(Vol.62 No.4 1998)
Separation, Characterization, and Specificity of α-Mannosidases
from Vigna umbellata
Patjaraporn WONGVITHOONYAPORN,1 Christopher BUCKE,2 and Jisnuson SVASTI1,3
613
NADH-Dependent Inhibition of Branched-Chain Fatty Acid
Synthesis in Bacillus subtilis
Hirosuke OKU＊, Keisuke FUJITA, Tomoko NOMOTO, Kiyoshi SUZUKI, Hironori
IWASAKI, and Isao CHINEN 622
Purification and Properties of Inulin Fructotransferase
(DFA III-producing) from Bacillus sp. snu-7
Su-Il KANG, Woo-Pyo KIM, Yung-Jin CHANG, and Su-Il KIM＊ 628
Effects of O-Glycosylation Inhibitors on the Differentiation
of HL-60 Cells
Yoshito SHIBUYA,＊ Tomomi SEKIGUCHI, Kiyofumi SUZUKI, Tetsuo TAKAHASHI,
and Yoshihisa NISHIKAWA 632
Phytotoxins from the Septoria spp. Plant Pathogenic Fungus
on Leafy Spurge
Fumio SUGAWARA,・＊ Jun UZAWA,・ Yasuaki ESUMI,・ Mayuko SUZUKI,・ Shigeo
YOSHIDA,・ Gary STROBEL,§ Jorge L. Ros STEINER・ and Jon CLARDY・＊ 638
Under-flame Reaction of Sulfur-containing Amino Acids by
a Hydrogen-Oxygen Flame
Shinya NOMOTO,1,＊ Akira SHIMOYAMA,1 Susumu SHIRAISHI,2 Tomoyuki SENO,3
and Denzo SAHARA3 643
Isolation and Primary Structure of a Methionine- and Cystine-rich
Seed Protein of Cannabis sativa
Sumiko ODANI＊ and Shoji ODANI＊＊ 650
Optimization for β-Mannanase Production of a Psychrophilic
Bacterium, Flavobacterium sp.
Mia Md. ZAKARIA, Makoto ASHIUCHI,＃ Shinpei YAMAMOTO, and Toshiharu YAGI＊
655
Novel DPPH Radical Scavengers, Bisorbicillinol and Demethyltrichodimerol,
from a Fungus
Naoki ABE, Takashi MURATA, and Akira HIROTA 661
Purification and Partial Characterization of Purine Nucleoside
Phosphorylase from Serratia marcescens
Hye-Seon CHOI 667
Chemical Composition fo the Glue From Appressoria of Magnaporthe
grisea
Yoshinobu EBATA, Hirotaka YAMAMOTO, and Takeo UCHIYAMA・ 672
Effects of Dietary α-Linolenic, Eicosapentaenoic and
Docosahexaenoic Acids on Hepatic Lipogenesis and β-Oxidation in Rats・
Ikuo IKEDA,1 Jae-Young CHA,2 Teruyoshi YANAGITA,2 Noriaki NAKATANI,1 Kazuhiro
OOGAMI,2 Katsumi IMAIZUMI,1 and Kazunaga YAZAWA3 675
Syntheses and Potato Tuber-inducing Activity of Coronafacic
Acid Analogues
Hiroaki TOSHIMA, Shinji NARA, Akitami ICHIHARA, Yasunori KODA,＊ and
Yoshio KIKUTA＊ 681
Syntheses of 3-Oxa-OPC and 2-Fluoro-OPC Homologues
Hiroaki TOSHIMA, Yumiko FUJINO, Akitami ICHIHARA, Yasunori KODA,＊ and
Yoshio KIKUTA＊ 689
Substrate Specificities of α-L-Arabinofuranosidases Produced
by Two Species of Aspergillus niger
Satoshi KANEKO,1,・・ Tadashi ISHII,2 Hideyuki KOBAYASHI,3 and Isao KUSAKABE1,・
695
Crystalline Features of Chitosan-L- and D-Lactic Acid
Salts
Junpei KAWADA, Toshifumi YUI,＊ Yasuo ABE, and Kozo OGAWA・ 700
Purification and Characterization of Sulfur Reductase
from a Moderately Thermophilic Bacterial Strain, TI-1, that Oxidizes Iron
Tsuyoshi SUGIO,1,・ Keiichi ODA,1 Keiko MATSUMOTO,1 Masaki TAKAI,2 Satoshi
WAKASA,2 and Kazuo KAMIMURA1 705
Prevention of Peroxidative Stress in Rats Fed on a Low
Vitamin E-containing Diet by Supplementing with a Fermented Bovine Milk
Whey Preparation: Effect of Lactic Acid and β-Lactoglobulin on the Antiperoxidative
Action
Mohsen ZOMMARA, Hirokazu TOUBO, Masanobu SAKONO,・ and Katsumi IMAIZUMI・・
710
Identification of Bombyx mori Midgut Receptor for Bacillus
thuringiensis Insecticidal CryIA(a) Toxin
Yasunori NAGAMATSU, Satoshi TODA, Fumihide YAMAGUCHI, Masashi OGO, Masato
KOGURE, Masahiro NAKAMURA, Yasuyo SHIBATA, and Tetsuo KATSUMOTO＊ 718
Cloning, Sequencing, and Expression of the Bombyx mori
Receptor for Bacillus thuringiensis Insecticidal CryIA(a) Toxin
Yasunori NAGAMATSU, Satoshi TODA, Takashi KOIKE, Yoko MIYOSHI, Sara SHIGEMATSU,
and Masato KOGURE 727
Trehalose 6-Phosphate Production with Energy Coupling
Fermentation by Yeast Cells
Junko DOI,・ Kumio YOKOIGAWA, Yuka ISOBE, and Hiroyasu KAWAI 735
Potent Antioxidative Isoflavones Isolated from Soybeans
Fermented with Aspergillus saitoi
Hideo ESAKI,・ Hiromichi ONOZAKI, Yasujiro MORIMITSU,＊ Shunro KAWAKISHI,＊
and Toshihiko OSAWA＊ 740
Purification, Characterization, and Gene Analysis of Catechol
2,3-Dioxygenase from the Aniline-Assimilating Bacterium Pseudomonas Species
AW-2
Shuichiro MURAKAMI, Yoko NAKANISHI,＊ Noriko KODAMA,＊ Shinji TAKENAKA,＊
Ryu SHINKE, and Kenji AOKI・ 747
Molecular Cloning and Sequence Analysis of Catalase cDNA
from Green Pepper Seedlings Elicited with Arachidonic Acid
Takashi YAMADA,＊ Hiromasa IMAISHI,＊＊ and Hideo OHKAWA＊,＊＊,・ 753
Purification and Characterization of Triacylglycerol Lipase
from Aspergillus oryzae
Jinichi TOIDA, Yukihiko ARIKAWA, Kimio KONDOU, Mikio FUKUZAWA, and Junichi
SEKIGUCHI＊ 759
Stereoselectivity in the Michael Addition Reaction of
Dialkylcuprates to the 2-Cyclohexenones with C-4 Ester Substituents
Satoshi YAMAUCHI,・ Makiko KADOYA, Masafumi KITAGAWA, and Yoshiro KINOSHITA
764
Rapid Discrimination of Mitochondrial DNA Type and Use
of Results to Study Mitochondrial Inheritance in Pleurotus spp.
