Toshihide Nishimura

Nippon Veterinary and Life Science University, Edo, Tōkyō, Japan

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Publications (24)36.74 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS-PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A(4) hydrolase (LTA(4)H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.
    No preview · Article · Jun 2011 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: In this study, we examined the contribution of the cathepsins (cathepsin D and crude cathepsins containing cathepsins B and L) to troponin T degradation during postmortem aging. The action of cathepsin D on troponin T was examined at various pHs (pH 4.0-6.5). The degradation of intact troponin T was observed at pH 4.0, but not observed at pH 5.5 and 6.5. As a result of the degradation of troponin T, the 30 kDa fragment was not generated in any pH condition. The action of the crude cathepsins on troponin T was also examined at various pHs (pH 4.0-6.5). The intact troponin T was degraded at pH 4.0 and the 30 kDa fragments were observed. These 30 kDa fragments disappeared during further incubation. On the other hand, at pH 5.5 and 6.5, the intact troponin T was degraded and the 30 kDa fragment was accumulated. These results suggested that the cathepsin D scarcely contributed to the degradation of troponin T during postmortem aging, but crude cathepsins containing cathepsins B and L were partially involved in the degradation of troponin T and the generation of 30 kDa fragments.
    No preview · Article · Aug 2010 · Animal Science Journal
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    ABSTRACT: Dimethyl trisulfide (DMTS) is involved in the unpalatable aroma of stale sake, called "hineka"; however, the mechanism underlying the formation of DMTS during the storage of sake has not been elucidated. This paper investigates the precursors of DMTS in sake. An experiment using [methyl-d(3)]-methionine showed that Strecker degradation of methionine plays a minor role in the formation of DMTS. Separation of components in sake by cation exchange resin revealed that DMTS precursors are present in the acidic/neutral fraction rather than in the basic one. Purification of the DMTS precursor compounds was carried out through several chromatographic steps, measuring DMTS-producing potential as an index. High-resolution ESI-MS and 1D/2D NMR experiments enabled the identification of one of the precursor compounds as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one.
    No preview · Article · Jan 2009 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: In this study, collagen extracted from chicken legs (which are the yellow keratin parts containing a nail) was hydrolyzed with various enzymes, and the angiotensin I-converting enzyme (ACE)-inhibitory activity of each hydrolysate was determined. The hydrolysate by treatment with an Aspergillus species-derived enzyme had the highest activity (IC 50 = 260 microg/mL). The fraction of this hydrolysate obtained by ultrafiltration with a molecular-weight cutoff of 3000 Da (low fraction) had a stronger activity (IC 50 = 130 microg/mL) than the fractionated one. This fraction was further fractionated by HPLC, and the peptides in the fraction with high ACE-inhibitory activity were identified. The amino acid sequences of the four peptides were identified using a protein sequencer. These peptides were synthesized to confirm their ACE-inhibitory activities; this showed that peptides with a Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro sequence had the highest activity (IC 50 = 29 microM). When the low fraction was administered to spontaneous hypertensive rats, a decrease in their blood pressure was observed after 2 h of administration, and a significant decrease in blood pressure (-50 mmHg) was observed after 6 h. Moreover, long-term administration studies indicated that the low fraction showed a significant suppression of increased blood pressure.
    Preview · Article · Oct 2008 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: Protein hydrolysates obtained by treatment with papain, trypsin, chymotrypsin and actinase all exhibited inhibitory activity (IC50: 3.4–41.8 mg%) toward angiotensin-converting enzyme (ACE) (EC In particular, the protein hydrolysate obtained by treatment with papain showed the highest inhibitory activity (3.7–5.3 mg%). The ACE inhibitory activity of the gluten hydrolysate obtained with actinase was mainly due to peptides of less than 500 Da in molecular mass. On the other hand, the ACE inhibitory activity of the myofibrillar protein hydrolysate obtained with papain was due to peptides of both less and more than 500 Da in molecular mass. The blood pressure of spontaneously hypertensive rats (SHR) administered the myofibrillar protein hydrolysate was significantly reduced at 2 h after administration. The blood pressure of SHR was also reduced at 2 h after administration of the gluten hydrolysate, and this effect continued until 6 h. These hydrolysates may potentially be useful as antihypertensive food materials.
