〔主要な業績〕Functional Expression and Mutant Analysis of Thioredoxin-Fused CEL-III, a Hemolytic Lectin from the Marine Invertebrate Cucumaria echinata
Yoshiki Shimizu; Masami Yonekura; and Yoshiaki Kouzuma, 責任著者, 日本農芸化学会
Bioscience, Biotechnology, and Biochemistry, 2022年05月22日, ［査読有り］

Purification, cDNA cloning and characterization of Kunitz-type protease inhibitors from Apios americana tubers
Liu J; Yonekura M; Kouzuma Y, ラスト(シニア)オーサー
Bioscience, Biotechnology, and Biochemistry, 2020年03月, ［査読有り］

Identification of an aldehyde oxidase involved in indole-3-acetic acid synthesis in Bombyx mori silk gland
Takei M; Kogure S; Yokoyama C; Kouzuma Y; Suzuki Y
Bioscience, Biotechnology, and Biochemistry, 2019年01月, ［査読有り］

Novel tryptophan metabolic pathways in auxin biosynthesis in silkworm
Chiaki Yokoyama; Mami Takei; Yoshiaki Kouzuma; Shinji Nagata; Yoshihito Suzuki, In the course of our study of the biosynthetic pathway of auxin, a class of phytohormones, in insects, we proposed the biosynthetic pathway tryptophan (Trp) -> indole-3-acetaldoxime (IAOx) -> indole-3-acetadehyde (IAA1d) -> indole-3-acetic acid (IAA). In this study, we identified two branches in the metabolic pathways in the silkworm, possibly affecting the efficiency of IAA production: Trp indole-3-pyruvic acid -> indole-3-lactic acid and IAAld -> indole-3-ethanol. We also determined the apparent conversion activities (2.05 x 10(-7) UmL(-1) for Trp -> IAA, 1.30 x 10(-5) U mL(-1) for IAOx -> IAA, and 3.91 x 10(-1) U mL(-1) for IAAld -> IAA), which explain why IAOx and IAAld are barely detectable as either endogenous compounds or metabolites of their precursors. The failure to detect IAAld, even in the presence of an inhibitor of the conversion IAAld -> IAA, is explained by a switch in the conversion from IAAld -> IAA to IAAld -> IEtOH., PERGAMON-ELSEVIER SCIENCE LTD
JOURNAL OF INSECT PHYSIOLOGY, 2017年08月, ［査読有り］

Seed Endosperm and Embryo Proteomics of the Lotus (Nelumbo Nucifera Gaertn.) by One-Dimensional Gel-Based Tandem Mass Spectrometry and a Comparison with the Mature Endosperm Proteome
C. F. Moro; Y. Fukao; J. Shibato; R. Rakwal; G. K. Agrawal; Y. Shioda; Y. Kouzuma; M. Yonekura, Lotus (Nelumbo nucifera Gaertn.) seed proteome has been the focus of our studies, and we have recently established the first proteome dataset for its mature seed endosperm. The current study unravels the immature endosperm, as well as the embryo proteome, to provide a comprehensive dataset of the lotus seed proteins and a comparison between the mature and immature endosperm tissues across the seed's development. One-dimensional gel electrophoresis (SDS-PAGE) linked with tandem mass spectrometry provided a protein inventory of the immature endosperm (122 non-redundant proteins) and embryo (141 non-redundant proteins) tissues. Comparing with the previous mature endosperm dataset (66 non-redundant proteins), a total of 206 non-redundant proteins were identified across all three tissues of the lotus seed. Results revealed some significant differences in proteome composition between the three lotus seed tissues, most notably between the mature endosperm and its immature developmental stage shifting the proteins from nutrient production to nutrient storage.
Proteomes, 2015年08月14日, ［査読有り］

Unraveling the seed endosperm proteome of the lotus (Nelumbo nucifera Gaernt.) utilizing 1DE and 2DE separation in conjunction with tandem mass spectrometry　　　　　　　　　　　　　　　
C. F. Moro; Y. Fukao; J. Shibato; R. Rakwal; A. M. Timperio; L. Zolla; G. K. Agrawal; Y. Shioda; Y. Kouzuma; M. Yonekura
Proteomics, 2015年02月12日, ［査読有り］

Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers
Yoshiaki Kouzuma; Satoshi Irie; Rikiya Yamazaki; Masami Yonekura, An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono-or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved., TAYLOR & FRANCIS LTD
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 2014年04月, ［査読有り］

Lotus – A source of food and medicine: Current status and future perspectives in context of the seed proteomics
Carlo F Moro; Masami Yonekura; Yoshiaki Kouzuma; Ganesh K Agrawal; Randeep Rakwal
International Journal of Life Sciences, 2013年, ［査読有り］

Molecular cloning, recombinant expression, and characterization of isolectin genes of hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata　　　　　　　　　　　　　　　
Y. Shimizu; H. Yamazaki; S. Yoshida; M. Yonekura; Y. Kouzuma
Bioscience, Biotechnology, and Biochemistry, 2012年02月07日, ［査読有り］

Effect of AATI, a Bowman-Birk type inhibitor from Apios americana, on proliferation of cancer cell lines
Y. Zhang; C. Zhou; S. Tang; X. Yu; Y. Kouzuma; and M. Yonekura, In this paper, the inhibitory effects of AATI on proliferation of four cancer cell lines were investigated. Compared with SBBI, AATI showed the same or stronger inhibitory effect on the proliferation rate among the cell lines tested. The morphology of the cell treated with AATI appeared irregular and the adhesion ability was changed among cells. The nuclei of the cells presented characters of apoptosis. Furthermore, the amount of phosphatidylserine in the outer leaflet of the cell membrane was increased significantly during the treatment. The genomic DNA test suggested that inhibitory effects on the proliferation of cells by AATI was caused by induction of apoptosis. Moreover, chemical modification of arginine or lysine residues in AATI resulted not only in the partial loses of protease-inhibitory activity, but also in the inhibitory effect on cancer cell lines, suggesting that the inhibitory effects on the proliferation are strongly correlated with its protease-inhibitory activity. (C) 2011 Elsevier Ltd. All rights reserved., ELSEVIER SCI LTD
Food Chemistry, 2011年10月15日, ［査読有り］

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain
T. Miyaji; S. Murayama; Y. Kouzuma; N. Kimura; M. R. Kanost; K. J. Kramer; and M. Yonekura, A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large ( kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu-Arg-MCA, Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA rapidly, whereas hydrolysis of Suc-Leu-Tyr-MCA and Z-Arg-Arg-MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K(i) values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases. (C) 2010 Elsevier Ltd. All rights reserved., PERGAMON-ELSEVIER SCIENCE LTD
Insect Biochemistry and Molecular Biology, 2010年12月

