Researchers Database

SHIMAZAKI, Yoji

    Graduate School of Science and Engineering Engineering for Production and Environment Associate Professor
Last Updated :2020/10/13

Researcher Information

J-Global ID

Research Interests

  • プロテオミックス   質量分析   電気泳動法   構造   活性   酵素   タンパク質   proteomics   mass spectrometry   electrophoresis   sequence structure   activity   enzyme   protein   

Research Areas

  • Nanotechnology/Materials / Analytical chemistry
  • Life sciences / Functional biochemistry

Academic & Professional Experience

  • 2007/04 - Today  - 愛媛大学大学院 理工学研究科准教授
  • 1996/01 - 2007  Ehime UniversityFaculty of Science助手
  • 1994/04 - 1995  三菱化学生命科学研究所特別研究員

Association Memberships

  • Society for Chromatographic Sciences   日本化学会   日本電気泳動学会   日本分析化学会   The Japanese Society for Comparative Physiology and Biochemistry   日本動物学会   

Published Papers

  • Youji Shimazaki, Aoshi Inoue, Haruka Ikeuchi
    Journal of Microbiological Methods 176 106028 - 106028 0167-7012 2020/09 [Peer-reviewed]
     Scientific journal
  • Youji Shimazaki, Hikari Nakamaru
    Journal of Electrophoresis 64 (1) 1 - 6 1349-9394 2020/08 [Peer-reviewed]
     Scientific journal
  • Kawano Risa, Nakanishi Ayaka, Zako Tamotsu, SHIMAZAKI Yoji
    Separation Science Plus (Wiley-VCH) 2 (9) 322 - 328 2019/09 [Peer-reviewed]
     Scientific journal
  • SHIMAZAKI Yoji, Inoue Suzuka
    Applied Biochemistry and Biotechnology 189 680 - 689 2019/09 [Peer-reviewed]
     Scientific journal
  • SHIMAZAKI Youji, Ayumi Takahashi
    Journal of Microbiological Methods 154 19 - 24 2018/11 [Peer-reviewed]
     Scientific journal
  • Youji Shimazaki, Yoshiko Ochi, Kennosuke Fujimura
    Electrophoresis 39 (8) 1054 - 1061 1522-2683 2018/04 [Peer-reviewed]
     Scientific journal 
    To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non-denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non-denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF-separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme-ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme-ovotransferrin complex, can be effectively separated and extracted using non-denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.
  • ShimazakiI Youji, Kosuke Tanaka, Keisuke Sakata
    CHROMATOGRAPHY 39 (1) 33 - 39 2018/02 [Peer-reviewed]
     Scientific journal
  • Youji Shimazaki, Rino Miyatsuka
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 182 (3) 1208 - 1217 0273-2289 2017/07 [Peer-reviewed]
     Scientific journal 
    Delipidation in biological samples is important for some diagnostic tests and protein analyses. Lipids in the samples can be hydrolyzed by native esterases (ESs) within gel capsules after ES, and ES-antibody complexes are specifically trapped, extracted, and separated. Acrylamide and agarose gel capsules containing complexes of ES antibody were produced after the complexes were extracted using protein A-immobilized membranes, separated by non-denaturing electrophoresis, and stained by colloidal silver using glucose as a reductant. ES activity of ES-antibody complexes within the gel capsule was significantly higher than that in the complexes with the control antibodies upon isolation, separation, and detection of the complex. In addition, lipids bound to human serum albumin decreased after human plasma was treated with gel capsules containing ES-antibody complexes. We demonstrate that the gel capsule containing ES-antibody complexes can be successfully isolated using techniques described in this study. Furthermore, delipidation of human plasma is obtained by incubation with the gel capsule. These results indicate that surplus materials such as lipids in biological samples can be removed or reduced by gel capsule containing enzymes.
  • Masaki Saito, Tamotsu Zako, Ryoji Takahashi, Youji Shimazaki
    CHEMISTRY LETTERS 45 (11) 1241 - 1243 0366-7022 2016/11 [Peer-reviewed]
     Scientific journal 
    In this study, we firstly report inhibition of amyloid beta(42) (A beta(42)) fibrillation, which is related to the progression of Alzheimer's disease, by digestion using proteases, combined with A beta(42) isolation using an immunoaffinity membrane. Especially, digestion of A beta(42) with carboxypeptidase Y (CPY) is effective for the inhibition of A beta(42) fibrillation, indicating that carboxy-terminal digestion of A beta(42) is efficient for the prevention of its fibrillation. The combinational method of the immunoaffinity membrane with CPY digestion is applicable to suppress A beta(42) fibrillation.
  • Youji Shimazaki, Yuki Sato
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 1021 108 - 113 1570-0232 2016/05 [Peer-reviewed]
     Scientific journal 
    The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5 mu L of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane. (C) 2015 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Yoko Takatsu
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 177 (7) 1565 - 1571 0273-2289 2015/12 [Peer-reviewed]
     Scientific journal 
    Amyloid beta 1-40 peptide was specifically isolated and analyzed from human plasma spiked with amyloid beta using a combined method of biotinylated anti-amyloid beta antibody binding to membrane-immobilized avidin (immunoaffinity membrane) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (MALDI-TOF MS). A solution of 10 mu L containing 13.6 ng to 2.9 mu g of amyloid beta peptide was examined in this method. After the isolated amyloid beta peptide from the spiked human plasma containing 2.9 mu g of amyloid beta peptide was incubated in the presence of trifluoroacetic acid, fibrillization of the peptides was observed using a thioflavin T assay. Furthermore, an immunoaffinity membrane present on the inner wall of a tube (diameter 2 mm) captured the amyloid beta peptide from the spiked human plasma. Our results indicate that the combination of the immunoaffinity membrane procedure and MALDI-TOF MS can be used to capture and analyze the target antigens such as amyloid beta in micro-spaces.
  • Youji Shimazaki, Yu Hirose
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 972 53 - 57 1570-0232 2014/12 [Peer-reviewed]
     Scientific journal 
    Monomeric molecules such as amyloid-beta can aggregate and transform into oligomeric and fibrous forms, which are implicated in the development and progression of Alzheimer's disease. Novel analytical techniques for the formation of oligomers are required to examine the neurotoxic amyloid-beta oligomers involving fibrils. After isolating amyloid-beta monomer 1-42 using a biotinylated antibody bound to membrane-immobilized avidin (immunoaffinity membrane), their masses were determined by MALDI-TOF MS. Fluorometric determination of more than 0.5 mu M of aggregated amyloid-beta in pipette droplets was performed after aggregation and dilution of 1 mM amyloid-beta. Thus, large (>105 nm) amyloid-beta oligomers in microliter volumes of fluids were isolated using the immunoaffinity membrane and quantitatively analyzed after removal of amyloid-beta monomers and small oligomers by non-denaturing electrophoresis. In addition, amyloid-beta oligomers were specifically isolated from a mixture of human plasma and aggregated amyloid-beta and then fluorometrically analyzed. Our results show that the combination of immunoaffinity membrane-binding and fluorescence determinations together with one drop analysis could be used to isolate and detect huge neurotoxic amyloid-beta oligomers such as fibrils in plasma samples. (C) 2014 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Ai Hashimoto