    No preview · Article · Feb 2007 · Journal of Food Biochemistry
  • Kaoru INDOH · Sadao NAGATA · Ken KANZAKI · Kiwamu SHIIBA · Toshihide NISHIMURA
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    ABSTRACT: A new method for producing fermented fish sauce was developed to improve the taste of the fish sauce. The method involved the production of a fermented sauce, seasoning liquid from salmon that was enzymatically hydrolyzed with the wheat gluten koji, lactic acid bacteria and yeast. During fermentation, the total nitrogen, lactic acid, and ethanol contents in the fish sauce increased continuously and then plateaued 3 months after fermentation had begun. We investigated the chemical composition and the sensory properties of the fish sauce prepared by this 3-month fermentation, and we compared these characteristics with those of the fish sauce from salmon similarly hydrolyzed with soy sauce koji prepared from soybean and wheat. The fish sauce produced using wheat gluten was very light-colored and had a higher content of free amino acids, especially glutamic acid. The peptides consisted mainly of Glx, Asx and Pro, compared with Asx, Glx, and Gly in the liquid from soy sauce koji. Sensory evaluation revealed that the fish sauce derived from the wheat gluten koji had an intense umami taste and a fine flavor better than that of soy sauce koji.
    No preview · Article · Aug 2006 · Food Science and Technology Research
  • Koshiro Migita · Toshihide Nishimura
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    ABSTRACT: To elucidate the mechanisms involved in the increase in free amino acids during postmortem storage of meat, a novel aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and successive chromatographies such as DEAE-cellulose, Sephacryl S-200, Hydroxyapatite, Phenyl-Sepharose, and Hi-Trap affinity column chromatography. The molecular mass of the enzyme was found to be 58 kDa on SDS-PAGE. This enzyme had optimum pH at around 7.5, and preferably hydrolyzed Ala-beta-naphthylamide (-NA) in amino acid-NAs. The activity was strongly inhibited by phenylmethansulfonyl fluoride (PMSF) and bestatin, suggesting that it is to be classified as a serine protease. Moreover, the activity was enhanced by chloride and nitrate ions, which is the most remarkable property of this enzyme. The enzyme appeared to be involved in the increase in free amino acids during postmortem storage of meat.
    No preview · Article · Jun 2006 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: In this study, 10 troponin T isoforms from adult porcine skeletal muscle messenger RNA were clarified. These were eight fast- and two slow-type isoforms. Fast-type isoforms had three and two variable exons in the N-terminal and the C-terminal region respectively. Slow-type isoforms had one variable exon in the N-terminal region.
    Full-text · Article · Apr 2006 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: In a previous study, we isolated the inhibitory peptide (P4 peptide, Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe) for angiotensin I-converting enzyme (ACE) from chicken breast muscle extract possessing hypotensive activity for spontaneously hypertensive rats (SHRs). This study was performed to elucidate the peptide's action mechanisms of inhibiting ACE. Intravenous administration of synthetic P4 peptide resulted in significant drops in the blood pressures of SHRs. As Dixon plots indicate, the P4 peptide showed high affinity toward ACE (K(i) = 11.48 microM) and only 10% of the total amount of the P4 peptide was decomposed. The analyses of the relationship between the ACE inhibitory activity and structure of the P4 peptide clarified that Hyp-Gly-Leu-Hyp-Gly-Phe showed a stronger activity (IC50 = 10 microM) than the P4 peptide (IC50 = 46 microM). When Phe at the C-terminus of the P4 peptide was deleted, IC50 changed to 25000 microM, indicating that Phe at the C-terminus of the peptide is very important for ACE inhibitory activity.
    Preview · Article · Mar 2006 · Journal of Agricultural and Food Chemistry
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    ABSTRACT: This study was performed to examine the effect of consumption of pork-liver protein hydrolysate (PLH) on body fat accumulation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a non-insulin-dependent diabetes mellitus model and in Long-Evans Tokushima Otsuka (LETO) rats as a control. Male 20-week-old OLETF and LETO rats were pair-fed either PLH or casein containing diet for 14 weeks. In the OLETF rats, dietary PLH significantly reduced the growth and weight of fat pad including perirenal and epididymal adipose tissues. Consumption of PLH markedly suppressed hepatic activities of lipogenesis enzymes such as glucose-6-phosphate dehydrogenase and fatty acid synthase and slightly elevated fecal excretion of total fat. In the LETO rats, growth and adipose tissue weight were unaffected by dietary treatment. The results suggest that PLH is a novel ingredient suppressing body fat in genetically obese rats by reducing lipogenesis.
    No preview · Article · Feb 2006 · Bioscience Biotechnology and Biochemistry