Structures and properties of antioxidative peptides derived from royal jelly protein
H. Guo; Y. Kouzuma; and M. Yonekura, We previously reported that royal jelly proteins (RJPs) hydrolyzed with protease N show the strong antioxidative activity against the peroxidation of linoleic acid. In this study, 29 antioxidative peptides were isolated from hydrolysate by membrane ultrafiltration, anion-exchange chromatography, gel filtration chromatography, and reverse-phase high performance liquid chromatography. We particularly focused on 12 small peptides with 2-4 amino acid residues: these structures were identified as Ala-Leu, Phe-Lys, Phe-Arg, Ile-Arg, Lys-Phe, Lys-Leu, Lys-Tyr, Arg-Tyr, Tyr-Asp, Tyr-Tyr, Leu-Asp-Arg, Lys-Asn-Tyr-Pro. Analysis of the antioxidative properties of these peptides revealed strong hydroxyl radical scavenging activity, but neither metal-chelating activity nor superoxide-anion radical scavenging activity differed significantly among these peptides. Moreover, three dipeptides (Lys-Tyr, Arg-Tyr, and Tyr-Tyr) containing Tyr residues at the C-terminal had strong hydroxyl-radical and hydrogen-peroxide scavenging activity. This suggests that the antioxidant properties of these peptides are due to a combination of these abilities to act as free-radical scavengers. Three tyrosyl dipeptides containing Tyr residues at their C-termini (Lys-Tyr, Arg-Tyr, and Tyr-Tyr) have phenolic hydroxyl groups, which scavenge the free radicals via the mechanism of donating a hydrogen atom from their hydroxyl group. (C) 2008 Elsevier Ltd. All rights reserved., ELSEVIER SCI LTD
Food chemistry, 2009年, ［査読有り］

Steady-State and Time-Resolved Fluorescence Spectroscopic Studies on Interaction of the N-terminal Region with the Hairpin Loop of the Phytocystain Scb
K. Doi-Kawano; E. Nishimoto; Y. Kouzuma; D. Takahashi; S. Yamashita; and M. Kimura, The steady-state and time-resolved fluorescece spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure., SPRINGER/PLENUM PUBLISHERS
Journal of Fluorescence, 2009年, ［査読有り］

Comprehensive royal jelly (RJ) proteomics using one- and two-dimensional proteomics platforms reveals novel RJ proteins and potential phospho/glycoproteins.
T. Furusawa; R. Rakwal; H. W. Nam; J. Shibato; G. K. Agrawal; Y. S. Kim; Y. Ogawa; Y. Yoshida; Y. Kouzuma; Y. Masuo; M. Yonekura., Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html)., AMER CHEMICAL SOC
Journal of Proteome Research, 2008年07月26日, ［査読有り］

Proteomics of two cultivated mushrooms Sparassis crispa and Hericium erinaceum provides insight into their numerous functional protein components and diversity
K. Horie; R. Rakwal; M. Hirano; J. Shibato; H. W. Nam; Y. S. Kim; Y. Kouzuma; G. K. Agrawal; Y. Masuo; and M. Yonekura, Mushroom can be defined as a macrofungus with a distinctive fruiting body. Mushrooms of class Basidiomycete are primarily wood degradation fungi, but serve as food and a part of traditional medicine used by humans. Although their life cycle is fairly well-established, the information on the molecular components, especially proteins are very limited. Here, we report proteomics analysis of two edible mushrooms (fruiting bodies) Sparassis crispa and Hericium erinaceum using one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) based complementary proteomics approaches. 1-DGE coupled with liquid chromatography and mass spectrometry identified 77 (60 nonredundant proteins) and 121 (88 nonredundant proteins) proteins from S. crispa and H. erinaceum, respectively. 2-DGE analysis revealed 480 and 570 protein spots stained with colloidal coomassie brilliant blue in S. crispa and H. erinaceum, respectively. Of the 71 and 115 selected protein spots from S. crispa and H. erinaceum 2D gel blots on polyvinyldifluoride (PVDF) membranes, respectively, 29 and 35 nonredundant proteins were identified by N-terminal amino acid sequencing. Identified nonredundant proteins from 1- or 2-DGE belonged to 19 functional categories. Twenty-one proteins were found common in both S. crispa and H. erinaceum proteomes, including 14-3-3 protein and septin. Together this study provides evidence for the presence of a large number of functionally diverse proteins, expressed in the fruiting body of two economically important mushrooms, S. crispa and H. erinaceum. Data obtained from 1-DGE and 2-DGE analyses is accessible through the mushroom proteomics portal http://foodfunc.agr.ibaraki.ac.jp/ mushprot.html., AMER CHEMICAL SOC
Journal of Proteome Research, 2008年04月02日, ［査読有り］

Systematic investigation of the hemolymph proteome of Manduca sexta at the fifth instar larvae stage using one- and two-dimensional proteomics platforms
T. Furusawa; R. Rakwal; H. W. Nam; M. Hirano; J. Shibato; Y. S. Kim; Y. Ogawa; Y. Yoshida; K. J. Kramer; Y. Kouzuma; G. K. Agrawal; and M. Yonekura, Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein IoIC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1 -DGE and 2-DGE analyses are accessible through the M.sexta proteomic sportal(http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae., AMER CHEMICAL SOC
Journal of Proteome Research, 2008年03月, ［査読有り］

Purification, characterization, and cDNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers
Y. Zhang; Y. Kouzuma; T. Miyaji and M. Yonekura, An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) m and 1.0 x 10(-6) m respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 2008年01月, ［査読有り］

C-type lectin-like carbohydrate-recognition of the hemolytic lectin CEL-III containing ricin-type beta-trefoil folds
T. Hatakeyama; H. Unno; Y. Kouzuma; T. Uchida; S. Eto; H. Hidemura; N. Kato; M. Yonekura; and M. Kusunoki, CEL-III is a Ca2+ dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/ methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca2+ located at the subdomains 1 alpha, 1 gamma, 2 alpha, 2 beta, and 2 gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca2+ ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2 alpha and 2 beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein., AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal of Biological Chemistry, 2007年12月, ［査読有り］

Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development
S. Yoshida; Y. Shimada; D. Kondoh; Y. Kouzuma; A. K. Ghosh; M. Jacobs-Lorena; R. E. Sinden, The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions., PUBLIC LIBRARY SCIENCE
PLoS Pathogens, 2007年12月, ［査読有り］

Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein　　　　　　　　　　　　　　　
T. Miyaji; Y. Kouzuma; J. Yaguchi; R. Matsumoto; M. R. Kanost; K. J. Kramer; M. Yonekura
Insect Biochemistry and Molecular Biology, 2007年07月, ［査読有り］

Contribution of conserved Asn residues to the inhibitory activities of Kunitz-type protease inhibitors from plants.
Iwanaga; S.; Yamasaki; N.; Kimura; M.; and Kouzuma; Y., Plant Kunitz-type protease inhibitors contain a conserved Asn residue in the N-terminal region. To investigate the role of Asn residue in protease inhibitory activities, Etythrina variegata trypsin inhibitor a (ETIa), E. variegata chymotrypsin inhibitor (ECI), and their mutants, ETIa-N12A and ECI-N13A, were used. Both mutants exhibit weaker inhibitory activities toward their cognate proteases than the wild-type proteins and were readily cleaved at reactive sites. Furthermore, kinetic analysis of the interactions of the mutated proteins with their cognate proteases by surface plasmon resonance (SPR) measurement indicated that replacements of the Asn residue mainly affected dissociation rate constants. The conserved Asn residues of Kunitz-type inhibitors play an important role in exhibiting effective inhibitory activity by stabilizing the structures of the primary binding loop and protease-inhibitor complex., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 2005年