Iwao SAGAWA,1 Yuji MORIYAMA,3 Sonoe O. YANAGI,2 Akikazu ANDO,3 and Yoshiho
NAGATA3・ 769
Hypercholesterolemic Effect in Rats of a Dietary Addition
of the Nitric Oxide Synthase Inhibitor, L-NωNitroarginine, by Less Synthesis
of Bile Acids
Abdelkrim KHEDARA, Tsuyoshi GOTO, Jun KAYASHITA,＊ and Norishisa KATO・
773
In Search of Circular Permuted Variants of Escherichia
coli Dihydrofolate Reductase
Masahiro IWAKURA 778
Purification and Characterization of Trehalose Phosphorylase
from Catellatospora ferruginea
Kazuo AISAKA,・ Tomomi MASUDA, Tadashi CHIKAMUNE, and Kazuyo KAMITORI・・
782
Note
Physiological Properties of a Neutralo-sensitive Mutant Derived from Facultative
Alkaliphilic Bacillus sp. C-125
Shinya MOROTOMI,1,2 Makio KITADA,1,・ Ron USAMI,2 Koki HORIKOSHI,2 and
Toshiaki KUDO1 788
Note
A Spin Trap, N-tert-Butyl-α-phenylnitrone Extends the Life Span of Mice
Kieko SAITO,＊,・ Hisashi YOSHIOKA,＊＊ and Richard G. CUTLER＊,・・ 792
Note
Purification and Properties of Acetylacetoin Synthase from Bacillus sp.
YUF-4
Sadaharu UI,・ Takeshi HOSAKA, Kimiya MIZUTANI, and Akio MIMURA 795
Note
Need for Aromatic Residue at Position 115 for Proteolytic Activity Found
by Site-directed Mutagenesis of Tryptophan 115 in Thermolysin
Kuniyo INOUYE,＊・ Nozomi MAZDA,＊＊ and Motoki KUBO＊＊・・ 798
Note
Xylooligosaccharide Production by Aspergillus oryzae I3 Immobilized on
a Nonwoven Fabric
Hiroharu TOKUDA, Keigo SATO, and Kotoyoshi NAKANISHI 801
Note
Okaramine G, a New Okaramine Congener from Penicillium simplicissimum ATCC
90288
Hideo HAYASHI・ and Atsushi SAKAGUCHI 804
Note
Purification of Pomacea canaliculata α-Fucosidase Isoforms with Different
Thermostabilities
Koji HIRATA, Yoichi ASO,・ Shoji YASUDA, and Masatsune ISHIGURO 807
Note
Autoxidation Reaction Mechanism for L-Ascorbic Acid-related Compounds in
Methanol without Metal Ion Catalysis
Noriko MIYAKE and Tadao KURATA 811
Note
Bacterial Reduction of 9-Fluorenone to 9-Hydroxyfluorene
Tomomi INOUE, Tetsuya TASAKA, Reiji MARUYAMA, Yasuteru SUMITA, Shin ONO,
and Masami INOUE・ 814
Note
Theanine-induced Reduction of Brain Serotonin Concentration in Rats
Hidehiko YOKOGOSHI,・ Mikiko MOCHIZUKI, and Kotomi SAITOH 816
Note
Optical Resolution by Preferential Crystallization of (RS)-2-Amino-3-(2-carboxyethylthio)propanoic
Acid
Tadashi SHIRAIWA,・ Motoki KUBO, Mitsuhiro WATANABE, Hidemasa NAKATANI,
Masanori OHKUBO, and Hidemoto KUROKAWA 818
Note
Binding of Paratropomyosin to β-Connectin from Chicken Skeletal Muscle
Minoru YAMANOUE, Sha FEI,＊ and Takahide OKAYAMA 821
Note
Complete Amino Acid Sequence of Chitinase-A from Leaves of Pokeweed (Phytolacca
americana)
Takeshi YAMAGAMI,・ Miho TANIGAWA, Masatsune ISHIGURO, and Gunki FUNATSU
825
Rapid Paper
Structure-related Emission Spectrometric Analysis of the Chemiluminescence
of Catechins, Theaflavins and Anthocyanins
Teruo MIYAZAWA・ and Kiyotaka NAKAGAWA 829
-1-
Separation, Characterization, and Specificity of α-Mannosidases
from Vigna umbellata
Patjaraporn WONGVITHOONYAPORN,1 Christopher BUCKE,2 and Jisnuson SVASTI1,3
1Department of Biochemistry, Faculty of Science, Mahidol University,
Rama VI Road, Bangkok 10400, Thailand
2Division of Biotechnology, School of Biological and Health Sciences, University
of Westminster, 115 New Cavendish Street, London W1M 8JS, UK
3Laboratory of Biochemistry, Chulabhorn Research Institute, Vibhavadee
Rangsit Road, Bangkok 10210, Thailand
Received July 22, 1997
Information about the specificity of glycosidase enzymes is important since
it affects their use for characterization and synthesis of oligosaccharides.
Two α-mannosidases (EC 3.2.1.24), I and II, were isolated from rice beans
(Vigna umbellata). The native molecular weight of both isozymes was estimated
to be 329,000, but pIs of form I were 5.03--5.34 and pIs of form II were
5.46--6.20. The two isozymes were characterized in terms of optimal pH
and temperature, effects of metal ions, inhibition by swainsonine and 1-deoxymannojirimycin,
and kinetic parameters for p-nitrophenyl-α-D-mannopyranoside and Manα(1--2)Man.