    TALANTA 125 400 - 404 0039-9140 2014/07 [Peer-reviewed]
     Scientific journal 
    A microfluidic device containing membrane-immobilized anti-esterase (ES) antibodies and anti-lactate dehydrogenase (LDH) antibodies was prepared. The membrane was prepared by transferring antibodies that had been separated by non-denaturing two-dimensional electrophoresis to a polyvinylidene difluoride membrane, which was then stained and cut into small pieces (16 mm(2)). In this microfluidic device, > 0.014 Unit mL(-1) of the purified porcine carboxylesterase was specifically captured by membrane-immobilized anti- ES antibodies and > 147 Unit mL(-1) of purified porcine LDH was specifically captured by membrane-immobilized anti-LDH antibodies. Furthermore, ES and LDH in micro-scale aliquots of porcine liver cytosol were successively captured by membrane-immobilized antibodies in the device, and the enzyme activities were quantitatively analyzed by spectrofluorometry. The results indicate that the microfluidic device containing membrane-immobilized antibodies can be used to investigate the activities of several types of intact enzymes. (C) 2014 Elsevier B.V. All rights reserved.
  • Ayaka Kimura, Youji Shimazaki
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 172 (8) 4053 - 4061 0273-2289 2014/04 [Peer-reviewed]
     Scientific journal 
    Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm(2)) of this membrane and eluted by rinsing with 5 mu L of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme's activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.
  • Youji Shimazaki, Shizuka Miki
    JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 28 (5) 894 - 899 1475-6366 2013/10 [Peer-reviewed]
     Scientific journal 
    Non-denaturing electrophoresis can be used to screen enzymes that self-regulate their activities by using a combination of enzymes and their inhibitors. Furthermore, this technique can be applied to develop enzyme reactors that self-regulate their activities. After separation of proteins from mouse liver cytosol by non-denaturing isoelectric focusing, lactate dehydrogense (LDH) and esterase activities were qualitatively and quantitatively examined using a combination of two-dimensional electrophoresis (2-DE) and non-denaturing stacking gel electrophoresis. Activities of mouse liver-derived LDH and carboxylesterase were reversibly inhibited by oxamate and 6,9-diamino-2-ethoxyacridine (acrinol), respectively, in the stacking gels and recovered when the enzymes migrated towards the separation gels. After separation and immobilization of the enzymes, their activities were inhibited by inhibitors and recovered after inhibitor removal. These results indicate that non-denaturing electrophoresis can be applied to select enzymes that self-regulate their activities and subsequently aid in the development of enzyme reactors that can control the enzyme activities.
  • Youji Shimazaki, Madoka Michhiro
    CLINICA CHIMICA ACTA 425 48 - 53 0009-8981 2013/10 [Peer-reviewed]
     Scientific journal 
    Background: The functions of proteins can be retained following separation by non-denaturing two-dimensional electrophoresis (2-DE). The trypsin inhibition activities can then be examined following the separation and immobilization of the proteins under non-denaturing conditions. Methods: Human plasma proteins were separated using 2-DE and transferred onto a polyvinylidene difluoride membrane and stained using Ponceau S. The trypsin inhibition activity of the membrane-bound proteins was qualitatively examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The activities were also quantitatively examined by analyzing the release of the azo-chromophore when azocasein was the substrate. Results: Ttypsin activity was inhibited by the haptoglobin and alpha(2)-macroglobulin spots located on the membrane, whereas the protease activity was retained for the spots containing albumin and transferrin. The inhibition activities of the alpha(2)-macroglobulin and haptoglobin spots were 4.81- and 4.83-fold higher, respectively, when compared with the inhibition activity of the albumin spot. An axis of the relative activities of trypsin inhibition was added to the 2-DE pattern of human plasma proteins to construct a non-denaturing 3-D map of human plasma proteins. Conclusion: This 3-D map should represent a suitable diagnostic tool for the qualitative and quantitative analyses of the ttypsin inhibition activities of proteins. (C) 2013 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Yuri Nishimura, Masaki Saito
    JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 83 293 - 298 0731-7085 2013/09 [Peer-reviewed]
     Scientific journal 
    A combination of methods is required to achieve separation of intact proteins and subsequently perform structure analysis to examine their unstable or external structures. The aim of this study was to develop a method of structure analysis in intact proteins after purification. Transferrin from human plasma was trapped by membrane-immobilized anti-transferrin antibody, which was produced by non-denaturing two-dimensional electrophoresis (2-DE), and transferred to a polyvinylidene fluoride (PVDF) membrane and stained with Ponceau S. The antigen transferrin was eluted by rinsing the membrane with trifluoroacetic acid (TFA) or aspartic acid. In addition, a method was established by which the purified human transferrin was enzymatically digested on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) target plate. Thus, after purification of the human transferrin antigen from tens of microlitres of human plasma using an immunoaffinity membrane, transferrin polypeptide fragments were obtained on the plate following digestion with pepsin in the presence of 0.1% TFA or endoproteinase Lys-C or Lys-C/trypsin with 0.001% sodium dodecyl sulphate (SDS). The results indicated that the combined methods of isolation using an immunoaffinity membrane and enzymatic digestion on a MALDI-TOF MS plate could be applied to the purification and microanalysis of antigens. This approach would be particularly applicable to the analysis of the primary structure and the less stable and highly accessible regions of antigens from limited sample volumes. (c) 2013 Elsevier B.V. All rights reserved.