Isolation, and properties of antioxidative peptides from water-soluble royal jelly protein hydrolysate.
Guo; H.; Kouzuma; Y.; and Yonekura; M., Several novel antioxidative peptides from the hydrolysate of water-soluble royal jelly protein (WSRJP) were isolated, identified, and characterized. WSRJP was extracted from royal jelly and hydrolyzed with protease N, and the resulting hydrolysate was fractionated by ultrafiltration with cutoff membranes of 1 and 3 kDa. Three fractions ( < 1 kDa, 1-3 kDa, and > 3kDa) were separated. The antioxidative activity of the fractions against the peroxidation of linoleic acid was determined in vitro, using the 1,3-diethyl-2-thiobarbituric acid method. The < 1 kDa fraction, which exhibited the highest antioxidative activity, was further purified using anion-exchange and reverse phase high performance liquid chromatography. Fourteen antioxidative peptides were identified using a protein sequencer and electron spray ionization-mass spectrometry. Among these, four dipeptides (Phe-Asp, Trp-Val, Leu-Trp, and Trp-Leu) were revealed to have potent antioxidative activity against lipid peroxidation. Moreover, three of these antioxidative dipeptides (Phe-Asp, Trp-Val, and Leu-Trp) were found to protect against oxidative stress-induced cell death in human cultured cells., KARGER
Food Science and Technology Research, 2005年

Characterization of functional domains of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata.
Kouzuma; Y.; Suzuki; Y.; Nakano; M.; Matsuyama; K.; Tojo; S.; Kimura; M.; Yamasaki; T.; Aoyagi; H.; and Hatakeyama; T., CEL-III is a Ca2+-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectin isolated from the marine invertebrate Cucumaria echinata. This lectin exhibits strong hemolytic activity and cytotoxicity through pore formation in target cell membranes. The amino acid sequence of CEL-III revealed the N-terminal two-thirds to have homology to the B-chains of ricin and abrin, which are galactose-specific plant toxic lectins; the C-terminal one-third shows no homology to any known proteins. To examine the carbohydrate-binding ability of the N-terminal region of CEL-III, the protein comprising Pyr1-Phe283 was expressed in Escherichia coli cells. The expressed protein showed both the ability to bind to a GalNAc-immobilized column as well as hemagglutinating activity for rabbit erythrocytes, confirming that the N-terminal region has binding activity for specific carbohydrates. Since the C-terminal region could not be expressed in E. coli cells, a fragment containing this region was produced by limited proteolysis of the native protein by trypsin. The resulting C-terminal 15 kDa fragment of CEL-III exhibited a tendency to self-associate, forming an oligomer. When mixed with erythrocytes, the oligomer of the C-terminal fragment caused hemagglutination, probably due to hydrophobic interaction with cell membranes, while the monomeric fragment did not. Chymotryptic digestion of the preformed CEL-III oligomer induced upon lactose binding also yielded an oligomer of the C-terminal fragment comprising six molecules of the 16 kDa fragment. These results suggest that after binding to cell surface carbohydrate chains, CEL-III oligomerizes through C-terminal domains, leading to the formation of ion-permeable pores by hydrophobic interaction with the cell membrane., JAPANESE BIOCHEMICAL SOC
Journal of Biochemistry, 2003年

Reconstitution of archaeal ribonuclease P from RNA and four protein components.
Kouzuma; Y.; Mizoguchi; M.; Takagi; H.; Fukuhara; H.; Tsukamoto; M.; Numata; T.; and Kimura; M., Ribonuclease P (RNase P) is an endonuclease responsible for generating the 5' end of matured tRNA molecules. A homology search of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 genome database revealed that the four genes, PH1481, PH1601, PH1771, and PH1877, have a significant homology to those encoding RNase P protein subunits, hpop5, Rpp21, Rpp29, and Rpp30, of human, respectively. These genes were expressed in Escherichia coli cells, and the resulting proteins Ph1481p, Ph1601p, Ph1771p, and Ph1877p were purified to apparent homogeneity in a set of column chromatographies. The four proteins were characterized in terms of their capability to bind the cognate RNase P RNA from P. horikoshii. All four proteins exhibited the binding activity to the RNase P RNA. In vitro reconstitution of four putative RNase P proteins with the in vitro transcripted P. horikoshii RNase P RNA revealed that three proteins Ph1481p, Ph1601p, and Ph1771p, and RNase P RNA are minimal components for the RNase P activity. However, addition of the fourth protein Ph1877p strongly stimulated enzymatic activity, indicating that all four proteins and RNase P RNA are essential for optimal RNase P activity. The present data will pave the way for the elucidation of the reaction mechanism for archaeal as well as eukaryotic RNase P. (C) 2003 Elsevier Science (USA). All rights reserved., ACADEMIC PRESS INC ELSEVIER SCIENCE
Biochemical and Biophysical Research Communincations, 2003年

Genomic cloning of ribonucleases in Nicotiana glutinosa leaves, as induced in response to wounding or to TMV-infection, and characterization of their promoters.
Hayashi; T.; Kobayashi; D.; Kariu; T.; Tahara; M.; Hada; K.; Kouzuma; Y.; and Kimura; M., We previously cloned two distinct cDNA clones, NGR1 and NGR3, encoding S-like ribonucleases (RNases) induced by wounding and tobacco mosaic virus (TNIV) infection, respectively, in Nicotiana glutinosa leaves. To gain insight into the regulatory mechanism of the RNase genes, we analyzed nucleotide sequences of the genes ngr1 (4.1 kbp) and ngr3 (5.3 kbp), containing their structural genes as well as 5'-flanking regions. The ngr1 gene is organized in three exons with two intervening introns, and ngr3 has four exons interrupted by three introns. Primer extension analyses localized single transcription initiation sites at - 32 and - 99 upstream of the translation initiation codons ATG in the genes ngr1 and ngr3, respectively. The beta-glucuronidase (GUS) reporter gene analysis with serial 5'-deletion mutants as well as a gel shift assay defined the wound-responsive region at residues - 509 to - 288 in gene ngr1 and a TMV-responsive region at the residues - 401 to - 174 in ngr3, respectively. Sequence search using PLACE and PlantCARE data bases showed that a wound-responsive element: the WUN-motif, occurs within the wound-responsive region in ngr1, while ngr3 contains several potential cis-regulating elements, such as the elicitor responsiveness element: the W-box, a TNIV responsive element: GT1, and the WUN-motif at positions between - 401 and - 174. These findings suggested that some of these cis-elements may be involved in inducible expressions of ngr1 and ngr3. Furthermore, the gel shift assay suggested that the dissociation of protein factor(s) upon TMV-infection from the regulatory region may cause an inducible expression of ngr3., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 2003年

On the interaction of ribosomal protein L5 with 5S RNA.
Iwasaki; K.; Kikukawa; S.; Kawamura; S.; Kouzuma; Y.; Tanaka; I.; and Kimura; M., Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.
Bioscience, Biotechnology, and Biochmistry, 2002年

Requirement for C-terminal extension to the RNA binding domain for efficient RNA binding by ribosomal protein L2.
Hayashi; T.; Tahara; M.; Iwasaki; K.; Kouzuma; Y.; and Kimura; M., Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.
Bioscience, Biotechnology, and Biochemistry, 2002年