Both enzymes were more specific towards Manα(1--2)Man in both hydrolysis
and synthesis, but their hydrolytic specificities towards Manα(1--3)[Manα(1--6)]Man
were different. enzyme specificity; HPLC; mannosidase; oligosaccharide;
synthesis
-2-
NADH-Dependent Inhibition of Branched-Chain Fatty Acid
Synthesis in Bacillus subtilis
Hirosuke OKU＊, Keisuke FUJITA, Tomoko NOMOTO, Kiyoshi SUZUKI, Hironori IWASAKI, and Isao CHINEN
Laboratory of Applied Biochemistry, Faculty of Agriculture, University of The Ryukyus, Nishihara-Cho, Okinawa-Ken 903-01, Japan
Received July 28, 1997
Addition of NADH to crude but not to pure branched-chain α-keto acid
decarboxylase decreased the CO2 production from α-keto-β-methylvalerate
(KMV) suggesting the presence of an NADH dependent inhibitor in the crude
enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor
was purified to homogeneity by a fast protein liquid chromatography system.
The purified inhibitor was identical with leucine dehydrogenase as to
N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed
the oxidative deamination of three branched chain amino acids (BCAAs),
valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore
identified as leucine dehydrogenase. A decreased substrate availability
caused by leucine dehydrogenase thus reasonably accounted for the NADH
dependent inhibition of the decarboxylation. In turn, the observation that
leucine dehydrogenase competes with the decarboxylase for branched-chain
α-keto acid (BCKA) suggested an involvement of this enzyme in the branched
chain fatty acid (BCFA) biosynthesis. This view was supported by the observation
that addition of NAD to crude fatty acid synthetase increased the incorporation
of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate,
cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine
in vitro, suggesting also the involvement of transaminase reaction in BCFA
biosynthesis.
branched-chain fatty acid; biosynthesis; decarboxylase; isoleucine dehydrogenase;
primer
-3-
Purification and Properties of Inulin Fructotransferase
(DFA III-producing) from Bacillus sp. snu-7
Su-Il KANG, Woo-Pyo KIM, Yung-Jin CHANG, and Su-Il KIM＊
Department of Agricultural Chemistry and Research Center for New Bio-Materials in Agriculture, College of Agriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea
Received July 29, 1997
Inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] secreted from
Bacillus sp. snu-7 was purified 60.3-fold with a yield of 11.6％ from a
culture supernatant by ammonium sulfate precipitation, preparative isoelectrofocusing,
anion exchange chromatography and preparative polyacrylamide gel electrophoresis.
The purified enzyme gave a single band on polyacrylamide gel electrophoresis.
The molecular mass of the enzyme was estimated to be 62kDa on SDS-polyacrylamide
gel electrophoresis. The N-ter||minal amino acid sequence was found to
be Ala-Asp-Gly - Gln - Asp - Gly - Ala - Pro - Leu - Asn -
Gln - Val - Asn - Thr -Tyr-Asp. The optimal pH and
temperature for the enzyme reaction were 6.0 and 40°C, respectively. The
enzyme was stable with a pH range of 4.0 to 7.0 and at up to 60°C. As
the production of di-D-fructose 1,2′:2,3′ dianhydride increased in the
course of enzyme reaction, the Km of the purified enzyme was estimated
to be 5.4 mM. One mM each of Cu2＋, Fe2＋ and Hg2＋ inhibited the enzyme
activity strongly. Exhaustive enzymatic digestion of inulin produced 1-kestose,
1-nystose, and 1-F-fructofuranosylnystose as well as di-D-fructose 1,2′:2,3′
dianhydride. inulin; Bacillus sp. snu-7; di-D-fructose 1,2′:2,3′ dianhydride;
IFTase (DFA III-producing).
-4-
Effects of O-Glycosylation Inhibitors on the Differentiation
of HL-60 Cells
Yoshito SHIBUYA,＊ Tomomi SEKIGUCHI, Kiyofumi SUZUKI, Tetsuo TAKAHASHI, and Yoshihisa NISHIKAWA
Laboratory for Glycobiology and Glycotechnology, Department of Industrial Chemistry, School of Engineering, Tokai University, 1117 Kitakaname, Hiratsuka City, Kanagawa 259-12, Japan
Received August 11, 1997
The effects of O-glycosylation inhibitors on the growth and differentiation
of the human acute promyeloblastic leukemia cell line HL-60 were studied
to examine whether the O-glycosylation is needed for HL-60 cells to differentiate
into granulocyte-like cells or monocyte-macrophage-like cells. N-Acetyl-α-D-galactosaminides,
inhibitors of mucin-type oligosaccharide synthesis, and N-acetyl-β-D-galactosaminides
did not affect either growth or differentiation. β-D-Xylosides, the artificial
initiators of glycosaminoglycan synthesis, also were tested. Only 4-methylumbelliferyl-β-D-xyloside
induced HL-60 cells, to differentiate, and they differentiated into granulocyte-like
cells, assessed by reduction of nitroblue tetrazolium, Giemsa staining,
and esterase double-staining. The aglycon portion of 4-methylumbelliferyl-β-D-xyloside,
4-methylumbelliferone, caused the differentiation. Thus we could find a
new drug that induces the differentiation of HL-60 cells.
HL-60 cell; differentiation; glycosaminoglycan; 4-methylumbelliferyl-β-D-xyloside;
glycosylation
-5-
Phytotoxins from the Septoria spp. Plant Pathogenic Fungus
on Leafy Spurge
Fumio SUGAWARA,・＊ Jun UZAWA,・ Yasuaki ESUMI,・ Mayuko SUZUKI,・ Shigeo YOSHIDA,・ Gary STROBEL,§ Jorge L. Ros STEINER・ and Jon CLARDY・＊
・Department of Applied Biological Science, Research Institutes for
Science and Technology, Science University of Tokyo, Noda, Chiba 278-8501,
Japan
・The Institute of Physical and Chemical Research, Wako, Saitama 351, Japan
§Department of Plant Pathology, Montana State University, Bozeman, MT
59717. U.S. A.
・Department of Chemistry-Baker Laboratory, Cornell University, Ithaca,
NY 14853-1301, U. S. A.
Received August 11, 1997
The fungal pathogen Septoria spp., that had been isolated from the infected
leaves of leafy spurge, produced two phytotoxic compounds that were identified
by X-ray diffraction and spectroscopic methods as 1 and 2.