  • Takahiro Sakikawa, Youji Shimazaki
    JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 71 179 - 182 0731-7085 2012/12 [Peer-reviewed]
     Scientific journal 
    Apolipoprotein A-1 (apo A-1), a major component of high density lipoprotein (HDL), was efficiently digested by membrane-immobilized trypsin after HDL was treated with membrane-immobilized esterase. Compared to treatment with membrane-immobilized trypsin alone, the relative amounts of apo A-1 polypeptides, m/z 1723.78 and m/z 1568.82, increased by 2.7- and 3.9-fold, respectively, when HDL was treated with membrane-immobilized esterase and trypsin. Furthermore, the efficient digestion of apo A-1 by trypsin was inhibited when HDL was treated with membrane-immobilized esterase in the presence of an esterase inhibitor, 6,9-diamino-2-ethoxyacridine (acrinol). The data indicate that the lipid components of lipoproteins are released by membrane-immobilized esterase. This method can be used to investigate the structure and function of other apolipoproteins. (C) 2012 Elsevier By. All rights reserved.
  • Youji Shimazaki, Yoshinori Kohno, Izumi Fukui, Toshiharu Koyashiki
    PROTEIN EXPRESSION AND PURIFICATION 83 (2) 177 - 181 1046-5928 2012/06 [Peer-reviewed]
     Scientific journal 
    Adrenocorticotropic hormone (ACTH) and transferrin were trapped by biotinylated anti-ACTH antibody and anti-transferrin antibody, respectively, bound to membrane-immobilized avidin. Polypeptides with the sequences SYSMEHFR. SYSMEHFRWGKPVGK and SYSMEHFRWGKPVGKK were bound to the biotinylated anti-ACTH antibody on the membrane-immobilized avidin after the trapped ACTH was digested with trypsin on the membrane and non-binding polypeptides were washed from the membrane. Further, the polypeptides with the sequence SYSMEHFRWGKPVGK and SYSMEHFRWGKPVGKK were trapped by anti-ACTH antibody bound to membrane-immobilized protein A. The antibody recognized the WGKPVGK region of the antigen, ACTH. Polypeptide with the sequence SMGGKEDLIWELLNQAQEHFGKDK was bound to the biotinylated anti-transferrin antibody on the membrane-immobilized avidin after the trapped transferrin was digested with trypsin on the membrane and non-binding polypeptides were washed from the membrane. Further, the polypeptide with the sequence SMGGKEDLIWELLNQAQEHFGKDK was trapped by anti-transferrin antibody bound to membrane-immobilized protein A. The antibody recognized the SMGGKEDLIWELLNQAQEHFGKDK region of the antigen, transferrin. These results thus indicate that the combined methods of membrane-immobilized avidin and protein A can be applied to examine the epitopes of antigens. (C) 2012 Elsevier Inc. All rights reserved.
  • Youji Shimazaki, Yoshinori Kohno
    ANALYTICAL BIOCHEMISTRY 422 (1) 55 - 57 0003-2697 2012/03 [Peer-reviewed]
     Scientific journal 
    Avidin from egg white was migrated toward a cathode of nondenaturing electrophoresis and then immobilized on a polyvinylidene difluoride membrane. Adrenocorticotropic hormone (ACTH) was specifically captured after the biotinylated anti-ACTH antibody was bound to the membrane-immobilized avidin, and the captured ACTH was digested by the biotinylated trypsin on the membrane after extraction. The digested polypeptides from the ACTH were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These results indicate that target substances can be specifically trapped and digested on membrane-immobilized avidin. (C) 2011 Elsevier Inc. All rights reserved.
  • Youji Shimazaki, Takahiro Sakikawa, Ayaka Kimura
    CLINICA CHIMICA ACTA 413 (1-2) 269 - 272 0009-8981 2012/01 [Peer-reviewed]
     Scientific journal 
    Background: Specific proteins in biological fluids can be captured on an immunoaffinity membrane after polyclonal anti-porcine liver esterase antibodies are separated by non-denaturing 2-dimensional electrophoresis (2-DE) and transferred onto the membrane. The enzymatic activities of these captured proteins can then be monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods: Polyclonal anti-porcine liver esterase antibody was separated by non-denaturing 2-DE, transferred onto a polyvinylidene difluoride membrane and stained with Ponceau S. Esterase activity was examined by enzyme activity staining and MALDI-TOF MS after antigens, including purified carboxylesterase from porcine liver and cytosolic esterase from porcine retina, were captured on the immunoaffinity membrane. Results: Esterase activity was detected on the immunoaffinity membrane after the enzyme was captured. Phosphatidylcholine hydrolysis by the esterase was monitored after the esterase was captured onto the membrane and attached to the target plate for MALDI-TOF MS. Conclusions: This method could be used to analyze changes in enzymatic activity under biological conditions such as health and disease conditions using immunoaffinity membranes and MALDI-TOF MS. (C) 2011 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Ayaka Kimura
    JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 56 (5) 1085 - 1088 0731-7085 2011/12 [Peer-reviewed]
     Scientific journal 
    Haptoglobin is known to bind to hemoglobin during intravascular hemolysis. Membrane-immobilized anti-haptoglobin antibody, which was produced after antibody was isolated by non-denaturing two-dimensional electrophoresis, was transferred to a polyvinylidene difluoride membrane and was stained using Ponceau S. The proteins bound to the membrane-immobilized anti-haptoglobin antibody were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Hemoglobin was specifically obtained when the membrane-immobilized anti-haptoglobin antibody was incubated with human serum obtained from hemolysis blood. Furthermore, hemoglobin in the flowing fluid was captured by the membrane-immobilized anti-haptoglobin antibody and analyzed directly. The results indicate that hemolysis can be examined by direct trapping and analysis of hemoglobin under physiological conditions. (C) 2011 Elsevier B.V. All rights reserved.
  • Takahiro Sakikawa, Youji Shimazaki
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 165 (1) 69 - 74 0273-2289 2011/09 [Peer-reviewed]
     Scientific journal 
    An inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), is a reversible inhibitor of esterases. The reversible inhibition of the enzyme activity is thought to be examined after separation and immobilization of the enzyme under non-denaturing conditions. Hydrolytic changes of phosphatidylcholine by carboxylesterase were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the esterase was separated by non-denaturing two-dimensional electrophoresis, was immobilized to membranes and was stained by Ponceau S. The changes were inhibited after the enzyme on the membrane was treated by tacrine. Furthermore, the hydrolytic activity of the esterase was recovered after the inhibitor was washed with aspartic acid solution. These results indicate that the phosphatidylcholine hydrolysis activity of the isolated and immobilized enzyme is reversibly inhibited under non-denaturing conditions. Furthermore, this method can be developed to the production of an enzyme reactor able to regulate amounts of lipids.