Protein engineering of novel proteinase inhibitors and their effect on the growth of Spodoptera exigua larvae.
Inanaga; H.; Kobayashi; D.; Kouzuma; Y.; Aoki-Yasunaga; C.; Iiyama; K.; and Kimura; M., Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds. Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) m and 1.75 x 10(-7) m), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) m and 1.52 x 10(-7) M), respectively. We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua. When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed. Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor. In contrast, BGIT had little effect on the growth of the S. exigua larvae. This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S. exigua larvae. Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 2001年

Calcium ions stabilize a protein structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata.
Sallay; I.; Tojo; S.; Nomiyama; K.; Kouzuma; Y*.; Kimura; M.; and Yamasaki; N., GEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc)-specific lectin purified from a marine invertebrate, Cucumaria echinata, has a strong hemolytic activity, especially toward human and rabbit erythrocytes in the presence of Ca2+. We evaluated the role of Ca2+ in hemagglutinating and hemolytic activities of CEL-III. We found that Ca2+ is closely associated with both activities of GEL-III. The fluorescence spectra of GEL-III upon binding to Ca2+ were measured. The result showed a structural change of GEL-III in the presence of Ca2+. The structural change of CEL-III upon Ca2+ binding was further demonstrated by stabilization against urea denaturation and by insusceptibility to protease digestions. GEL-III was completely unfolded at a low concentration of 2 M urea, while GEL-III complexed with Ca2+ was stable in 6 M urea. As for protease digestions, GEL-III monomer and oligomer were readily digested by trypsin, chymotrypsin, and papain in the absence of Ca2+, while they were insusceptible to the three proteases in the presence of Ca2+. The papain digestion of the decalcified oligomer produced a large C-terminal peptide, suggestting that the C-terminal region of GEL-III may participate in oligomerization of GEL-III as a core domain., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 2001年

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor Sca from sunflower seeds.
Kouzuma; Y.; Tsukigata; K.; Inanaga; H.; Doi-Kawano; K.; Yamasaki; N.; and Kimura; M., Sunflower cystatin a (Sca) is distinguished from other photocystatins by its lack of the N-terminal about 20 amino acids, resulting in the absence of the evolutionarily; conserved Gly: residue. The cDNA encoding Sea was amplified by PCR methods. The cDNA consists of 520 nucleotides and includes an open reading frame encoding a polypeptide of 98 amino acids. Comparison of the deduced amino acid sequence with; the Sea protein sequence indicated that the deduced sequence has an extra 15 amino acids and one amino acid at the N- and C-termini, respectively. This result suggests that Sea is synthesized as a preprotein (preSca) and proteolytic cleavages at peptide bonds may give rise to the mature Sea. To address this assumption and also to investigate the significance of the N-terminal er;tension sequence to Sea for inhibitory activity, a recombinant pre-Sca (rpre-Sca), in which the N-terminal extension was fused to the matured Sea, and a recombinant matured Sea (rSca) mere overproduced in Escherichia coli cells. Incubation of the rpre-Sca with a seed extract resulted in a mobility by SDS-PAGE; that was the same air rSca, demonstrating a proteolytic cleavage by endogenous proteinases. The rSca and rpre-Sca proteins were further characterized with respect to inhibitory activity and sensorgrams of the interaction with papain. The result showed that rpre-Sca had stronger inhibitory activity than rSca, and that the increased activity toward papain was due to a lower dissociation rate constant. This finding indicates that the N-terminal region of rpre-Sca increases the inhibitory activity by stabilizing the rpre-Sca and papain complex., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 2001年

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds.
Kouzuma; Y.; Inanaga; H.; Doi-Kawano; K.; Yamasaki; N.; and Kimura; M., Two cysteine proteinase inhibitors, cystatins Sea and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J, Biochem, 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sea cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sea, The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins., OXFORD UNIV PRESS
Journal of Biochemistry, 2000年

Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA : structural similarity to the B-chain from plant lectin, ricin.
Nakano; M.; Tabata; S.; Sugihara; K.; Kouzuma; Y.; Kimura; M.; and Yamasaki; N., CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M-r of 47 457 and has six internal tandem repeats, each with of 40-50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane. (C) 1999 Elsevier Science B.V. All rights reserved., ELSEVIER SCIENCE BV
Biochimica et Biophysica Acta, 1999年

Conformation of the primary binding loop folded through an intramolecular interaction contributes to the strong chymotrypsin inhibitory activity of the chymothrypsin inhibitor from Erythrina variegata seeds.
Iwanaga; S.; Nagata; R.; Miyamoto; A.; Kouzuma; Y.; Yamasaki; N.; and Kimura; M., We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (K-i, 5.6 X 10(-7) M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the "scaffold." A chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (K-i, 1.3 X 10(-6) M) than ECI (K-i, 9.8 X 10(-8) M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (K-i, 5.7 X 10(-7) M) than ETIa (K-i, 1.3 X 10(-6) M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (k(on)) and a decrease in the dissociation rate constant (k(off)) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant., JAPANESE BIOCHEMICAL SOC
Journal of Biochemistry, 1999年

Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibiter from sunflower seeds.
Doi-Kawano; K.; Kouzuma; Y.; Yamasaki; N.; and Kimura; M., Sunflower cystatin Scb differs from other phytocystatins in that; it is a highly basic protein with a pi value of 9.6 and includes six additional amino acids (Arg(30)-Leu-Gln-Arg-Thr(34), Thr(37)) in the middle region as compared with other phytocystatins [Kouzuma ct al, (1996) J. Biochem. 119, 1106-1113], We identified and sequenced a complete cDNA encoding the Scb; the cDNA of Scb consists of 645 nucleotides and includes an open reading frame encoding a polypeptide of 123 amino acids. On the basis of these findings, Scb appears to be synthesized as a prepeptide consisting of a signal sequence of 22 amino acids and a mature protein of 101 amino acids. A recombinant Scb (rScb) was produced by expression in Escherichia coli and purified by gel filtration on Sephacryl S-200 followed by ion-exchange column chromatography on a S-Sepharose column. rScb exhibited almost the same inhibitory activity toward papain as the authentic Scb did, but its inhibition profile toward cathepsins B, L, and H was slightly different, Scb mutant proteins, in which selected N-terminal residues or the additional amino acids were deleted, were subsequently constructed and characterized with respect to their inhibitory activities toward papain, The result revealed that the additional sequence (Arg(30)-Leu-Gln-Arg-Thr(34)) in Scb is not essential for papain-inhibitory activity, while the N-terminal amino acids (Ile(1)-Pro(2)) as well as the N-terminal glycine residues Gly(3) and/or Gly(4) play an important role in manifesting the inhibitory activity toward papain., JAPANESE BIOCHEMICAL SOC
Journal of Biochemistry, 1998年