Septoria spp.; phytotoxin; X-ray diffraction; ACTG toxin D
-6-
Under-flame Reaction of Sulfur-containing Amino Acids by
a Hydrogen-Oxygen Flame
Shinya NOMOTO,1,＊ Akira SHIMOYAMA,1 Susumu SHIRAISHI,2 Tomoyuki SENO,3 and Denzo SAHARA3
1Institute of Chemistry, The University of Tsukuba, Tsukuba, Ibaraki
305-8571, Japan
2Matsuyama Shinonome Junior College, Matsuyama, Ehime 790-8531, Japan
3Institute of Agricultural and Forest Engineering, The University of Tsukuba,
Tsukuba, Ibaraki 305-8572, Japan
Received August 12, 1997
Methionine was subjected to a flame-induced reaction in water or in an
aqueous formic acid solution by using a hydrogen (50％)-oxygen (50％),
hydrogen (87％)-oxygen (13％) and hydrogen diffusion flame. Besides the
already-known stepwise oxidation by a hydroxyl radical, the contribution
of a hydrogen atom from the flame to the reaction was recognized when the
hydrogen-rich mixtures were employed. Homoserine was obtained under all
the reaction conditions employed here, and glutamic acid when employing
aqueous formic acid as a solvent. A common intermediate, the 3-carboxy-3-aminopropyl
radical, appeared to exist in the reaction pathway. A coupling reaction
of this radical with a hydrogen atom, hydroxyl radical and hydroxycarbonyl
radical afforded 2-aminobutyric acid, homoserine and glutamic acid, respectively.
Lanthionine and S-methylcysteine underwent the same reactions. Increasing
the hydrogen content of the fuel and adding formic acid to the solvent
resulted in retarding the reaction rate. The latter modification of the
reaction system also brought about greater stability of the reaction products.
＊ To whom correspondence should be addressed.
Abbreviations: homoserine, 4-hydroxy-2-aminobutyric acid; homocysteic
acid, 4-sulfo-2-aminobutyric acid flame-induced reaction; hydroxyl radical;
hydrogen atom; hydroxycarbonyl radical
-7-
Isolation and Primary Structure of a Methionine- and Cystine-rich
Seed Protein of Cannabis sativa
Sumiko ODANI＊ and Shoji ODANI＊＊
＊Department of Home Economics, Faculty of Education, Niigata University,
Ikarashi, Niigata 950-21, Japan
＊＊Department of Biology, Faculty of Science, Niigata University, Ikarashi,
Niigata 950-21, Japan
Received August 18, 1997
A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa)
by buffer extraction, gel filtration, ion-exchange chromatography, and
reversed-phase high-pressure liquid chromatography. The protein did not
inhibit bovine trypsin. It consisted of subunits composed of 27 and 61
residues and was held together by two disulfide bonds. The complete amino
acid sequence was identified by protein analysis, and had 20 mole％ of
amino acids containing sulfur. The protein was most similar to a methionine-rich
protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing
protein of Capparis masaikai. The high methionine content and the absence
of trypsin inhibitory activity suggested that the seed protein can be used
to improve the nutritional quality of plant foodstuffs.
Cannabis sativa; seed protein; amino acid sequence; methionine-rich protein;
protein purification
-8-
Optimization for β-Mannanase Production of a Psychrophilic
Bacterium, Flavobacterium sp.
Mia Md. ZAKARIA, Makoto ASHIUCHI,＃ Shinpei YAMAMOTO, and Toshiharu YAGI＊
Department of Bioresources Science, Faculty of Agriculture, Kochi University,
Nankoku, Kochi 783, Japan
＃Research Institute of Molecular Genetics, Kochi University, Nankoku,
Kochi 783, Japan
Received August 22, 1997
We found that a psychrophilic bacterium, Flavobacterium sp., characterized
in this study, has a β-mannanase (EC 3.2.1.78) activity in the culture
medium. The mannanase activity was the highest in the culture medium, containing
1.0％ (w/v) guar gum (as a carbon source), 0.3％ (NH4)2SO4 (as a nitrogen
source), and 0.06％ (w/v) yeast extract, of five-days cultivation at 4°C.
No mannanase activity was found in the medium containing a monosaccharide
or a disaccharide as a carbon source, although the psychrophile could use
them. The enzyme activity was found only when the bacterium was cultured
in the medium containing a polysaccharide. The enzyme preparation showed
a single activity band on a washed gel of SDS-PAGE. The optimal temperature
for the enzyme activity was 35°C. When the reaction was done at 10°C,
the enzyme showed 25％ of the optimal activity. The β-mannanase preparation
efficiently hydrolyzed guar gum, locust bean gum, and glucomannan as well
as β-mannan.
β-mannanase; psychrophile; Flavobacterium; optimized enzyme production
-9-
Novel DPPH Radical Scavengers, Bisorbicillinol and Demethyltrichodimerol,
from a Fungus
Naoki ABE, Takashi MURATA, and Akira HIROTA
Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422, Japan
Received August 22, 1997
In our screening program for antioxidants with DPPH radical scavenging
activity, we isolated four yellowish compounds from the fermentation broth
of Trichoderma sp. USF-2690 strain isolated from a soil sample: two were
novel compounds designated bisorbicillinol (1) and demethyltrichodimerol
(2), and two were known compounds bisvertinolone (3) and trichodimerol
(4). The structures of 1 and 2 were elucidated by spectroscopic evidence
and chemical modification. Two compounds seemed to be the oxidized dimers
of sorbicillin. In the evaluation of DPPH radical scavenging activity,
bisorbicillinol gave the lowest ED50 value (31.4 μM) among the four compounds,
equal to that of BHT (27.0 μM).
DPPH radical scavenging compound; antioxidant; Trichoderma sp. USF-2690;
bisorbicillinol; demethyltrichodimerol
-10-
Purification and Partial Characterization of Purine Nucleoside
Phosphorylase from Serratia marcescens
Hye-Seon CHOI
Department of Microbiology, University of Ulsan, Ulsan 680--749, Korea
Received August 25, 1997
Purine nucleoside phosphorylase (PNP) was purified to homogeneity. The
molecular weight of the enzyme was 170,000. The enzyme consisted of six
subunits, each with a molecular weight of 27,000. Serratia PNP had ten
times the affinity for adenosine and deoxyadenosine than for inosine and
deoxyinosine in a pattern characteristic of bacterial PNP. 1-Methylinosine
and 1-methylguanosine, which have no affinity for mammalian PNP, bound
Serratia PNP. In terms of kcat/Km, the substrate specificities were in
the descending order of guanosine, inosine, and adenosine. When inosine
or deoxyinosine was used as a variable substrate, a biphasic reciprocal
plot with upward curvature was observed. The values of the Hill coefficient
were 1.2 and 1.1 for inosine and deoxyinosine, respectively. Positive cooperativity
seemed to be involved in the binding of inosine and deoxyinosine to the
enzyme.
bacterial purine nucleoside phosphorylase; Serratia marcescens; substrate
specificity; positive cooperativity
-11-
Chemical Composition fo the Glue From Appressoria of Magnaporthe
grisea
Yoshinobu EBATA, Hirotaka YAMAMOTO, and Takeo UCHIYAMA・
Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-21, Japan
Received September 1, 1997
The chemical composition of the glue substance that attaches the appressoria
of Magnaporthe grisea bar artificial leaf wax was investigated. As a percentage
of fresh weights, the glue was crude lipids 29.0％, proteins 12.2％, sugars
7.6％, water 26.5％ and other substances 24.7％. The major fatty acid components
of the crude lipids were hexadecanoic and octadecanoic acids. Unsaturated
fatty acids and branched fatty acids were also detected as minor components.