  • Youji Shimazaki, Masayuki Miyamoto
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 878 (28) 2852 - 2856 1570-0232 2010/10 [Peer-reviewed]
     Scientific journal 
    We simultaneously separated antibodies for transferrin, the third component of complement (C3), haptoglobin and transthyretin by multi-sample non-denaturing two-dimensional electrophoresis (2-DE), transferred them to a polyvinylidene difluoride (PVDF) membrane and then stained them using direct blue 71 to obtain membrane-immobilized antibodies. The antigens, transferrin, C3. haptoglobin and transthyretin were specifically bound to the membrane-immobilized antibodies and were eluted only after rinsing the membrane with acid solution. The antigens specifically bound to the membrane-immobilized antibodies were separated by SDS-PAGE and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, transferrin and transthyretin were trapped and eluted by each membrane-immobilized antibody and detected by MALDI-TOF MS directly without separations. Using membrane-immobilized anti-transferrin antibody, transferrin in flowing blood was directly trapped and analyzed. The results indicated that membrane-immobilized antibodies are simultaneously produced, and that the immunoaffinity membranes can capture specific substances in flowing fluids. (C) 2010 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Kenta Shimizu, Suzuna Masaoka
    TALANTA 82 (3) 1063 - 1067 0039-9140 2010/08 [Peer-reviewed]
     Scientific journal 
    Carboxylesterase and sorbitol dehydrogenase are separated by non-denaturing two-dimensional electrophoresis (2-DE) of isoelectric focusing separation using 5% carrier ampholyte (pH 6-8) and 1.25% carrier ampholyte (pH 3-10) and size separation. Furthermore, activities of sorbitol, malate and lactate dehydrogenases are sequentially examined when the enzymes are separated by 2-DE and are sequentially reacted to sorbitol, malic and lactic acid, respectively, in the presence of nicotinamide adenine dinucleotide, nitro blue tetrazolium and phenazine methosulphate. Several kinds of enzymes including lactate dehydrogenize isozymes can be simultaneously separated using 2-DE. Furthermore, the binding differences between lactate dehydrogenase isozymes and concanavalin A (con A) can be examined using a combination of 2-DE and non-denaturing stacking gel electrophoresis. The results of this study indicate that non-denaturing 2-DE can be applied to both enzyme separation and isozyme heterogeneity analysis. (C) 2010 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Seigo Horikawa
    CLINICA CHIMICA ACTA 411 (13-14) 992 - 993 0009-8981 2010/07 [Peer-reviewed]
     Scientific journal
  • Youji Shimazaki, Takahiro Sakikawa
    PROTEIN AND PEPTIDE LETTERS 17 (8) 1048 - 1052 0929-8665 2010 [Peer-reviewed]
     Scientific journal 
    Mouse liver cytosolic malate dehydrogenase was separated by non-denaturing two-dimensional electrophoresis and identified. Furthermore, the activity of the enzyme was preserved even after separation, electroblotting onto a membrane and staining with Ponceau S in acidic buffer solution (pH 5.1). Using the membrane-immobilized enzyme, the malic acid content was estimated by measuring absorbance changes due to the conversion of nicotinamide adenine dinucleotide (NAD) to NADH. These results indicate that enzyme reactors can be systematically produced after purification, immobilization and staining with Ponceau S.
  • Youji Shimazaki, Takahiro Kuroda
    BIOTECHNOLOGY LETTERS 31 (10) 1545 - 1549 0141-5492 2009/10 [Peer-reviewed]
     Scientific journal 
    Activities of carboxylesterase and malate dehydrogenase on membranes were retained after enzymes of mouse liver cytosol were separated by non-denaturing, two-dimensional electrophoresis (2-DE), stained using imidazole and zinc salts and electroblotted on to membranes. Furthermore, hydrolytic changes of phosphatidylcholine by the esterase were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after separation, and reversible staining and immobilization to membranes. Hydrolytic activity of the esterase on the membranes was 20% of the original activity of the tissue homogenate. The present method can be applied to the production of several types of enzyme reactors on membranes.
  • Youji Shimazaki, Azusa Kodama
    ANALYTICA CHIMICA ACTA 643 (1-2) 61 - 66 0003-2670 2009/06 [Peer-reviewed]
     Scientific journal 
    Membrane-immobilized anti-transferrin antibody, which was produced after antibody was separated using non-denaturing two-dimensional electrophoresis (2DE), was transferred to a polyvinylidene difluoride (PVDF) membrane and was stained by direct blue 71. The antigen, transferrin, specifically bound to the membrane-unmobilized antibody and was eluted only after rinsing the membrane with glutamic acid or aspartic acid Solution. The antigen was analyzed directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) when the membrane was incubated with human plasma after the removal Of human serum albumin using polyethylene glycol. The transferrin extracted by glutamic acid or aspartic acid solution retained the binding of Fe(3+) in the presence of the carbonate anion. We found that immunoaffinity membranes can be useful for simple and rapid removal and extraction of intact proteins after antibody separation and blotting under non-denaturing conditions. (C) 2009 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Mariko Kadota
    ANALYTICA CHIMICA ACTA 618 (1) 116 - 119 0003-2670 2008/06 [Peer-reviewed]
     Scientific journal 
    We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins. (C) 2008 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Sono Watanabe
    CLINICA CHIMICA ACTA 390 (1-2) 145 - 147 0009-8981 2008/04 [Peer-reviewed]
     Scientific journal 
    Background: Enzyme activity is normally lost when formaldehyde is used as a reductant for silver staining after separation by electrophoresis. Hydrolytic activity of esterases can be examined on membranes without impairing enzyme activity when another reductant such as glucose is used for silver staining of the enzyme after separation by non-denaturing two-dimensional electrophoresis (2-DE) and subsequent transfer. Methods: The hydrolysis of lipids in human high density lipoprotein (HDL) by esterases first separated on a polyvinylidene fluoride membrane using non-denaturing 2-DE and silver stained using glucose as a reductant was examined. Results: Esterase activity was retained after glucose was used as a silver reductant for silver staining after separation using non-denaturing 2-DE. Lipids of HDL were removed by the esterases retained on the membrane after esterases were separated by 2-DE. Conclusion: The results indicated that hydrolytic enzyme activity is retained after separation, staining and immobilization. (C) 2008 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Takahiro Kuroda