Cloning, expression, and mutagenesis of trypsin inhibitor ETIb from Erythrina variegata seeds.
Kouzuma; Y.; Kimura; M.; and Yamasaki; N., Erythrina variegata trypsin inhibitors designated ETIa and ETIb belong to the Kunitz family trypsin inhibitor, but ETIa is unique in its ability to inhibit tissue-type plasminogen activator, while ETIb is not, The cDNA clone encoding ETIb was isolated from the seed cDNA library constructed in the lambda phage lambda gt11. The ETIb cDNA insert consists of 765 bp, including an open reading frame of 606 ph from ATG to TGA codons, The deduced amino acid sequence consists of 202 amino acids, having the signal peptides of 22 amino acids in the N-terminus and 2 amino acids in C-terminus, The cDNA fragment encoding the mature form of ETIb was introduced into an expression vector, pET-22b, and expressed in Escherichia coli BL21 (ED3) in a functional form. Furthermore, the ETIb mutant bP61R/F62L, in which Pro(61) and Phe(62) in ETIb were changed to the corresponding amino acid residues Arg and Leu, respectively, as in ETIa, was constructed, and its inhibitory potency toward tPA was assayed, This mutant showed significant tPA inhibitory activity, albeit less than ETIa, The result demonstrates that the Arg(61) and Leu(62) residues in ETIa are important in inhibiting tPA, and also suggests that beside these two residues, the other amino acid(s) or other structural element may be involved in interaction of ETIa with tPA., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 1997年

The tissue-type plasminogen activator inhibitor ETIa from Erythrina variegata : Structural basis for the inhibitory activity by cloning, expression, and mutagenesis of the cDNA encoding ETIa.
Kouzuma; Y.; Kimura; M.; and Yamasaki; N., Erythrina variegata trypsin inhibitor ETIa belongs to the Kunitz inhibitor family, but is unique in its ability to bind and inhibit tissue-type plasminogen activator (tPA), A cDNA clone encoding ETIa was isolated from the lambda gt11 cDNA library using specific antiserum as a probe and characterized by nucleotide sequencing, The cloned ETIa cDNA consists of 762 nucleotides and includes an open reading frame encoding a polypeptide of 198 amino acids, Comparison of the deduced protein sequence and the determined protein sequence indicated the presence of two signal peptides composed of 24 and 2 amino acids at the N- and C-termini, respectively, The cDNA encoding mature ETIa was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3), The recombinant ETIa (rETIa) was expressed in E. coli as inclusion bodies; it was purified to homogeneity by gel filtration on Sephadex G-75, The rETIa exhibited almost the same inhibitory activity toward trypsin and tPA as ETIa, Six mutants, in which the amino acids Arg(61), Leu(62), Arg(63), and Ala(65) were replaced by Pro, Phe, Leu/Asp, and Tyr, respectively, were constructed by site-specific mutagenesis and expressed in E. coli, The site-specific mutation of Arg(63) to Leu (aR63L) or Asp (aR63D) in ETIa resulted in abolition of the inhibitory activities toward both trypsin and tPA, The mutants aR61P and aL62F showed significantly reduced tPA-inhibitory activity, and furthermore the double mutant aR61P/L62F lacked tPA-inhibitory activity, despite retaining the trypsin-inhibitory activity, In contrast, the mutant aA65Y exhibited tPA-inhibitory activity to the same extent as rETIa, This result suggests that Arg(61) and Leu(62) in ETIa, in addition to Arg(63), may play an important role in the interaction with tPA., JAPANESE BIOCHEMICAL SOC
Journal of Biochemistry, 1997年

Purification, Characterization, and Sequencing of Two Cysteine Proteinase Inhibitors, Sca and Sub, from Sunflower (Helianthus annuus) Seeds.
Kouzuma; Y.; Kawano; K.; Kimura; M.; Yamasaki; N.; Kadowaki; T.; and Yamamoto; K., Two proteinaceous cysteine proteinase inhibitors (cystatins) referred to as Sea and Scb were purified to homogeneity from the seeds of sunflower (Heliantas annuus) by gel filtration on Sephadex G-75 followed by a series of ion-exchange column chromatographies and reverse-phase HPLC (RP-HPLC), The isoelectric points (pi) of Sea and Scb were estimated to 5.0 and 9.6, respectively. The inhibitory potencies of these two cystatins were examined with cysteine proteinases from various sources, such as plants, mammals, and bacteria, Papain was strongly inhibited by both Sea and Scb with K-i, values of 5.6x10(-9) and 1.7x10(-10) M, respectively. Sea and Scb were also found to be potent inhibitors of ficin (K-i values of 1.9x10(-6) and 2.8x10(-9) M, respectively), Rat cathepsin H was inhibited strongly by Scb and slightly by Sca, Although rat cathepsins B and L were significantly inhibited by Scb, they were scarcely affected by Sea. Neither Sea nor Scb inhibited Arg-gingipain, an arginine-specific cysteine proteinase of Porphyromonas gingivalis. The. complete amino acid sequences of the two inhibitors were determined by protein chemical methods, The proteins Sea and Scb consist of 83 and 101 amino acid residues with M(r) of 9,330 and 11,187, respectively, and there are identical residues at 34 positions in the two sequences, that is at 42% of the residues compared, Comparison of their sequences with those of other cystatins revealed that Sca shares 59-73% identical residues with other phytocystatins, while Scb shows less identity to other phytocystatins, sharing only 28-38%, identical residues, Furthermore, only 20-27% of the residues of both cystatins, Sea and Scb, are identical to those of the animal cystatins., OXFORD UNIV PRESS
Journal of Biochemistry, 1996年

Inhibitory potency of Erythrina variegata proteinase inhibitors toward serine proteinases in the blood coagulation and fibrinolytic systems.
T Nakagaki; Y Shibuya; Y Kouzuma; N Yamasaki; M Kimura, The Erythrina variegata Kunitz family trypsin inhibitors, ETIa and ETIb, prolonged the activated partial thromboplastin time (APTT) and also the prothrombin time (PT) of human plasma, but the Kunitz family chymotrypsin inhibitor, ECI, and Bowman-Birk family inhibitor, EBI, from E. variegata hardly prolonged these times. Trypsin inhibitors ETIa and ETIb inhibited the amidolytic activity of factor Xa, and ETIb but not ETIa inhibited plasma kallikrein. Neither Ena nor ETIb exhibited any inhibitory activity toward beta-factor XIIa and thrombin. Furthermore, trypsin inhibitors ETIa and ETIb inhibited plasmin, a serine proteinase in the fibrinolytic system, whereas ECI and EBI did not. These results indicate that Erythrina Kunitz proteinase inhibitors possess different potency toward serine proteinases in the blood coagulation and fibrinolytic systems, in spite of their high similarity in amino acid sequence., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 1996年

On a Bowman-Birk family proteinase inhibitor from Erythrina variegata seeds.
Kimura; M.; Kouzuma; Y.; Abe; K.; and Yamasaki; N., A Ban man-Birk family proteinose inhibitor (EBI) was isolated from the seeds of Erythrina variegata. The protein was purified by ion-exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-75. The stoichiometry with trypsin was estimated to be 1:1, while that with chymotrypsin was not obvious, as determined from the titration patterns of its inhibitory activities. The complete amino acid sequence of EBI was determined by sequencing tryptic and chymotryptic peptides. The EBI protein consists of 61 amino acid residues, which is the shortest among the Bowman-Birk family inhibitors sequenced to date, and has a M(r) of 6,689. Comparison of this sequence with those of other leguminous Bowman-Birk family inhibitors revealed that EBI could be classified as a group II inhibitor, showing the best homology (67%) to the Bowman-Birk proteinase inhibitor from soybeans., JAPANESE BIOCHEMICAL SOC
Journal of Biochemistry, 1994年