The protein was abundant in glycine, glutamic acid, and serine. Glucose,
xylose, and mannose were the major monosaccharides.
appressorium; glue substance; leaf wax; Magnaporthe grisea; Pyricularia
oryzae
-12-
Effects of Dietary α-Linolenic, Eicosapentaenoic and
Docosahexaenoic Acids on Hepatic Lipogenesis and β-Oxidation in Rats・
Ikuo IKEDA,1 Jae-Young CHA,2 Teruyoshi YANAGITA,2 Noriaki NAKATANI,1 Kazuhiro OOGAMI,2 Katsumi IMAIZUMI,1 and Kazunaga YAZAWA3
1Laboratory of Nutrition Chemistry, Faculty of Agriculture, Kyushu University,
Fukuoka 812-8581, Japan
2Department of Applied Biological Sciences, Saga University, Saga 840-8502,
Japan
3Sagami Chemical Research Center, Sagamihara, Kanagawa 229-0012, Japan
Revised September 5, 1997
The effects of dietary α-linolenic, eicosapentaenoic and docosahexaenoic
acids on the enzyme activities related to hepatic lipogenesis and β-oxidation
were compared under constant polyunsaturated/monounsaturated/saturated
fatty acids and n-6/n-3 ratios of dietary fats in rats. Dietary fat containing
linoleic acid as the sole polyunsaturated fatty acid (PUFA) was also given
as a control. The concentration of serum triglyceride and phospholipid
in the three n-3 PUFA groups was lower than in the linoleic acid group.
The hepatic triglyceride concentration was lower and the phospholipid concentration
was higher in the three n-3 PUFA groups than in the linoleic acid group.
Cytosolic fatty acid synthase (FAS) activity was lower in the n-3 PUFA
groups than in the linoleic acid group, the reduction being more predominant
in the eicosapentaenoic acid and docosahexaenoic acid groups than in the
α-linolenic acid group. The cytosolic activities of the NADPH-generating
enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and the malic enzyme,
were lower in the three n-3 PUFA groups. The activity of carnitine palmitoyltransferase
(CPT) in mitochondria was higher only in the eicosapentaenoic acid group
than in the other groups. The activity of Mg2＋-dependent phosphatidate
phosphohydrolase (PAP) in microsomes and cytosol was lower in the eicosapentaenoic
and docosahexaenoic acid groups than in the linoleic acid group, while
there was no effect of dietary fats on the activities of diacylglycerol
acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (G3PAT)
in microsomes. The CTP: phosphocholine cytidylyltransferase (CT) activity
in the homogenate was lower in the n-3 PUFA groups, the reduction being
more prominent in the eicosapentaenoic and docosahexaenoic acid groups
than in the α-linolenic acid group. The choline kinase (CK) activity in
cytosol was lower in the eicosapentaenoic acid group than in the linoleic
acid group. These results showed that dietary α-linolenic, eicosapentaenoic
and docosahexaenoic acids differently influenced hepatic lipogenesis and
the partition of fatty acid into oxidation or glycerolipid synthesis.
docosahexaenoic acid; eicosapentaenoic acid; α-linolenic acid; lipogenesis;
β-oxidation
-13-
Syntheses and Potato Tuber-inducing Activity of Coronafacic
Acid Analogues
Hiroaki TOSHIMA, Shinji NARA, Akitami ICHIHARA, Yasunori KODA,＊ and Yoshio KIKUTA＊
Department of Bioscience and Chemistry, and ＊Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Received September 5, 1997
Coronafacic acid (1) is an acid component of coronatine, and has been isolated
from several pathovars of Pseudomonas syringae. Syntheses of C6-non- and
C6-alkyl-substituted analogues of 1 were accomplished via intramolecular
1,6-conjugate addition as the key step. Among them, 1 and four C6-alkyl-substituted
analogues exhibited potato tuber-inducing activity, but the C6-non-substituted
analogue did not. It was revealed that a certain length of the C6-alkyl
group was necessary to exhibit activity.
coronafacic acid; coronatine; jasmonic acid; 1,6-conjugate addition; tuber-inducing
activity
-14-
Syntheses of 3-Oxa-OPC and 2-Fluoro-OPC Homologues
Hiroaki TOSHIMA, Yumiko FUJINO, Akitami ICHIHARA, Yasunori KODA,＊ and Yoshio KIKUTA＊
Department of Bioscience and Chemistry, ＊Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Received October 17, 1997
Synopsis: 3-Oxa-OPC and 2-fluoro-OPC homologues which do not undergo β-oxidation
were synthesized from esters of odd-numbered OPC homologues by a short-step
procedure. The 3-oxa-OPC homologues were synthesized via etherification
of an alcohol with tert-butyl bromoacetate under phase-transfer conditions.