  • No preview · Article · Jan 2006 · Nihon Chikusan Gakkaiho
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    ABSTRACT: Troponin T (TnT) is one of the myofibrillar proteins that is easily degraded during postmortem aging of pork. In this study, we determined the N-terminal amino acid sequences of TnT degradation fragments produced during postmortem aging and by m-calpain hydrolysis. The N-terminal amino acid sequences of TnT fragments produced during postmortem aging were EVHEPEEKPRPKLTAP, EKPRPKLTAPKIPEG, and APKIPEGEKVDF. On the other hand, the N-terminal amino acid sequences of TnT fragments produced by the action of m-calpain were APPPPAEV, EVHEPEEK, and APK. These sequences of degradation fragments could be mapped on fast type TnT isoform 2. The peptide bonds of His37-Glu38 and Thr51-Ala52 in fTnT2 were cleaved during postmortem aging as well as by the calpain hydrolysis; therefore, calpain was concluded to have an important role in TnT degradation during postmortem aging. It was also found that the sourness-suppressing peptide APPPPAEVHEVHEEVH (Okumura et al. Biosci. Biotechnol. Biochem. 2004, 68, 1657-1662) derived from TnT degradation could be produced by the action of calpains on Glu21-Ala22 and His37-Glu38 sites.
    No preview · Article · Jun 2005 · Journal of Agricultural and Food Chemistry
  • Tomoyuki Okumura · Ryoji Yamada · Toshihide Nishimura
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    ABSTRACT: This study was conducted to identify the sourness-suppressing peptides in cooked pork and to clarify the mechanism of sour taste suppression by the peptides. An extract prepared from pork loins vacuum-cooked at 60 degrees C for 6 hours after conditioning at 4 degrees C for 20 days was separated into three fractions: under MW 500 (Fraction I), MW 500-1,000 (Fraction II), and over MW 1,000 (Fraction III). The Fraction I content was largest. As judged by sensory evaluation, the addition of Fraction II was capable of suppressing stronger sourness than the other fractions. Fraction II also enhanced umami and saltiness. Three peptides (APPPPAEVHEVV, APPPPAEVHEVVE, and APPPPAEVHEVHEEVH) in Fraction II increased greatly during conditioning. A common peptide, APPPPAEVHEV, in the amino acid sequences of the three peptides suppressed the sour taste. The mechanism of sourness suppression by the peptide was concluded to comprise inhibition of the binding of sour taste substances to the membranes of the tongue.
    No preview · Article · Sep 2004 · Bioscience Biotechnology and Biochemistry
  • Soichi Tanabe · Rumiko Shibata · Toshihide Nishimura
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    ABSTRACT: We recently identified IgE-binding epitopes of bovine serum albumin (BSA), the major beef allergen, and found that the major epitope structure contains an EXXV motif. Here we report hypoallergenic analogue peptides of BSA that preserve T cell reactivity to retain the capacity to induce immunotolerance. Substitution of the glutamic acid in the EXXV sequence to aspartic acid leads to a remarkable reduction of IgE-binding ability and induces potent T cell proliferation. Moreover, in response to the substitutions, the production of INF-gamma is markedly increased compared with that elicited by the native peptides. Therefore, these analogue peptides represent potential candidates for specific immunotherapy.
    No preview · Article · Aug 2004 · Molecular Immunology
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    ABSTRACT: We have determined the amino (N)-terminal amino acid (AA) sequences of five troponin T (TnT) fragments produced during postmortem aging of bovine longissimus muscle. Western blot analysis showed that 32.1, 28.8, 27, and 25.8 kDa anti-fast-type TnT (fTnT)-positive fragments and a 31 kDa anti-slow-type TnT (sTnT)-positive fragment were present at 14 d postmortem. The N-terminal AA sequences of the 32.1, 28.8 (conventional 30 kDa), 27, and 25.8 kDa fragments were APPPPAEV, EVHEPEEK, EKPRPRLT, and APKIPEGE, respectively, and they were mapped to the N-terminal region of bovine fTnT isoforms. The N-terminal sequences of the 31 kDa fragment, EAPEEPEP, were mapped to the sTnT isoforms. These findings indicate that the two isoform types of fTnT predominantly expressed in the longissimus muscle are cleaved specifically at Glu(21)-Ala(22) and Glu(15)-Ala(16), His(37)-Glu(38) and His(31)-Glu(32), Glu(43)-Glu(44) and Glu(37)-Glu(38), and/or Thr(51)-Ala(52) and Thr(45)-Ala(46), respectively, and that a sTnT isoform is cleaved specifically at Glu(23)-Glu(24).
    Full-text · Article · May 2004 · Meat Science
  • Tomoyuki Okumura · Ryoji Yamada · Toshihide Nishimura
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    ABSTRACT: This study was performed to examine the changes in myofibrils, proteins and peptides during postmortem conditioning of vacuum-packed pork loins at 4 °C for 30 days. The fragmentation of myofibrils has been observed during postmortem aging for 20 days and its ratio increased until 20 days. The 32 kDa component derived from troponin T increased during storage for 20 days, while a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) among sarcoplasmic proteins was significantly degraded during storage for 15 days. Some oligopeptides increased during such storage, two peptides (peptides P1 and P2) significantly increasing during storage for 20 days. Their sequences were APPPPAEVHVHEEVH (P1) and VPTPNVSVVDLT (P2). Homology analysis showed that peptides P1 and P2 were derived from troponin T and GAPDH, respectively. It was concluded that the fragmentation of myofibrils, the increases in the 32 kDa component and peptides P1 and P2, and the decrease in GAPDH were useful as conditioning indicators of progressive degree during the storage of pork loins stored at 4 °C.
    No preview · Article · Aug 2003 · Meat Science
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    ABSTRACT: Physicochemical and conformational changes, water-holding capacity (WHC) and the structure of heat-induced gel of porcine myosin were investigated to elucidate the relationship between denaturation of myosin and gelation properties during postmortem aging. The turbidity of porcine myosin upon heating increased as the period of postmortem aging increased. During postmortem aging, the increased velocity in values of aliphatic hydrophobicity of porcine myosin by heating was prominent, which suggested that porcine head was susceptible to conformational changes during storage. On the other hand, according to the changes in circular dichroism (CD) spectra of heated porcine myosin, the decrease in α-helix content was almost the same during postmortem aging, indicating no conformational changes in porcine myosin rod during aging. WHC values of porcine myosin gels showed a gradual decrease during storage. This coincided with the progressive loosening in its three dimensional ordering with increased postmortem aging period, as was revealed by SEM studies. In conclusion, conformational changes of myosin head by denaturation during postmortem aging of pork could cause a progressive loosening of heat-induced gel of porcine myosin. This result could be, in part, helpful in the subsequent use of pork in meat processing.
    No preview · Article · Aug 2003 · Food Science and Technology Research
  • Ai Saiga · Soichi Tanabe · Toshihide Nishimura
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    ABSTRACT: Hydrolysates obtained from porcine myofibrillar proteins by protease treatment (papain or actinase E) exhibited high antioxidant activity in a linolenic acid peroxidation system induced by Fe(2+). Hydrolysates produced by both papain and actinase E showed higher activities at pH 7.1 than at pH 5.4. The antioxidant activity of the papain hydrolysate was almost the same as that of vitamin E at pH 7.0. These hydrolysates possessed 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and chelating activity toward metal ions. Antioxidant peptides were separated from the papain hydrolysate by ion exchange chromatography. The acidic fraction obtained by this method exhibited higher activity than the neutral or basic fractions. Antioxidant peptides in the acidic fraction were isolated by high-performance liquid chromatography on an ODS column and shown to possess the structures DSGVT, IEAEGE, DAQEKLE, EELDNALN, and VPSIDDQEELM. The DAQEKLE peptide showed the highest activity among these peptides.
    No preview · Article · Jul 2003 · Journal of Agricultural and Food Chemistry
  • Ai Saiga · Toshihide Nishimura