Amino acid sequence of chymotrypsin inhibitor ECI from the seeds of Erythrina variegata (LINN.) var. Orientalis.
Kimura; M.; Kouzuma; Y.; and Yamasaki; N., The amino acids of the chymotrypsin inhibitor (ECI) from the Erythrina variegata seeds have been sequenced. The sequence was solved by analysis of peptides derived from the protein by enzymatic digestions with trypsin and Staphylococcus aureus V8 proteinase, as well as by chemical cleavage with o-iodosobenzoic acid. The ECI consists of 179 amino acid residues with a pyroglutamic acid as the N-terminal residue and has a calculated molecular weight of 19,791. Comparison of this sequence with the sequences of the two trypsin inhibitors, ETIa and ETIb, from the E. variegata seeds shows that about 60% of the residues of ECI are identical to those of ETIa and ETIb and that the reactive sites, Arg63, in ETIa and ETIb change to Leu64 in ECI., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 1993年

Isolation and primary structure of proteinase inhibitors from Erythrina variegata (LINN.) var. Orientalis seeds.
Kouzuma; Y.; Suetake; M.; Kimura; M.; and Yamasaki; N., The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1 : 1, while that of chymotrypsin inhibitor with chymotrypsin was 1 : 2, judging from the titration patterns of their inhibitory activities.
The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues., TAYLOR & FRANCIS LTD
Bioscience, Biotechnology, and Biochemistry, 1992年

Comprehemsive analysis of wound-inducible genes from the Nicotiana glutinosa leaves using a full-length cDNA microarray
Y. Kouzuma; Y. Tsutsumi; M. Abe; T. Hayashi; K. Hada; K. Uehashi; Y. Shimada; K. Tashiro; S. Kuhara; and M. Kimura
Journal of the Faculty of Agriculture, Kyushu University, 2005年

超好熱古細菌Pyrococcus horikoshii由来リボヌクレアーゼPの再構成
上妻由章; 木村誠
Radioisotopes, 2003年

植物プロテアーゼの活性制御に関する研究
上妻由章
日本農芸化学会誌, 2001年06月

多機能プロテアーゼインヒビターの創製と応用
上妻由章、木村誠
バイオサイエンスとインダストリー, 2001年

Characterization of two cysteine proteinase inhibitors (Sca and Scb) from sunflower seeds
Kouzuma, Y; Kawano, K; Harada, R; Kariu, T; Kimura, M; Yamasaki, N
The Second Symposium on Frontier of Protein Chemistry and Biotechnology (Yangzhou Univ. (Yangzhou, China)), 1995年08月19日

Characterization of serine proteinase inhibitors from the Erythrina variegata seeds
Kimura, M; Kouzuma, Y; Kuramitu, J; Iwanaga, S; Yamasaki, N
The Second Symposium on Frontier of Protein Chemistry and Biotechnology (Yangzhou Univ. (Yangzhou, China)), 1995年08月19日

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生物機能の新展開-人類の生存をかけて-「生理活性タンパク質の分子認識機構の解析」　　　　　　　　　　　　　　　
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多重変異導入による溶血性レクチンCEL-IIIの溶血活性の向上　　　　　　　　　　　　　　　
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日本蛋白質科学会（あわぎんホール（徳島））, 2015年06月24日, 日本蛋白質科学会

アピオス塊茎由来ポリガラクツロナーゼインヒビター（PGIP）の分離精製、性質とcDNAクローニング　　　　　　　　　　　　　　　
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溶血性レクチンCEL-IIIの溶血活性向上にむけた糖結合部位の変異体解析　　　　　　　　　　　　　　　
上妻由章、山田麻由、劉佳、清水良樹、米倉政実
第14回日本蛋白質科学会年会（ワークピア横浜 / 横浜産貿ホール マリネリア）, 2014年06月25日, 日本蛋白質科学会

デキストラン硫酸ナトリウムで誘発したヒト腸管上皮細胞株Caco-2の細胞傷害に対するローヤルゼリータンパク質の影響　　　　　　　　　　　　　　　
松浦 遼太郎、村松 和香、上妻 由章、米倉 政実
日本農芸化学会大会（明治大学）, 2014年03月28日, 日本農芸化学会

アピオス塊茎のβ－アミラーゼの分離精製、性質および構造解析　　　　　　　　　　　　　　　
日野 ちはる、井下 智香子、上妻 由章、米倉 政実
日本農芸化学会大会（東北大学）, 2013年03月25日, 日本農芸化学会

ローヤルゼリータンパク質アピシン及びその構成成分のヒト組織球性リンパ腫細胞株 ( U937 ) の増殖に対する作用　　　　　　　　　　　　　　　
仙波 美紗、上妻 由章、米倉 政実
日本農芸化学会大会（東北大学）, 2013年03月25日, 日本農芸化学会

溶血性レクチンCEL-IIIの糖結合サブドメイン2βへの変異導入による溶血活性の向上　　　　　　　　　　　　　　　
上妻 由章、清水 良樹、米倉 政実
日本農芸化学会大会（東北大学）, 2013年03月25日, 日本農芸化学会

タバコスズメガ由来システインプロテアーゼインヒビター前駆体中のシスタチン様ドメイン3の発現とその特徴　　　　　　　　　　　　　　　
上妻 由章、 檜山 嵩央、村山 智史、 宮地 崇之、米倉 政実
日本生化学会大会（マリンメッセ福岡）, 2012年12月15日, 日本生化学会

アピオス塊茎由来レクチンの分離精製と性質　　　　　　　　　　　　　　　
入江 聡、上妻 由章、米倉 政実
日本農芸化学会大会（京都女子大学）, 2012年03月24日, 日本農芸化学会

糖結合サブドメインへの変異導入による溶血性レクチンCEL-IIIの機能向上　　　　　　　　　　　　　　　
清水 良樹、米倉 政実、上妻 由章
日本農芸化学会大会（京都女子大学）, 2012年03月24日, 日本農芸化学会

海産無脊椎動物グミ由来溶血性レクチンCEL-IIIの糖結合部位への変異導入による糖結合活性及び溶血活性に対する影響　　　　　　　　　　　　　　　
清水 良樹、米倉 政実、上妻 由章
日本蛋白質科学会年会（ホテル阪急エキスポパーク（大阪））, 2011年06月07日

溶血性レクチンCEL-IIIの糖結合部位の溶血活性への役割　　　　　　　　　　　　　　　
清水 良樹、米倉 政実、上妻 由章
日本農芸化学会大会（京都女子大学）, 2011年03月26日

チオレドキシン融合型溶血性レクチンCEL-IIIの発現及びその特徴　　　　　　　　　　　　　　　
清水良樹、米倉政実、上妻由章
日本分子生物学会年会・日本生化学会大会 合同大会（神戸ポートアイランド）, 2010年12月10日

アピオス塊茎由来レクチンのcDNAクローニング　　　　　　　　　　　　　　　
入江 聡、山崎 力也、小倉 恵生、上妻 由章、米倉 政実
日本農芸化学会大会（東京大学）, 2010年03月29日, 日本農芸化学会

タバコスズメガ由来システインプロテアーゼインヒビター（MsCPI）前駆体中のシスタチン様ドメインの機能解析　　　　　　　　　　　　　　　
村山 智史、宮地 崇之、上妻 由章、米倉 政実
日本農芸化学会大会（東京大学）, 2010年03月28日, 日本農芸化学会