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 860 (2) 180 - 184 1570-0232 2007/12 [Peer-reviewed]
     Scientific journal 
    The reaction from retinal to retinoic acid catalyzed by retinal dehydrogenase on a polyvinylidene difluoride (PVDF) membrane was examined using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS) when the enzyme was separated by non-denaturing two-dimensional electrophoresis (2-DE), transferred onto the membrane, and stained without impairing the enzyme activity. Furthermore, the enzyme was analyzed by de novo sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after proteins from mouse liver were separated by non-denaturing 2-DE, blotted onto the membrane, and stained. The results indicated that the reported methods could be applied for the direct examination of changes in retinoid catalyzed by enzymes on such membranes. (C) 2007 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Masayuki Miyamoto
    BIOTECHNOLOGY AND BIOENGINEERING 98 (4) 732 - 736 0006-3592 2007/11 [Peer-reviewed]
     Scientific journal 
    After separation by microscale non-denaturing two-dimensional gel electrophoresis (2DE) and transferring to a blotting membrane, major proteins are detected by a staining of direct blue 71 in a neutral solution. The carboxylesterase on the membrane hydrolyzes phosphatidylcholine after the spot of carboxylesterase is excised from the membrane, and incubated with phosphatidylcholine. Lipids of human serum proteins and the purified human high density lipoprotein (HDL) are removed by enzymatic hydrolysis when human serum proteins and the purified HDL are respectively incubated with the spot of carboxylesterase on the membrane. These results indicate that carboxylesterase on the membrane hydrolyzes not only lipids such as phosphatidylcholine but also lipids of lipoproteins such as HDL after separation by the 2DE, transferring to the membrane and staining without impairing the activity. These results also indicate that a micro-immobilized enzyme reactor on the membrane can be produced when biological enzymes are separated by microscale 2DE, transferred to the membrane and stained without impairing their activities.
  • Youji Shimazaki
    JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 70 (3) 487 - 491 0165-022X 2007/04 [Peer-reviewed]
     Scientific journal 
    This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using alpha-naphthyl acetate in a 2-DE gel. A spot of phospholipase Calpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell (TM) Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column. (c) 2006 Elsevier B.V. All rights reserved.
  • Youji Shimazaki, Haruka Kishi, Masami Mori, Chiyo Yasuda, Takashi Manabe
    JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES 843 (1) 42 - 46 1570-0232 2006/10 [Peer-reviewed]
     Scientific journal 
    Hydrolysis of retinyl esters and phospholipids is important for visual functions of the animal retina. This study aimed to examine hydrolytic activity of an enzyme with native substrates such as retinyl esters and phospholipids responsible for this function in porcine retina. After cytosolic proteins were extracted from porcine retina, the proteins were separated using non-denaturing two-dimensional electrophoresis (2DE). Some major proteins and phospholipase Cot were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) or electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). The phospholipase C alpha showed hydrolytic activities with not only alpha-naphtyl acetate but also with retinyl palmitate and phosphatidylcholine when effects of different substrates were investigated using enzyme activity staining on 2DE or MALDI-TOF-MS. Results indicated that hydrolytic activity of the enzyme with non-native and native substrates could be examined using a combination of non-denaturing 2DE and MALDI-TOF-MS. (c) 2006 Elsevier B.V. All rights reserved.
  • Y Shimazaki, T Manabe
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1749 (1) 95 - 101 1570-9639 2005/05 [Peer-reviewed]
     Scientific journal 
    To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n -octyl β-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0. 1 % SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry. © 2005 Elsevier B.V. All rights reserved.
  • Y Shimazaki, Y Sugawara
    ANALYTICAL BIOCHEMISTRY 328 (1) 87 - 89 0003-2697 2004/05 [Peer-reviewed]
     Scientific journal
  • Y Shimazaki, Y Sugawara, T Manabe
    PROTEOMICS 4 (5) 1406 - 1411 1615-9853 2004/05 [Peer-reviewed]
     Scientific journal 
    After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.
  • Y Shimazaki, Y Hiraka, M Uesugi, T Manabe
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1696 (1) 51 - 57 1570-9639 2004/01 [Peer-reviewed]
     Scientific journal 
    Esterase and transferase activities were analyzed simultaneously after cytosol proteins in the bovine retina were separated by microscale non-denaturing two-dimensional electrophoresis (2-DE). Esterase activity was specifically inhibited by an esterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), and transferase activity was specifically inhibited by a glutathione S-transterase (GST) inhibitor, 2-phenyl-1,2-benziso selenazol-3(2H)-one (ebselen). Both esterase and transferase were precipitated when ammonium sulfate was added to the cytosol up to 50% saturation (50% AS fraction), and were detected in the 50% AS fraction by using the 2-DE. After the cytosol proteins in the 50% AS fraction were separated by using non-denaturing 2-DE, polypeptides of the separated proteins were identified by peptide mass fingerprinting and post-source decay analysis by using MALDI-MS, or by immunoreactivity by using a specific antibody. The spots of esterase and transferase activities in the 2-DE pattern were identified as phosphodiesterase and GST, respectively. This simultaneous analysis of enzyme activities can be applied to screen-specific or non-specific medicines which affect enzyme activities. (C) 2003 Elsevier B.V. All rights reserved.
  • Y Shimazaki, Y Sugawara, Y Ohtsuka, T Manabe
    PROTEOMICS 3 (10) 2002 - 2007 1615-9853 2003/10 [Peer-reviewed]
     Scientific journal 
    Enzyme activities such as of fructose bisphosphatase, malate dehydrogenase and carbonic anhydrase were analyzed after cytosol proteins in the mouse liver and were separated using nondenaturing two-dimensional electrophoresis (2-DE). The activities of both fructose bisphosphatase and malate dehydrogenase were inhibited by thyroxine, and fructose bisphosphatase activity was specifically inhibited by adenosine monophosphate in nondenaturing 2-DE. Furthermore, polypeptides of the separated proteins were analyzed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by peptide sequencing using electrospray ionization-tandem mass spectrometry, or both. Proteins separated by 2-DE were identified. These results indicate that the function of proteins such as enzyme activity, and their sequence structure can be analyzed, for example by peptide mapping and peptide sequencing, after the proteins have been separated by nondenaturing 2-DE. Present results also indicate analysis of enzyme activity using nondenaturing 2-DE can be applied to screen substances which affect enzyme activity.