The 2-fluoro-OPC homologues were synthesized via the addition of the trichloromethyl
anion to an aldehyde and subsequent fluorination.
jasmonic acid; jasmonoid; octadecanoid; OPC; β-oxidation
-15-
Substrate Specificities of α-L-Arabinofuranosidases Produced
by Two Species of Aspergillus niger
Satoshi KANEKO,1,・・ Tadashi ISHII,2 Hideyuki KOBAYASHI,3 and Isao KUSAKABE1,・
1Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoodai,
Tsukuba, Ibaraki 305, Japan
2Forestry and Forest Products Research Institute, P.O. Box 16, Tsukuba
Norin Kenkyu Danchinai, Ibaraki 305, Japan
3National Food Research Institute, Ministry of Agriculture, Forestry, and
Fisheries, 2-1-2 Kannondai, Tsukuba Ibaraki 305, Japan
Received September 8, 1997
Precise substrate specificities of α-L-arabinofuranosidases from Aspergillus
niger 5--16 and Aspergillus niger (Megazyme) were investigated. Both enzymes
hydrolyzed arabinan and debranched-arabinan at almost the same rate. The
α-L-Arabinofuranosidase from A. niger (Megazyme) preferentially released
arabinosyl side-chains of arabinan. The enzyme tore off both arabinoses
attached to||O - α - L - arabinofuranosyl - (1→3) - O -
β - D - xylopyranosyl - (1→||4)-D-xylopyranose and
O-β-D-xylopyranosyl-(1→4)-[O-α-||L - arabinofuranosyl - (1→3)]
- O - β - D - xylopyranosyl - (1→4) -||||D-xylopyranose,
but did not tear off xylosyl-arabinose||||from O-β-D-xylopyranosyl-(1→2)-O-α-L-arabinofuranosyl-||||(1→3)
- O - β - D - xylopyranosyl - (1→4) - O - β -
D - xylopyranosyl -||(1→4)-D-xylopyranose. The enzyme from A.
niger (Megazyme) hydrolyzed methyl 2-O-, methyl 3-O- and methyl 5-O-α-L-arabinofuranosyl-α-L-arabinofuranosides
to arabinose and methyl α-L-arabinofuranoside in the order of (1→5)->(1→2)->(1→3)-linkages.
On the other hand, α-L-arabinofuranosidase from A. niger 5--16 successively
liberated the arabinose of arabinan from non-reducing terminals. The enzyme
hydrolyzed in the order of (1→2)->(1→3)->(1→5)-linkages. Both
of the enzymes hydrolyzed the (1→3)-linkage more than the (1→5)-linkage
of methyl 3,5-di-O-α-L-arabinofuranosyl-α-L-arabinofuranoside. α-L-arabinofuranosidase;
Aspergillus niger; α-L-arabinofuranobiosides; α-L-arabinofuranotrioside.
-16-
Crystalline Features of Chitosan-L- and D-Lactic Acid
Salts
Junpei KAWADA, Toshifumi YUI,＊ Yasuo ABE, and Kozo OGAWA・
Research Institute for Advanced Science and Technology, Osaka Prefecture
University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan
＊Faculty of Engineering, Miyazaki University, Miyazaki 889-2192, Japan
Received September 22, 1997
The crystal structures of chitosan-L- and D-lactate salts were studied
by X-ray diffraction measurements on fiber diagrams. In each lactate, chitosan
took on a different crystalline polymorph depending on the preparation
temperature. At low temperature, they gave a similar fiber pattern to that
of the type II salt which has been found to be one of the two forms of
chitosan acid salts in which the backbone chitosan molecules take on an
eight-fold helix. At high temperature, however, the fiber pattern was that
of the type I salt, another form of chitosan salt in which the backbone
chains apparently retain the 21 symmetry of chitosan itself. The high-temperature
polymorph of the L-lactate was a monoclinic (pseudoorthorhombic) unit cell
whose lattice parameters were a＝10.51, b＝10.85, c(fiber axis)＝10.34
・ and γ＝90°. That of the D-lactate was also a monoclinic cell having
parameters a＝11.20, b＝11.60, c(fiber axis)＝10.38 ・ and γ＝93.0°.
Their unit cell volumes coupled with their observed density values indicate
that two chains of chitosan lactate were accommodated in each unit cell,
that the L-lactate was an anhydrous crystal, but that the D-lactate was
hydrated. The preparation temperature at which the salt changed from type
II to type I was different between the D- and L-lactate, suggesting that
these acids had different affinity to the chitosan molecule. When chitosan
powder was suspended in a racemic lactic acid solution, the resultant solution
always showed a minus sign for the rotation angle, indicating that D-lactic
acid had higher affinity to chitosan than the L-isomer.
X-ray fiber pattern; crystal structure; chitosan-L-lactate; chitosan-D-lactate;
chitosan-lactate conformation
-17-
Purification and Characterization of Sulfur Reductase
from a Moderately Thermophilic Bacterial Strain, TI-1, that Oxidizes Iron
Tsuyoshi SUGIO,1,・ Keiichi ODA,1 Keiko MATSUMOTO,1 Masaki TAKAI,2 Satoshi WAKASA,2 and Kazuo KAMIMURA1
1Division of Biological Function and Genetic Resources Science, Faculty
of Agriculture, Okayama University, 1-1-1, Tsushima Naka, Okayama 700,
Japan
2Miura Institute of Research and Development, 7 Horie-cho Matsuyama-shi,
Ehime 799-26, Japan
Received September 24, 1997
A moderately thermophilic bacterium, strain TI-1, produces H2S outside
of the cells when grown at 45°C on Fe2＋-medium (pH 1.8) containing elemental
sulfur and L-glutamic acid. A newly identified sulfur reductase was present
in the cytosol of this strain and was purified to an electrophoretically
homogeneous state from strain TI--1. The apparent molecular weight of sulfur
reductase was 86,000 by gel filtration and 48,000 by SDS-PAGE, so the enzyme
was a homodimer. The enzyme was most active at pH 9.0 and 60 to 70°C,
and it catalyzed the reduction of 1 mol of elemental sulfur with 1 mol
of NADH to give 1 mol of H2S and 1 mol of NAD＋. Elemental sulfur was a
specific electron acceptor of this enzyme. Thiosulfate, sulfite, and tetrathionate
were not electron acceptors, but inhibited sulfur reductase activity. NADPH
was not used as an electron donor.
sulfur reductase; moderate thermophile; iron-oxidizing bacterium; hydrogen
sulfide
-18-
Prevention of Peroxidative Stress in Rats Fed on a Low
Vitamin E-containing Diet by Supplementing with a Fermented Bovine Milk
Whey Preparation: Effect of Lactic Acid and β-Lactoglobulin on the Antiperoxidative
Action
Mohsen ZOMMARA, Hirokazu TOUBO, Masanobu SAKONO,・ and Katsumi IMAIZUMI・・
Laboratory of Nutrition Chemistry, Department of Food Science and Technology, Faculty of Agriculture (46-09), Kyushu University, Fukuoka 812--8581, Japan
Received September 26, 1997
We examined the antiperoxidative properties of a fermented bovine milk
whey preparation in rats fed on a low vitamin E-containing diet and identified
the active principle in the preparation. An exogenous supply of either
lactic acid or an amino acid mixture simulated the unfermented whey proteins
to prevent red blood cell (RBC) hemolysis and to lower liver thiobarbituric
acid reactive substances (TBARS). The supply of either whey proteins or
β-lactoglobulin resulted in an increase in liver GSH and prevented iron-mediated
lipoprotein peroxidation. These protein effects were reproduced in rats
orally administered with either GSH or its precursor, γ-glutamylcysteine.