タバコスズメガ由来システインプロテアーゼインヒビター前駆体のゲノムクローニングとシスタチン様ドメインの機能解析　　　　　　　　　　　　　　　
村山智史、宮地祟之、木村信忠、上妻由章、米倉政実
日本生化学会関東支部例会（つくば国際会議場）, 2009年06月20日, 日本生化学会関東支部

組換え型溶血性レクチンCEL-IIIの発現、特徴付けおよび変異体を用いた溶血機構の解析　　　　　　　　　　　　　　　
清水良樹、米倉政実、上妻由章
日本生化学会関東支部例会（つくば国際会議場）, 2009年06月20日, 日本生化学会関東支部

ローヤルゼリーの抗酸化作用　　　　　　　　　　　　　　　
上村知広、上妻由章、米倉政実
日本農芸化学会大会（マリンメッセ福岡）, 2009年03月29日, 日本農芸化学会

タバコスズメガ由来システインプロテアーゼインヒビター前駆体中のカテプシンF様酵素の特徴と機能解析　　　　　　　　　　　　　　　
宮地崇之、上妻由章、Kanost; Michael、Kramer; Karl、米倉政実
日本農芸化学会大会（マリンメッセ福岡）, 2009年03月29日, 日本農芸化学会

溶血性レクチンCEL-IIIのイソレクチン遺伝子のcDNAクローニングと大腸菌内での発現　　　　　　　　　　　　　　　
山崎寛司、米倉政実、上妻由章
日本農芸化学会大会（マリンメッセ福岡）, 2009年03月28日, 日本農芸化学会

溶血性レクチンCEL-IIIの糖認識部位の変異導入に対する溶血活性への影響とその速度論的解析　　　　　　　　　　　　　　　
清水良樹、吉田栄人、米倉政実、上妻由章
日本農芸化学会大会（マリンメッセ福岡）, 2009年03月28日, 日本農芸化学会

生ローヤルゼリー及び調製ローヤルゼリー中のタンパク質の性状比較　　　　　　　　　　　　　　　
高橋英輝、室崎伸二、上妻由章、米倉政実
日本食品科学工学会（京都大学）, 2008年09月07日

鶏卵卵殻膜分解物のアンジオテンシンI変換酵素阻害作用と高血圧自然発症ラットに対する血圧降下作用　　　　　　　　　　　　　　　
石井真梨、山本亜弥子、上妻由章、藤田悠記、池田賢次郎、○米倉政実
日本食品科学工学会（京都大学）, 2008年09月07日

アピオス抽出物の高血圧自然発症ラットにおける血圧降下作用　　　　　　　　　　　　　　　
石井真梨、上妻由章、米倉政実
日本食品科学工学会（京都大学）, 2008年09月07日

活性酸素によるコラーゲン分解に対するローヤルゼリータンパク質加水分解物の抑制作用　　　　　　　　　　　　　　　
泉宏樹、上妻由章、小山洋一、楠畑雅、米倉政実
日本食品科学工学会（京都大学）, 2008年09月07日

タバコスズメガ由来システインプロテアーゼインヒビター前駆体中のカテプシンF様酵素の発現とその特徴　　　　　　　　　　　　　　　
宮地崇之、上妻由章、Kanost; Michael、Kramer; Karl、米倉政実
日本農芸化学会大会（名城大学（名古屋））, 2008年03月28日, 日本農芸化学会

バキュロウイルス発現系を用いた溶血性レクチンCEL-IIIの発現およびその特徴　　　　　　　　　　　　　　　
清水良樹、吉田栄人、上妻由章、米倉政実
日本農芸化学会大会（名城大学（名古屋））, 2008年03月28日, 日本農芸化学会

マクロファージ様培養細胞（U937及びJ774.1）に対するアピオス塊茎タンパク質の影響　　　　　　　　　　　　　　　
小倉恵生、上妻由章、米倉政実
日本農芸化学会大会（名城大学（名古屋））, 2008年03月28日, 日本農芸化学会

溶血性レクチン CEL-III の糖複合体結晶構造解析　　　　　　　　　　　　　　　
海野英昭,衛藤誠一朗,秀村晴樹,上妻由章,米倉政実,内田達也,楠木正巳,畠山智充
日本結晶学会2007年会, 2007年12月01日

Prevention effects of antioxidative peptides derived from royal jelly proteins on protein degradation by reactive oxygen species　　　　　　　　　　　　　　　
Hiroki Izumi; Yoshiaki Kouzuma; Masami Yonekura
International Conference on Food Factors for Health Promotion -ICoFF2007, 2007年11月28日

Comprehensive investigation of royal jelly proteome by one- and two-dimensional proteomics platforms　　　　　　　　　　　　　　　
Takako Furusawa; Randeep Rakwal; Yoshiaki Kouzuma; Sinji Itoh; Mika Terakawa; Masami Yonekura
International Conference on Food Factors for Health Promotion -ICoFF2007, 2007年11月28日

Proteomics of two mushrooms Sparassis crispa and Hericium erinaceum provides insight into their functional Protein components and diversity　　　　　　　　　　　　　　　
Kiyotaka Horie; Randeep Rakwal; Misato Hirano; Junko Shibato; Hyung W. Nam; Yu S. Kim; Yoshiaki Kouzuma; Ganesh K. Agrawal; Masami Yonekura
International Conference on Food Factors for Health Promotion -ICoFF2007, 2007年11月28日

アピオス由来トリプシンインヒビターの白血病株化細胞に対する増殖抑制作用　　　　　　　　　　　　　　　
張有做、上妻由章、米倉政実
日本食品科学工学会年会（中村学園大学（福岡））, 2007年09月08日, 日本食品科学工学会

溶血性レクチンCEL-IIIの部位特異的変異体の発現とその自己会合性　　　　　　　　　　　　　　　
久松啓伍、海野英昭、郷田秀一郎、上妻由章、畠山智充
日本蛋白質科学会年会（仙台国際センター）, 2007年05月24日, 日本蛋白質科学会年会

培養ヒト細胞株に対するローヤルゼリー由来ペプチドの抗酸化作用　　　　　　　　　　　　　　　
金久保智史; 上妻由章; 米倉政実
日本農芸化学会大会（東京農業大学）, 2007年03月26日

ダイズ由来グルタミンシクロトランスフェラーゼの遺伝子クローニングと発現　　　　　　　　　　　　　　　
岡野有里; 上妻由章; 米倉政実
日本農芸化学会大会（東京農業大学）, 2007年03月26日

海洋生物由来のレクチンCEL-IIIを発現するトランスジェニックハマダラカのマラリア伝播阻止効果　　　　　　　　　　　　　　　
嶋田 陽平、近藤 大介 、上妻 由章、吉田 栄人
第58回日本衛生動物学会東日本支部大会（自治医科大学）, 2006年10月27日

Expression and X-ray crystallographic analysis of the recombinant hemolytic lectin CEL-III　　　　　　　　　　　　　　　
Haruki Hidemura; Yoshiaki Kouzuma; Tomomitsu Hatakeyama
20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress (Kyoto International Conference Hall), 2006年06月

カルシウム依存性リシン様レクチンCEL-IIIの糖認識部位に関する研究　　　　　　　　　　　　　　　
上妻由章; 大原浩司; 米倉政実
日本蛋白質科学会年会（京都国際会館会議場）, 2006年04月25日