    No preview · Chapter · Jun 2003
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    ABSTRACT: The blood pressure of spontaneously hypertensive rats (SHRs) decreased after oral administration of an extract prepared from chicken breast muscle, falling maximally to 50 mmHg lower than before. This effect continued for at least 4 h after administration. The peptides possessing hypotensive activity in the chicken extract were examined by measuring the inhibitory activity (IC(50)) against angiotensin I-converting enzyme (ACE). The inhibitory activity of the chicken extract was 1060 mg%, whereas the activity of the extract treated with an Aspergillus protease and gastric proteases (trypsin, chymotrypsin, and intestinal juice) became stronger, reaching 1.1 mg%. Peptides in this hydrolysate of the extract were isolated by HPLC on a reversed-phase column, and their N-terminal sequences were analyzed. Three peptides possessed a common sequence, Gly-X-X-Gly-X-X-Gly-X-X, which was homologous with that of collagen. The peptide Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe showed the strongest inhibitory activity (IC(50) = 42 microM).
    No preview · Article · Apr 2003 · Journal of Agricultural and Food Chemistry

Publication Stats

735 Citations
36.74 Total Impact Points


  • 2010-2011
    • Nippon Veterinary and Life Science University
      Edo, Tōkyō, Japan
  • 2002-2009
    • Hiroshima University
      • • Graduate School of Biosphere Sciences
      • • Faculty of Applied Biological Science
      Hirosima, Hiroshima, Japan
  • 1995
    • The University of Tokyo
      Tōkyō, Japan