The amount of TBARS formed during in vitro lipoprotein peroxidation were
positively correlated with liver TBARS. These results suggest that fermented
milk products containing lactic acid and bovine milk whey proteins can
ameliorate peroxidative stress in tissues subjected to vitamin E deficiency.
fermented milk; γ-glutamylcysteine; lactic acid; β-lactoglobulin; vitamin
E deficiency
-19-
Identification of Bombyx mori Midgut Receptor for Bacillus
thuringiensis Insecticidal CryIA(a) Toxin
Yasunori NAGAMATSU, Satoshi TODA, Fumihide YAMAGUCHI, Masashi OGO, Masato KOGURE, Masahiro NAKAMURA, Yasuyo SHIBATA, and Tetsuo KATSUMOTO＊
Department of Applied Biochemistry, Faculty of Applied Biological Science,
Hiroshima University, Higashi-Hiroshima, Hiroshima 739, Japan
＊Medical School, Tottori University, Yonago, Tottori 683, Japan
Received September 26, 1997
As part of a study of the mechanism by which Bacillus thuringiensis insecticidal
crystal protein acts, a Bombyx mori receptor to the CryIA(a) toxin specific
for lepidopterans was examined. Histological examination showed that the
toxin acted on the brush-border membrane of the midgut columnar cells and
broke its infolding structure, causing cell lysis. The membrane vesicles
were purified, and a 175-kDa protein binding the toxin was found that accounted
for some 0.015％ of membrane proteins. The protein, designated BtR175,
was a glycoprotein that reacted with concanavalin A. Anti-BtR antibodies
inhibited the binding of toxin to membrane vesicles in vitro and decreased
the effect of the toxin to silkworms in vivo. BtR175, although found in
the gut, was not found in fat bodies, integument, or silk glands. These
results indicated that BtR175 was the receptor protein for the insecticidal
toxin. Proteins (137 and 107 kDa) binding the CryIA(a) toxin also were
found in the gut membranes of Tenebrio moritor larvae, a coleopteran not
sensitive to the toxin.
The specificity of the toxin could not be explained only in term of the
existence of its binding protein.
Bacillus thuringiensis; biopesticide; toxin; silkworm; receptor
-20-
Cloning, Sequencing, and Expression of the Bombyx mori
Receptor for Bacillus thuringiensis Insecticidal CryIA(a) Toxin
Yasunori NAGAMATSU, Satoshi TODA, Takashi KOIKE, Yoko MIYOSHI, Sara SHIGEMATSU, and Masato KOGURE
Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739, Japan
Received October 20, 1997
Bacillus thuringiensis strains produce insect-specific Bt toxins. Bt CryIA(a)
toxin binds to a 175-kDa glycoprotein (BtR175) on the microvillus membranes
of columnar cells in the Bombyx mori midgut and causes lysis of the cells.
BtR175 was purified, and its cDNA was cloned. The cDNA encodes a newly
identified 193.3-kDa preproprotein form of BtR175 that includes nine extracellular
cadherin repeats, a 23.5-kDa membrane-proximal domain, a membrane-spanning
region, and a 13.6-kDa cytoplasmic domain. Spodoptera frugiperda cells
transfected with a recombinant baculovirus DNA carrying the cDNA produced
a 175-kDa protein that reacted with anti-BtR antibodies and the Bt CryIA(a)
toxin.
Bacillus thuringiensis; biopesticide; toxin; receptor; cadherin repeat
-21-
Trehalose 6-Phosphate Production with Energy Coupling
Fermentation by Yeast Cells
Junko DOI,・ Kumio YOKOIGAWA, Yuka ISOBE, and Hiroyasu KAWAI
Department of Food Science and Nutrition, Nara Women′s University, Kitauoyanishi-machi, Nara 630, Japan
Received September 29, 1997
We tried a method for the production of trehalose 6-phosphate (T6P) with
energy-coupling fermentation by baker′s yeast. T6P was produced in a reaction
mixture containing glucose, 5′-UMP, MgSO4, inorganic phosphate, and dried
cells of baker′s yeast as the enzyme preparation. T6P was isolated from
the reaction mixture and identified by TLC, HPLC, GC-MS, and enzymatic
methods. The reaction conditions suitable for T6P production were investigated.
The formation of T6P and its precursors, glucose 6-phosphate and UDPglucose,
at various pHs and concentrations of substrates was examined. Accumulation
of T6P was maximum with a reaction mixture containing 1 M glucose, 20 mM
5′-UMP, 20 mM MgSO4, 400 mM sodium phosphate buffer (pH 6.2), and 100
mg/ml dried cells of baker′s yeast shaken at 37°C for 6 h. The yield
of T6P as a percentage of glucose was 11％ (mol/mol) under these reaction
conditions.
trehalose 6-phosphate; energy-coupling fermentation; baker′s yeast
-22-
Potent Antioxidative Isoflavones Isolated from Soybeans
Fermented with Aspergillus saitoi
Hideo ESAKI,・ Hiromichi ONOZAKI, Yasujiro MORIMITSU,＊ Shunro KAWAKISHI,＊ and Toshihiko OSAWA＊
Department of Food and Nutrition, Sugiyama Jogakuen University, 17--3
Hoshigaoka-motomachi, Chikusa-ku Nagoya 464, Japan
＊Department of Applied Biological Sciences, Nagoya University, Nagoya
464-01, Japan
Received September 29, 1997
Two potent antioxidative isoflavones were isolated from soybeans fermented
with Aspergillus saitoi by silica gel column chromatography and preparative
HPLC, using ODS column, or in addition, Toyopearl HW-40 column chromatography.
The purified AS-13 and AS-9B compounds were identified as 8-hydroxydaidzein
(8-OHD) and 8-hydroxygenistein (8-OHG), respectively, by MS, and 1H-NMR,
13C-NMR and HMBC spectra.