  • Y Shimazaki, H Ohnishi, S Matsuura, T Manabe
    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1571 (3) 245 - 248 0304-4165 2002/07 [Peer-reviewed]
     Scientific journal 
    The Cu,Zn-superoxide dismutase (SOD) activity in bovine retina cytosol was separated from retinal pigment using short-length non-denaturing isoelectric focusing (IEF) (15-mm long x 1.3-mm i.d. column) and detected using non-denaturing two-dimensional electrophoresis (2-DE), After the SOD and pigment in the retina cytosol are separated, SOD activity can be quantified by water-soluble tetrazolium salt. We also found that SOD separated by this IEF retained its native function. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Y Shimazaki, Y Hiraka, H Ohnishi, T Manabe
    BUNSEKI KAGAKU 51 (6) 367 - 371 0525-1931 2002/06 [Peer-reviewed]
     Scientific journal 
    Non-denaturing two-dimensional electrophoresis (2-DE) can be applied to the separation of soluble proteins with high resolution. The separated proteins retain their physiological functions. The present study indicates that soluble proteins of the bovine retina were separated by non-denaturing 2-DE, and the enzyme activities of esterase, dehydrogenase, superoxide dismutase and transferase were detected in the presence of each enzyme-specific substrate and chromophore. While the activities of esterase and dehydrogenase were inhibited by a cholinesterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), the transferase activity was not affected. The enzymes separated by non-denaturing 2-DE were analyzed by matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS). These results indicate that non-denaturing 2-DE is applicable to analyzing of the functions and structures of soluble proteins.
  • 島崎洋次, 平加容子, 大西弘美, 真鍋敬
    分析化学 51 (6) 367 - 371 2002 [Peer-reviewed]
  • Y Shimazaki, K Maeyama, T Fujii
    DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 25 (5-6) 467 - 474 0145-305X 2001/06 [Peer-reviewed]
     Scientific journal 
    The third component of complement (C3) of a newt, Cynops pyrrhogaster, was purified using a fast protein liquid chromatography technique. The purified newt C3 consists of two polypeptide chains (the molecular masses of the alpha and beta -chains of C3 were 120,000 and 70,000. respectively) linked by disulfide: bonds. The alpha -chain retained an internal thiolester bond that was cleaved with methylamine, acid the N-terminal amino acid sequence of the alpha -chain was XVQLIDAKAGKAAKF. Digestion of newt C3 with trypsin yielded fragments that induced significant histamine release from newt peritoneal cells. These results: indicate that newt C3 retains structural and functional properties shared with mammalian C3. (C) 2001 Elsevier science Ltd. All rights reserved.
  • Youji Shimazaki, Masayoshi Muro, Takashi Manabe
    Seibutsu butsuri kagaku 45 (1) 89 - 92 0031-9082 2001 [Peer-reviewed]
  • Y Shimazaki, M Muro, T Manabe
    CLINICA CHIMICA ACTA 302 (1-2) 221 - 224 0009-8981 2000/12 [Peer-reviewed]
     Scientific journal
  • S Iwasaki, M Kataoka, M Sekiguchi, Y Shimazaki, K Sato, M Takahashi
    JOURNAL OF BIOCHEMISTRY 128 (3) 407 - 414 0021-924X 2000/09 [Peer-reviewed]
     Scientific journal 
    Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation. of Ca2+-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an. anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187), DA and ACh release, assayed in low-K+ as well as high-K+ solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K+-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K+-solution, The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K+-dependent neurotransmitter release. The potentiation of high-K+-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min, PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K+-dependent PA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.
  • Youji Shimazaki, Mai Fujiwara, Takashi Manabe
    Seibutsu butsuri kagaku 44 (1) 21 - 25 0031-9082 2000 [Peer-reviewed]
     Scientific journal
  • Y Shimazaki, S Ohara, T Manabe
    JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 39 (3) 179 - 184 0165-022X 1999/05 [Peer-reviewed]
     Scientific journal 
    Specific proteins in small amounts of human plasma were subtracted from patterns of non-denaturing two-dimensional electrophoresis (non-denaturing 2-DE) by layering a 7 mu l aliquot of Protein A agarose-antibody complex on the top of an isoelectric focusing (IEF) gel before the loading of a plasma sample. The Protein A agarose suspension was recovered after non-denaturing IEF and was mixed with 8 M urea-5% 2-mercaptoethanol-1% NP-40 to extract the antibody and the specific plasma protein from Protein A agarose. The extract was then subjected to denaturing two-dimensional electrophoresis (denaturing 2-DE) and the location of the specific polypeptide was determined. The technique can be applied to the extraction and analysis of proteins in small amounts of samples. (C) 1999 Elsevier Science BN. All rights reserved.
  • Y Miyako-Shimazaki, Y Shimazaki, K Ohtsu, M Yamamoto
    CELL AND TISSUE RESEARCH 296 (2) 427 - 431 0302-766X 1999/05 [Peer-reviewed]
     Scientific journal 
    Heterotrimeric GTP-binding proteins (G proteins) play an important role in phototransduction. The presence of G-protein subclasses has been reported in photoreceptive membranes, e.g., the Gi subgroup (transducin) in vertebrate rods, and the Gq subgroup in the eyes of the Arthropoda and the Mollusca. We examined the immunoreactivity and distribution of a Gq homologue in the cerebral ocelli of Perinereis brevicirris (Polychaeta, Annelida) using an anti-GqC antibody raised against a conserved sequence at the C-terminal of the a-subunit of Gq (Gq-alpha). The anti-GqC antibody labeled a 48-kDa band on the Western blot of proteins from the Perinereis ocelli. The anti-GtC antibody, which is raised against the C-terminal sequence of bovine transducin cc-subunit (Gt-alpha), did not cross-react to the ocellar proteins of Perinereis. The rhabdomeric layers of the anterior and posterior ocelli were strongly labeled by anti-GqC on light-microscopic immunohistology. Immunoelectron microscopy showed that the Gq molecules were specifically localized in the photoreceptive membrane of the rhabdomeric microvilli. These results suggest that the Gq protein plays a role in the phototransduction of the Perinereis ocelli.
  • Y Shimazaki, S Ohara, T Manabe
    JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 37 (1-2) 1 - 4 0165-022X 1998/09 [Peer-reviewed]
     Scientific journal 
    Protein spots, such as immunoglobulin G (IgG), A (IgA) or alpha(2)-macroglobulin, on the patterns of non-denaturing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) were selectively removed by the application of protein A or antibody;conjugated protein A agarose to the first-dimension gel top at the cathode side. A protein residing at pI 5.8 and with a molecular mass of about 200 kDa was clearly detected when the IgG and IgA spots were removed from the 2-D PAGE pattern. (C) 1998 Elsevier Science B.V. All rights reserved.
  • Youji Shimazaki, Takashi Manabe
    Seibutsu busturi kagaku 42 (1) 13 - 18 0031-9082 1998 [Peer-reviewed]
     Scientific journal
  • Youji Shimazaki, Takashi Manabe
    Seibutsu butsuri kagaku 42 (3) 155 - 159 0031-9082 1998 [Peer-reviewed]
     Scientific journal
  • Y MiyakoShimazaki, Y Shimazaki, E Eguchi
    ZOOLOGICAL SCIENCE 14 (1) 29 - 35 0289-0003 1997/02 [Peer-reviewed]
     Scientific journal 
    Formoguanamine hydrochloride (FG) is known as a potent chemical to induce blindness in chick eyes by disrupting the pigment epithelium and visual cells in the retina. In this study, we examined the effect of FG on the structure and function of :he compound eyes of the butterfly, Papilio xuthus (Lepidoptera, Insecta). We administrated 2 mg FG per 1 g body weight of the pupa at about the first one third of the whole pupal period, because accomplishment of morphogenesis of the compound eye occurs in the last half of the pupal period. As a result, unusual membranous structures such as trophospongium-like structures and myeloid bodies were observed in the cytoplasm of the retinular cells besides the normal rhabdom. This result suggests that FG treatment influences on some steps in the formation of rhabdom membranes. However, the amount of chromophore, 3-hydroxyretinal and the responses to white light recorded by the ERG method from FG-treated specimens were not different from the control animals.
  • Y Shimazaki, T Nishiki, A Omori, M Sekiguchi, Y Kamata, S Kozaki, M Takahashi
    JOURNAL OF BIOLOGICAL CHEMISTRY 271 (24) 14548 - 14553 0021-9258 1996/06 [Peer-reviewed]
     Scientific journal 
    Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells, Phorbol 12-myristate 13 acetate treatment enhanced high potassium-induced [H-3]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25, A phosphorylation is likely to occur at Ser(187), as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins, SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co immunoprecipitated with SNAP-25 was decreased by phosphorylation, These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.
  • Y SHIMAZAKI, E EGUCHI
    JOURNAL OF COMPARATIVE PHYSIOLOGY A-SENSORY NEURAL AND BEHAVIORAL PHYSIOLOGY 176 (5) 661 - 671 0340-7594 1995/05 [Peer-reviewed]
     Scientific journal 
    1. After the intact compound eyes of the butterfly Papilio xuthus were adapted to darkness, white, blue (lambda max 460 nm) or orange light (lambda max 580 nm), the eyes were separated into the distal (primary pigment cells, the dioptric apparatus and ca. 30% of retinal tissue) and the proximal layers (the rest of the retinal tissues). Each layer was separated into a supernatant and a precipitate. Both in white and blue light-adapted eyes, the amount of 11-cis 3-hydroxyretinal increased in the supernatant of the distal layer (Sup-DL) much more than it did in dark-adapted eyes. No increase was observed in the Sup-DL of orange light-adapted eyes. 2. When all-trans retinol (non-native chemical) was added to the Sup-DL, it was converted to all-trans retinal under the darkness, and to all-trans and 11-cis retinal by blue light irradiation. When all-trans retinal was added to the Sup-DL, the isomerization of all-trans retinal to 11-cis retinal was accelerated by the blue light. 3. The Sup-DL was separated into ammonium sulfate soluble (AS-sup) and insoluble (AS-ppt) fractions. The AS-ppt fraction contained 3-hydroxyretinal but no 3-hydroxyretinol. Blue light irradiation to the AS-ppt fraction induced an increase in 11-cis 3-hydroxyretinal, with a concomitant decrease in all-trans 3-hydroxyretinal. These results indicate that both the oxidation of all-trans 3-hydroxyretinol to all-trans 3-hydroxyretinal and the light-dependent isomerization of all-trans 3-hydroxyretinal to 11-cis isomer take place in the tissues of the distal layer of the eyes.
  • Y SHIMAZAKI, E EGUCHI
    VISION RESEARCH 33 (2) 155 - 163 0042-6989 1993/01 [Peer-reviewed]
     Scientific journal 
    The metabolism of 3-hydroxyretinoids in the cytosol of the compound eyes of a species of butterfly, Papilio xuthus, was investigated. The cytosol was found to contain 25-30% of the total 3-hydroxy-retinal and 70-82% of the total 3-hydroxyretinol in the eye. These percentages of 3-hydroxyretinoids in the cytosol were found to be constant regardless of whether the eyes are light-adapted or dark-adapted. 3-Hydroxyretinal can be newly synthesized in the cytosol of light-adapted eves. Blue light specifically increases the amount of 11-cis and all-trans 3-hydroxyretinal ca 2.5 and 1.8 times respectively, compared to pre-irradiation. When 3-hydroxyretinal was synthesized, 3-hydroxyretinol was decreased or disappeared in the cytosol. When retinol (non-native chemical) was added to the cytosol, it was converted into retinal. This result indicates that an oxidative system exists in the compound eye which can convert 3-hydroxyretinol to 3-hydroxyretinal.
  • E EGUCHI, Y SHIMAZAKI, T SUZUKI
    ZOOLOGICAL SCIENCE 8 (3) 453 - 460 0289-0003 1991/06 [Peer-reviewed]
     Scientific journal 
    As is known from the literature, the intact compound eyes of the grapsid crab, Hemigrapsus sanguineus, show clear circadian changes in rhabdom sizes even in constant darkness. In order to identify the location controlling the circadian changes in rhabdom sizes, we incubated isolated compound eyes (distal to the basement membranes) and eye-stalks (compound eye with optic ganglions) for 6 to 8 hr in physiological saline at 20-degrees-C under a LD 12:12 light regime and in continuous darkness (DD). The rhabdom sizes were compared at different times using the electron microscope. There were no significant differences in rhabdom sizes between isolated eye-stalks and compound eyes at each time examined during a day. This indicates that optic ganglions do not directly participate in regulations of rhabdom sizes. The isolated compound eyes showed significant decreases in rhabdom sizes at the subjective dawn under DD. This fact indicates that the breakdown of rhabdoms was endogenously controlled by the biological clock within the retina. However, no distinct increases of rhabdom sizes in the isolated compound eyes were observed at the subjective dusk under DD.

Conference Activities & Talks

  • 色素結合型タンパク質のネイティブ電気泳動分離とその活性解析  [Not invited]
    井上蒼士, 足利諒, 池内晴佳, 中丸ひかり, 島崎洋次
    2019年日本化学会中国四国支部大会 徳島大会  2019/11
  • 種々色素とネイティブ電気泳動法の組み合わせによるタンパク質及びタンパク質凝集体のネイティブ分離分析法の検討  [Not invited]
    O島崎洋次, 井上蒼士, 足利諒, 座古保
    日本分析化学会第68年会  2019/09
  • 非変性条件アガロースゲル電気泳動法とコンゴーレッドによるアミロイドβ凝集体の分離分析法の構築  [Not invited]
    島崎洋次, 川野莉沙, 足利諒, 座古保
    第70回日本電気泳動学会総会  2019/07
  • ウズラ卵白タンパク質の大腸菌に対する抗菌活性分析法の開発  [Not invited]
    原田靖大, 井上涼香, 島崎洋次
    2018年日本化学会中国四国支部大会(愛媛大会)  2018/11
  • 生体酵素によるアミロイドβ凝集体の脱凝集過程分析法の構築  [Not invited]
    川野莉沙, 中西文香, 福永隼大, 座古保, 島﨑 洋次
    2018年日本化学会中国四国支部大会(愛媛大会)  2018/11
  • ニワトリとウズラ卵白リゾチーム存在状態の解析  [Not invited]
    原田靖大, 井上涼香, 島﨑 洋次
    第69回日本電気泳動学会総会  2018/08
  • 卵白中のリゾチーム複合体の電気泳動分離とその活性分析法の構築  [Not invited]
    高橋歩実, O島﨑 洋次
    第78回分析化学討論会  2018/05
  • ミクロ非変性等電点電気泳動法と培養技術を組み合わせたリゾチーム複合体の活性解析  [Not invited]
    高橋歩実, 中野令菜, 李海月, 島崎洋次
    第68回日本電気泳動学会総会  2017/11
  • 非変性条件の等電点電気泳動法による卵白リゾチームのインタクト分離溶出法の検討  [Not invited]
    o島崎洋次, 藤村建之介
    第77回分析化学討論会  2017/05

MISC

  • プロテオーム解析の医薬品や化粧品開発への応用を目指して
    島﨑洋次  SCAS NEWS  2020-  (1)  3  -6  2020/03  [Invited]
     Introduction commerce magazine
  • 分離. 電気泳動
    島崎洋次, 真鍋敬  ぶんせき(日本分析化学会)  (6)  301  -302  2001

Research Grants & Projects

  • 標的タンパク質凝集体分析システムの構築
    日本学術振興会:基盤研究C
    Date (from‐to) : 2013/04 -2016/03 
    Author : 島﨑 洋次
  • 電気泳動法による水容性酵素の機能-構造一斎分析
    日本学術振興会:若手研究(B)
    Date (from‐to) : 2002 -2004 
    Author : 島崎洋次
  • Global analysis of structure and function of cytosal enzymes by usiong electrophoresis.
    Date (from‐to) : 2002
  • Development of protein analytical method using electrophoresis and mass spectorometry

Social Contribution

  • 第9回CSJ化学フェスタ2019
    Date (from-to) : 2019/10/15-2019/10/17
    Role : Planner
    Sponser, Organizer, Publisher  : 日本化学会
  • グローバルサイエンスキャンパス(基盤学習)
    Date (from-to) : 2019/07/21-2019/07/21
    Role : Lecturer
    Sponser, Organizer, Publisher  : 科学技術振興機構、愛媛大学
  • 化学グランプリ2019
    Date (from-to) : 2019/07/15-2019/07/15
    Role : Planner
    Sponser, Organizer, Publisher  : 日本化学会
  • グロ-バルサイエンスキャンパス(基盤学習)
    Date (from-to) : 2018/10/20-2018/10/20
    Role : Lecturer
    Sponser, Organizer, Publisher  : 科学技術振興機構、愛媛大学

愛媛大学教員活動実績

教育活動(B)

担当授業科目(B01)

  • 2019, the first semester, under graduate, 新入生セミナーA
  • 2019, the first semester, under graduate, 卒業研究Ⅰ
  • 2019, the first semester, under graduate, 基礎化学実験
  • 2019, the first semester, under graduate, 基礎化学実験
  • 2019, the first semester, under graduate, 分析化学演習
  • 2019, the first semester, under graduate, 化学実験Ⅲ
  • 2019, the first semester, master course, 分子科学高等実習Ⅱ
  • 2019, the first semester, under graduate, 基礎化学実験
  • 2019, the first semester, under graduate, 化学実験Ⅲ


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