ナマコのレクチンCEL-IIIを導入した遺伝子操作蚊によるマラリア伝搬阻止　　　　　　　　　　　　　　　
嶋田 陽平; 近藤 大介; 上妻 由章; 吉田 栄人
日本衛生動物学会大会（長崎大学）, 2006年04月08日

タバコスズメガ由来の新規シスタチンの分離精製とその特徴　　　　　　　　　　　　　　　
宮地崇之; 上妻由章; Michael Kanost; Kramer Karl J.; 米倉政実
日本農芸化学会大会（京都女子大学）, 2006年03月26日

溶血性レクチンCEL-IIIの組換え体の発現及びその立体構造について　　　　　　　　　　　　　　　
秀村晴樹; 上妻由章; 畠山智充
日本農芸化学会大会（京都女子大学）, 2006年03月26日

シスタチン結晶化における制御要因としての N 末端揺動運動　　　　　　　　　　　　　　　
高橋 大輔; 上妻 由章; 西本 悦子; 山下 昭二
日本生物物理学会（札幌コンベンションセンター）, 2005年11月25日

海洋生物由来のレクチンを発現するトランスジェニックハマダラカのマラリア伝播阻止　　　　　　　　　　　　　　　
嶋田陽平; 近藤大介; 上妻由章; 吉田栄人
日本熱帯医学会大会（京都国際会館会議場）, 2005年10月15日

ローヤルゼリータンパク質由来の抗酸化性ペプチドの抗酸化特性　　　　　　　　　　　　　　　
Guo Hang; 上妻由章; 米倉政実
日本食品科学工学会（北海道大学）, 2005年08月30日

カルシウム依存性を示すリシン様レクチンの糖結合に関する研究　　　　　　　　　　　　　　　
上妻由章; 加藤紀久; 大成駿介; 米倉政実
日本蛋白質科学会年会（福岡国際会議場）, 2005年07月12日

大豆ホエイタンパク質分解物からのアンジオテンシンI変換酵素阻害ペプチドの単離・同定　　　　　　　　　　　　　　　
市村妙子; 山本亜弥子; 上妻由章; 佐本将彦; 釘宮渉; 米倉政実
日本農芸化学会大会（札幌コンベンションセンター）, 2005年03月30日

海産無脊椎動物由来溶血性レクチンCEL-IIIの糖結合に関する研究　　　　　　　　　　　　　　　
上妻由章; 加藤紀久; 大成駿介; 米倉政実
日本農芸化学会大会（札幌コンベンションセンター）, 2005年03月12日

Purification and characterization of a trypsin inhibitor from Apios americana Medikus tubers　　　　　　　　　　　　　　　
Youzuo Zhang; Yoshiaki Kouzuma; Masami Yonekura
日本生化学会大会（パシフィコ横浜）, 2004年10月14日

ローヤルゼリータンパク質由来の抗酸化性ペプチドの単離、構造及び作用解析　　　　　　　　　　　　　　　
Guo Hang; 上妻由章; 米倉政実
日本食品科学工学会（岩手大学）, 2004年09月02日

タバコスズメガ血液由来システインプロテアーゼインヒビターの性質とcDNAクローニング　　　　　　　　　　　　　　　
宮地崇之; 矢口純; 松本りか; 上妻由章; Kramer Karl J.; 米倉政実
日本農芸化学会大会（広島大学）, 2004年03月30日

A comprehensive analysis of wound- and TMV-induced genes in Nicotiana glutinosa leaves using a full-length cDNA microarray　　　　　　　　　　　　　　　
Takeshi Hayashi; Kazumasa Hada; Yusuke Tsutsumi; Yoshiaki Kouzuma; Kousuke Tashiro; Satoru Kuhara; Makoto, Kimura
日本生化学会大会（パシフィコ横浜）, 2003年10月18日

Reconstitution and functional analysis of ribonuclease p of the hyperthermophilic archaeon Pyrococcus horikoshii OT3　　　　　　　　　　　　　　　
Tomoyuki Numata; Kazumi Kimura; Mitsutoshi Watanabe; Yoshiaki Kouzuma; Makoto Kimura
日本生化学会大会（パシフィコ横浜）, 2003年10月18日, パシフィコ横浜

DNAマイクロアレイを用いたタバコ葉傷害及びTMV感染誘導遺伝子の網羅的検索　　　　　　　　　　　　　　　
堤優介、林毅、上妻由章、田代康介、久原哲、木村誠
日本農芸化学会大会（日本大学（神奈川））, 2003年04月02日

超好熱古細菌Pyrococcus horikoshii OT3リボヌクレアーゼP（RNase P）サブユニットタンパク質の活性への寄与　　　　　　　　　　　　　　　
上妻由章、塚本雅代、福原秀雄、木村誠
日本農芸化学会大会（日本大学（神奈川））, 2003年04月01日

The progress of the structural genomics of Pyrococcus horikoshii　　　　　　　　　　　　　　　
N. Sakai; Y. Tajika; Y. Tanaka; A. Okada; I. Ishikawa; T. Kawamura; M. Sokabe; H. Ito; T. Nakashima; H. Hosaka; K. Tsumoto; I. Kumagai; Y. Kouzuma; M. Kimura; M. Yao; N. Watanabe; I. Tanaka
International Conference on Structural Genomics ICSG 2002 (Congress Center of the Max Delbruk Center for Molecular Medicine (Berlin, Germany)), 2002年10月11日

Crystal structure of type 2 ribonuclease H from Pyrococcus horikoshii　　　　　　　　　　　　　　　
Tomonori Hata; Yoshimitsu Kakuta; Yoshiaki Kouzuma; Makoto Kimura
6th International Meeting On Ribonuclease-2002 (University of Bath (Bath, United Kingdom)), 2002年06月20日

Analysis of the interaction of ribosomal proteins with RNA by the surface plasmon resonance method　　　　　　　　　　　　　　　
Kenta Iwasaki; Takeshi Hayashi; Yoshiaki Kouzuma; Isao Tanaka; Makoto Kimura
The Dynamics of Ribosome Structure and Function (Rydges Hotel (Queenstown, New Zealand)), 2002年01月29日

Functional domains of hemolytic lectin CEL-III　　　　　　　　　　　　　　　
Kouzuma; M. Nakano; M. Kimura; N. Yamasaki
Pore-Forming Toxins Meeting (CentroPolifunzionaleOratorio del Duomo (Trento, Italy)), 2000年09月15日

Characterization of two cysteine proteinase inhibitors (Sca and Scb) from sunflower seeds　　　　　　　　　　　　　　　
Kouzuma, Y; Kawano, K; Harada, R; Kariu, T; Kimura, M; Yamasaki, N
The Second Symposium on Frontier of Protein Chemistry and Biotechnology (Yangzhou Univ. (Yangzhou, China)), 1995年08月19日

Characterization of serine proteinase inhibitors from the Erythrina variegata seeds　　　　　　　　　　　　　　　
Kimura, M; Kouzuma, Y; Kuramitu, J; Iwanaga, S; Yamasaki, N
The Second Symposium on Frontier of Protein Chemistry and Biotechnology (Yangzhou Univ. (Yangzhou, China)), 1995年08月19日

2007年06月, 日本食品科学工学会

2002年04月, 日本蛋白質科学会

1997年05月, 日本生化学会

1992年12月, 日本農芸化学会