These isoflavones, which have an o-dihydroxy structure between the 7- and
8-position, each exhibited significantly stronger antioxidative activity
than daidzein and genistein in both oil and lipid/aqueous systems. Furthermore,
the antioxidative activity and the content of each isoflavone analog in
soybeans with different fermentation periods were investigated. It is suggested
from these results that AS-13 and AS-9B were produced from daidzein and
genistein, respectively, by hydroxylation at the 8-position of each isoflavone
structure. In addition, it is concluded that these isoflavones were also
the principal antioxidants in potent antioxidative soybeans fermented with
A. saitoi.
antioxidant; 8-hydroxydaidzein; 8-hydroxygenistein; fermented soybeans;
Aspergillus saitoi
-23-
Purification, Characterization, and Gene Analysis of Catechol
2,3-Dioxygenase from the Aniline-Assimilating Bacterium Pseudomonas Species
AW-2
Shuichiro MURAKAMI, Yoko NAKANISHI,＊ Noriko KODAMA,＊ Shinji TAKENAKA,＊ Ryu SHINKE, and Kenji AOKI・
Department of Biofunctional Chemistry, Faculty of Agriculture
＊Division of Science of Biological Resources, Graduate School of Science
and Technology, Kobe University, Rokko, Kobe 657-8501, Japan
Received September 29, 1997
Catechol 2,3-dioxygenase (C23D; EC 1.13.1.2) was purified to homogeneity
from a cell extract of Pseudomonas sp. AW-2 grown on aniline, and the purified
C23D was characterized. The molecular mass estimated by gel filtration
was 110 kDa. The enzyme dissociated into four identical subunits each with
the molecular mass of 33 kDa. The enzyme had high activity for 3-methylcatechol
as well as catechol, and differed from the enzyme from Pseudomonas putida
mt-2, which carries the TOL plasmid, in optimal pH for catechol, extradiol
cleavage activities for 3-methylcatechol and 4-methylcatechol, and immunochemical
properties. The amino acid sequence deduced from a C23D gene, alnE, from
Pseudomonas sp. AW-2 was 85.7％ identical to that of 3-methylcatechol 2,3-dioxygenase
from toluidine-assimilating Pseudomonas putida UCC22. AlnE was 44.1％ identical
to the C23D encoded by xylE in P. putida mt-2. Because XylE has low activity
for 3-methylcatechol, these results suggest that the differences in substrate
specificity for 3-methylcatechol among the C23Ds reflected their sequence
similarity.
aniline-assimilating bacterium; catechol 2,3-dioxygenase; substrate specificity;
gene analysis; alnE gene
-24-
Molecular Cloning and Sequence Analysis of Catalase cDNA
from Green Pepper Seedlings Elicited with Arachidonic Acid
Takashi YAMADA,＊ Hiromasa IMAISHI,＊＊ and Hideo OHKAWA＊,＊＊,・
＊The Graduate School of Science and Technology, and ＊＊Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
Received October 1, 1997
We isolated a cDNA encoding catalase from green pepper seedlings elicited
with arachidonic acid, based on the amino acid sequences of the purified
protein. The nucleotide sequence of the isolated cDNA contained a single
open reading frame predicted to encode 492 amino acid residues with a calculated
molecular mass of 56439.0 daltons. The deduced amino acid sequence contained
the amino acid sequences found by sequencing of the peptides. The total
deduced amino acid sequence showed high similarity with those of the other
plant catalases reported so far and was found to possess the peroxisomal
targeting sequence conserved among plant catalases. Transcription of the
catalase gene in green pepper seedlings was found to be induced by treatment
with arachidonic acid.
green pepper; catalase; cDNA cloning; arachidonic acid; elicitation
-25-
Purification and Characterization of Triacylglycerol Lipase
from Aspergillus oryzae
Jinichi TOIDA, Yukihiko ARIKAWA, Kimio KONDOU, Mikio FUKUZAWA, and Junichi SEKIGUCHI＊
Food Technology Research Institute of Nagano Prefecture, 205-1 Nishibanba
Kurita, Nagano-shi, Nagano 380-0921, Japan
＊Department of Applied Biology, Faculty of Textile Science and Technology,
Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano 386-0018, Japan
Received October 2, 1997
Triacylglycerol lipase (L3) was purified from Aspergillus oryzae RIB128
by ammonium sulfate fractionation, acetone precipitation, anion-exchange
chromatography, and gel filtration. The purified enzyme was formed from
a glycoprotein and a monomeric protein with molecular masses of 25 and
29 kDa, by SDS-PAGE and gel filtration, respectively. The optimum pH at
40°C was 5.5 and the optimum temperature at pH 5.5 was 40°C. The enzyme
was stable between a pH range of 4.0--7.5 at 30°C for 24 h, and at up
to 30°C at pH 5.5 for 1 h. Heavy metal ions, detergents, DFP, and DEP
strongly inhibited the enzyme activity. The lipase hydrolyzed not only
triacylglycerols but also monoacylglycerols and diacylglycerols. The enzyme
had higher specificity toward triacylglycerols of middle-chain saturated
fatty acids than short-chain or long-chain fatty acids. The enzyme had
1,3-positional specificity. The N-terminal amino acid sequence of the enzyme
was not significantly similar to that of other lipases with published sequences.
Aspergillus oryzae; triacylglycerol lipase; enzyme characterization
-26-
Stereoselectivity in the Michael Addition Reaction of
Dialkylcuprates to the 2-Cyclohexenones with C-4 Ester Substituents
Satoshi YAMAUCHI,・ Makiko KADOYA, Masafumi KITAGAWA, and Yoshiro KINOSHITA
College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790, Japan
Received October 3, 1997
Stereoselectivity in the Michael addition of (Me2C＝CH)2CuMgBr and (Me2C＝CH)2CuLi
to 3-alkyl/H-4-[(tert-butoxycarbonyl)alkyl]-2-cyclohexenone was studied.
(Me2C＝CH)2CuMgBr showed stereoselectivity in all cases (3-H, Me: anti;
3-n-Bu: syn). This stereoselectivity disappeared in the reaction of (Me2C＝CH)2CuLi
with 4-[(tert-butoxycarbonyl)methyl]-3-butyl-2-cyclohexenone. However,
the stereoselectivity was recovered by elongating the 4-alkyl chain of
2-cyclohexenone to show the same selectivity as that of (Me2C＝CH)2CuMgBr.
Michael addition
-27-
Rapid Discrimination of Mitochondrial DNA Type and Use
of Results to Study Mitochondrial Inheritance in Pleurotus spp.
Iwao SAGAWA,1 Yuji MORIYAMA,3 Sonoe O. YANAGI,2 Akikazu ANDO,3 and Yoshiho NAGATA3・
＾{1}Industrial Research Institute of Chiba Prefecture, Wakaba-ku, Chiba,
Japan
＾{2}National Food Research Institute, Kannondai, Tukuba, Japan
＾{3}Faculty of Horticulture, