The above assignments were confirmed by both amino acid analysis and immunoblotting using rabbit antisera raised against counterparts of scallop striated muscle troponin.
  • T Ojima, M Maita, A Inoue, K Nishita  FISHERIES SCIENCE  63-  (1)  137  -141  1997/02  [Not refereed][Not invited]
     
    To investigate structure-function relationship for akazara scallop troponin C (TnC), we constructed a bacterial expression system using a cDNA clone which we isolated previously (Ojima, T., Tanaka, H., and Nishita, K. Arch. Biochem. Biophys., 311, 272-276 (1994)). The cDNA for akazara scallop TnC was subcloned into the expression plasmid pET-16b and expressed in Escherichia coli BL21 (DE3) by the induction with isopropyl-beta-D(-)-thiogalactopyranoside. Amount of the expressed TnC increased up to approximately 10% of total protein during 4 h incubation. The TnC was then extracted from the bacterial pellet and purified by subsequent column chromatographies on DEAE-Toyopearl 650M and Phenyl-Sepharose CL-4B. Although the expressed TnC possessed an extra moiety of 6 amino acids at N-terminus, which was derived from adaptor DNA of lambda gt11 and 5'-untranslated region of the cDNA clone, it showed basically the same properties as those of native TnC with respect to the Ca2+-induced difference UV-absorption spectrum and Ca2+-regulatory ability when reconstituted with native troponin T and troponin I.
  • T Ojima, M Maita, A Inoue, K Nishita  FISHERIES SCIENCE  63-  (1)  137  -141  1997/02  [Not refereed][Not invited]
     
    To investigate structure-function relationship for akazara scallop troponin C (TnC), we constructed a bacterial expression system using a cDNA clone which we isolated previously (Ojima, T., Tanaka, H., and Nishita, K. Arch. Biochem. Biophys., 311, 272-276 (1994)). The cDNA for akazara scallop TnC was subcloned into the expression plasmid pET-16b and expressed in Escherichia coli BL21 (DE3) by the induction with isopropyl-beta-D(-)-thiogalactopyranoside. Amount of the expressed TnC increased up to approximately 10% of total protein during 4 h incubation. The TnC was then extracted from the bacterial pellet and purified by subsequent column chromatographies on DEAE-Toyopearl 650M and Phenyl-Sepharose CL-4B. Although the expressed TnC possessed an extra moiety of 6 amino acids at N-terminus, which was derived from adaptor DNA of lambda gt11 and 5'-untranslated region of the cDNA clone, it showed basically the same properties as those of native TnC with respect to the Ca2+-induced difference UV-absorption spectrum and Ca2+-regulatory ability when reconstituted with native troponin T and troponin I.
  • INOUE Akira, OJIMA Takao, NISHITA Kiyoyoshi  J. Biochem.  120-  (4)  834  -837  1996/10/01  [Not refereed][Not invited]
  • A Inoue, T Ojima, K Nishita  JOURNAL OF BIOCHEMISTRY  120-  (4)  834  -837  1996/10  [Not refereed][Not invited]
     
    A cDNA clone encoding troponin T of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle has been isolated and sequenced, The complete sequence deduced consists of 314 amino acid residues with a molecular weight of 37,206, Akazara scallop troponin T contains 55 amino acid residues more and 82 residues fewer than rabbit skeletal muscle troponin T and Drosophila melanogaster troponin T, respectively, showing almost the lowest sequence homology with rabbit troponin T (26%) but the highest homology with Drosophila troponin T (33%), Further, high sequence homology was seen in the functional regions: residues 33-120 and 174-227, corresponding respectively to residues 71-158 and 197-250 of rabbit troponin T (tropomyosin-binding regions); and residues 200-204, corresponding to 223-227 of rabbit troponin T (troponin I-binding region), In residues 1-70 (tropomyosin-binding region), however, only six residues are identical with rabbit troponin T.
  • 西川 真史, 尾島 孝男, 西田 清義  日本分子生物学会年会プログラム・講演要旨集  19-  1996/08/01  [Not refereed][Not invited]
  • 井上 晶, 尾島 孝男, 西田 清義  日本分子生物学会年会プログラム・講演要旨集  19-  1996/08/01  [Not refereed][Not invited]
  • T OJIMA, T TOYOGUCHI, K NISHITA  FISHERIES SCIENCE  61-  (5)  871  -875  1995/10  [Not refereed][Not invited]
     
    Troponin, which confers high Ca2+-sensitivity to rabbit reconstituted actomyosin, was isolated from the abdominal muscle of prawn Penaeus japonicus. SDS-polyacrylamide gel electrophoresis showed that the prawn troponin consists of three components of Mr 17,000, 30,000-32,000 (doublet), and 42,000 in a molar ratio of 1:1:1, but Mr 52,000-59,000 component, which had been reported to be a troponin-T-like protein in arthropod troponin, was not found. The three components were separated from each other by DEAE-Toyopearl column chromatography in the presence of 6 M urea, and assigned as troponin-C, troponin-I, and troponin-T in increasing order of molecular weight. These features were basically identical with those of American lobster troponin of our previous study (Nishita and Ojima, J. Biochem., 108, 677-683 (1990)).
  • T OJIMA, H TANAKA, K NISHITA  JOURNAL OF BIOCHEMISTRY  117-  (1)  158  -162  1995/01  [Not refereed][Not invited]
     
    The M(r) 52,000 subunit of Akazara scallop striated muscle troponin, which was tentatively identified as troponin I, was cleaved into two major fragments with CNBr: C-terminal 17 kDa fragment (CN17K) and N-terminal 35 kDa fragment (CN35K) [J. Biochem. 108, 519-521 (1990)]. CN17K inhibits rabbit reconstituted actomyosin Mg-ATPase activity, weakly in the absence of troponin T but strongly in its presence, together with Akazara tropomyosin. CN35K, however, hardly shows such inhibition. Thus, the amino acid sequence of the CN17K was determined by the Edman method. CN17K comprises 135 amino acid residues and its calculated molecular mass is 15,732 Da. A computer search of the SWISS-PROT data base revealed the TnIs of crayfish tail muscle, rabbit skeletal muscle, and bovine cardiac muscle to be homologous proteins with total sequence homologies of 39, 30, and 30%, respectively, to CN17K. Significantly high homology was observed among these TnIs in the regions around residues 75-95, 99-114, and 135-151 of the rabbit TnI. From these facts, we conclude that the 52K subunit is a TnI.
  • T OJIMA, H TANAKA, K NISHITA  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS  311-  (2)  272  -276  1994/06  [Not refereed][Not invited]
     
    A cDNA clone encoding troponin C of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle was isolated and sequenced. The cDNA is composed of 1987 bp and has an open reading frame of 462 bp at nucleotide positions 12-473. The amino acid sequence deduced from the cDNA showed 89% homology with that of Ezo-giant scallop (Patinopecten yessoensis) troponin C (K. Nishita, H. Tanaka, and T. Ojima, 1994, J. Biol. Chem. 269, 3464-3468). However, it showed lower homology with those of troponin Cs from the other invertebrates (35-40%) and vertebrates (30%). A single functional Ca2+-binding site was found in site IV of the four potential Ca2+-binding sites as in the case of Ezo-giant scallop troponin C. Three amino acid deletions in the central D/E helix were required to align the scallop sequences with those of other troponin Cs. (C) 1994 Academic Press,Inc.
  • K NISHITA, H TANAKA, T OJIMA  JOURNAL OF BIOLOGICAL CHEMISTRY  269-  (5)  3464  -3468  1994/02  [Not refereed][Not invited]
     
    Troponin and its Ca2+-binding subunit troponin C (TnC) of the Ezo-giant scallop, Patinopecten yessoensis, have been revealed to bind only 1 mol of Ca2+/mol irrespective of the presence and absence of Mg2+. The amino acid sequence of the TnC has been determined by the automated Edman degradation. TnC is composed of 152 residues including 3 tryptophans at positions 53, 70, and 109, 4 cysteines at positions 19, 31, 67, and 145, and no proline. The molecular weight is calculated as 17,410. The NH2 terminus of TnC is blocked by an acetyl group. The sequence of scallop TnC required deletion of three residues in the D/E linker region to maximize sequence homology to other TnCs and shows considerably lower homology to vertebrate skeletal TnCs (27-30%), ascidian TnC (26%), arthropoda TnCs (30-37%), and chicken calmodulin (37%). Further, we conclude that Ezo-giant scallop TnC binds Ca2+ at site IV, a site specific for Ca2+. The other sites I, II, and III appeared to lose the Ca2+ binding ability due to substitutions of some important residues.
  • S CHIBA, T OJIMA, H TANAKA, K NISHITA  NIPPON SUISAN GAKKAISHI  59-  (10)  1783  -1791  1993/10  [Not refereed][Not invited]
     
    A 160 kDa actin-binding protein was isolated from surf clam foot muscle by DEAE-Toyopearl column chromatography. The protein was found to complex readily with F-actin to form turbidity. The turbidity reached a maximum when bound in a ratio of approx. 0.3:1 (w/w). The binding ratio as well as turbidity decreased steeply as the KCl increased concentration up to 80-100 mM. In these events, F-actin filaments were aggregated side by side and formed complicated networks, whose morphological features revealed close similarity to those reported for F-actin bundles formed by caldesmon. However, the binding features of the 160 kDa protein to F-actin are different from those with caldesmon in the presence of Ca2+-calmodulin. Besides, further comparison of molecular weights, amino acid compositions, and alpha-helix contents revealed that the 160 kDa protein is different from caldesmon, alpha-actinin, and several other actin-binding proteins. Thus, we consider that the 160 kDa protein is a novel actin-binding protein.
  • T OJIMA, K NISHITA  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS  299-  (2)  344  -349  1992/12  [Not refereed][Not invited]
  • O MAEDA, T OJIMA, K NISHITA  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY  102-  (1)  155  -157  1992/05  [Not refereed][Not invited]
     
    1. The scallop calpain-like proteinase is about five times more labile than the rabbit calpain II upon heat treatment at 35-degrees-C. 2. By autolysis of the scallop proteinase of two 100 kDa subunits, 90, 45 and 30 kDa fragments were formed. Thereby the activity decreased monophasically in the presence of millimolar order of Ca2+, but did not increase in the presence of micromolar order of Ca2+ unlike the rabbit calpain II.
  • O MAEDA, T OJIMA, K NISHITA  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY  102-  (1)  149  -153  1992/05  [Not refereed][Not invited]
     
    1. A Ca2+-dependent cysteine proteinase was purified from scallop striated adductor muscle by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephacryl S-300. 2. The enzyme is of M(r) approximately 200,000, composed of two M(r) 100,000 subunits. 3. The enzyme is a cysteine proteinase with optimum activity at pH 6.8 and about 18-degrees-C. In addition, it requires 1.7 mM Ca2+ for half-maximal activity and more than 10 mM Ca2+ for maximal activity. Thus the enzyme can be classified as calpain II.
  • Absence of troponin in surf-clam foot muscle
    Nippon Suisan Gakkaishi  58-  1919  -1923  1992  [Not refereed][Not invited]
  • Comparative studies on biochemical properties of troponins from ezo-giant scallop (Patinopecten yessoensis) and akazara scallop (Chlamys nipponensis akazara).
    Comparative Biochemistry and Physiology  103B-  727  -732  1992  [Not refereed][Not invited]
  • T OJIMA, K NISHITA  JOURNAL OF BIOCHEMISTRY  110-  (6)  847  -850  1991/12  [Not refereed][Not invited]
     
    A binary complex consisting of M(r) 19,000 and M(r) 40,000 components was co-purified with troponin from a crude troponin fraction of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle. This complex is incapable of conferring Ca2+-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, rather strongly inhibiting it, but became capable on further complexing with Akazara scallop troponin-C. To examine the effects of the M(r) 19,000 and M(r) 40,000 components on the ATPase activity, they were separated from each other by CM-Toyopearl column chromatography. The M(r) 19,000 component strongly inhibited the Mg-ATPase activity of actomyosin-tropomyosin and the inhibition was reversed by further addition of the Akazara scallop troponin-C. On the other hand, the M(r) 40,000 component slightly increased it. On hybridization with the Akazara scallop troponin subunits, the M(r) 19,000 and M(r) 40,000 components were shown to be able to substitute for troponin-I and troponin-T, respectively. The amino acid compositions of the M(r) 40,000 component and troponin-T were almost identical, and those of the M(r) 19,000 component and M(r) 17,000 C-terminal fragment of the troponin-I resembled each other fairly well. From these results, it may be concluded that the M(r) 19,000-40,000 binary complex is the troponin-I-troponin-T complex.
  • K NISHITA, T OJIMA  JOURNAL OF BIOCHEMISTRY  108-  (4)  677  -683  1990/10  [Not refereed][Not invited]
  • T OJIMA, H TANAKA, K NISHITA  JOURNAL OF BIOCHEMISTRY  108-  (4)  519  -521  1990/10  [Not refereed][Not invited]
  • 森 建太, 尾島 孝男, 西田 清義  日本水産学会誌  56-  (4)  663  -670  1990  [Not refereed][Not invited]
  • Kenta Mori, Takao Ojima, Kiyoyoshi Nishita  Nippon Suisan Gakkaishi  56-  (4)  663  -670  1990  [Not refereed][Not invited]
  • T OJIMA, K NISHITA  NIPPON SUISAN GAKKAISHI  55-  (10)  1857  -1863  1989/10  [Not refereed][Not invited]
  • T OJIMA, K YOKOMOTO, K NISHITA  NIPPON SUISAN GAKKAISHI  55-  (3)  567  -574  1989/03  [Not refereed][Not invited]
  • 森 建太, 尾島 孝男, 西田 清義  日本水産学会誌  55-  (10)  1851  -1856  1989  [Not refereed][Not invited]
  • 辻 正之, 尾島 孝男, 西田 清義  日本水産学会誌  55-  (4)  681  -687  1989  [Not refereed][Not invited]
  • 杉野 英彦, 小島 直也, 西田 清義, 尾島 孝男  日本水産学会誌  55-  (2)  333  -340  1989  [Not refereed][Not invited]
  • Kenta Mori, Takao Ojima, Kiyoyoshi Nishita  Nippon Suisan Gakkaishi  55-  (10)  1851  -1856  1989  [Not refereed][Not invited]
  • Hybridization between molluscan myosins and regulatory light chains from rabbit
    Nippon Suisan Gakkaishi  55-  681  -687  1989  [Not refereed][Not invited]
  • Properties of CNBr fragments of scallop troponin C and their pertial amino-acid sequences
    Nippon Suisan Gakkaishi  55-  333  -340  1989  [Not refereed][Not invited]
  • T OJIMA, K NISHITA  JOURNAL OF BIOCHEMISTRY  104-  (2)  207  -210  1988/08  [Not refereed][Not invited]
  • T OJIMA, K NISHITA  JOURNAL OF BIOCHEMISTRY  104-  (1)  9  -11  1988/07  [Not refereed][Not invited]
  • H TANAKA, T MAITA, T OJIMA, K NISHITA, G MATSUDA  JOURNAL OF BIOCHEMISTRY  103-  (3)  572  -580  1988/03  [Not refereed][Not invited]
  • 尾島 孝男, 西田 清義  日本水産学会誌  53-  (1)  93  -98  1987  [Not refereed][Not invited]
  • Takao Ojima, Kiyoyoshi Nishita  Nippon Suisan Gakkaishi  53-  (1)  93  -98  1987  [Not refereed][Not invited]
  • T OJIMA, K NISHITA  JOURNAL OF BIOCHEMISTRY  100-  (3)  821  -824  1986/09  [Not refereed][Not invited]
  • Troponin from akazara scallop striated adductor muscles
    Journal of Biological Chemistry  261-  16749  -16754  1986  [Not refereed][Not invited]
  • 尾島 孝男, 市川 寿, 西田 清義  日本水産学会誌  52-  (5)  839  -845  1986  [Not refereed][Not invited]
  • 岸村 栄毅, 尾島 孝男, 西田 清義  日本水産学会誌  52-  (8)  1469  -1472  1986  [Not refereed][Not invited]
  • 日本水産学会誌  52-  847  -851  1986  [Not refereed][Not invited]
  • Releasing properties of regulatory light chains from smooth and striated adductor muscles of bivalves
    Nippon Suisan Gakkaishi  52-  839  -845  1986  [Not refereed][Not invited]
  • Ca2+-binding and Ca2+-sensitivity of akazara scallop myosin and its hybrid myosins.
    Nippon Suisan Gakkaishi  52-  1469  -1472  1986  [Not refereed][Not invited]
  • Hybridization between fish regulatory light chains and akazara scallop desensitized myosin
    Nippon Suisan Gakkaishi  52-  847  -851  1986  [Not refereed][Not invited]
  • T OJIMA, K NISHITA, S WATANABE  JOURNAL OF BIOCHEMISTRY  96-  (4)  1109  -1115  1984  [Not refereed][Not invited]
  • T MAITA, K KONNO, T OJIMA, G MATSUDA  JOURNAL OF BIOCHEMISTRY  95-  (1)  167  -177  1984  [Not refereed][Not invited]
  • T OJIMA, K NISHITA, S WATANABE  JOURNAL OF BIOCHEMISTRY  94-  (1)  307  -310  1983  [Not refereed][Not invited]
  • 引地 聰, 尾島 孝男, 角舘 覚, 西田 清義  日本水産学会誌  49-  (1)  141  -148  1983  [Not refereed][Not invited]
  • 尾島 孝男, 西田 清義  日本水産学会誌  49-  (8)  1257  -1264  1983  [Not refereed][Not invited]
  • T OJIMA, K NISHITA, S WATANABE  JOURNAL OF BIOCHEMISTRY  93-  (2)  607  -613  1983  [Not refereed][Not invited]
  • Biochemical properties and Ca2+-sensitivities of akazara scallop and surf-clam smooth muscles.
    Nippon Suisan Gakkaishi  49-  141  -148  1983  [Not refereed][Not invited]
  • Reversible release of regulatory light chains from akazara striated adductor myosin by heat treatment
    Nippon Suisan Gakkaishi  49-  1257  -1264  1983  [Not refereed][Not invited]
  • T OJIMA, K NISHITA, S WATANABE  JOURNAL OF BIOCHEMISTRY  89-  (4)  1333  -1335  1981  [Not refereed][Not invited]
  • Myosin from striated adductor muscle of Chlamys nipponensis akazara.
    Journal of Biochemistry  86-  663  -673  1979  [Not refereed][Not invited]

Industrial Property Rights

  • 新規のFlavobacterium属細菌、その細菌が産出するアルギン酸を分解する酵素およびそれらを用いてオリゴ糖、不飽和単糖、ないしα-ケト酸を製造する方法
    特願2011-66721
  • アメフラシ由来キシログルカナーゼ
    2010-033922
  • アメフラシ由来マンナナーゼ
    2009-214412
  • 「巻貝類由来のセルラーゼ及びその製造方法」
    2002-34852
  • Xyloglucanase from sea hare
    2010-033922
  • Mannanase from sea hare
    2009-214412

Awards & Honors

  • 2010/03 日本水産学会 Award for excellent paper in Fisheries Science
     Comparative study on general properties of alginate lyases from some marine gastropod mollusks 
    受賞者: M. Hata;Y. Kumagai;M. M. Rahman;S. Chiba;H. Tanaka;A. Inoue;T. Ojima
  • 2002/03 日本水産学会 日本水産学会進歩賞
     水産軟体動物トロポニンの構造と機能に関する研究 
    受賞者: 尾島 孝男

Research Grants & Projects

  • Biochemical studies and protein engineerings on muscular proteins, glucanases, and proteases from marine organisms.
    Date (from‐to) : 2000


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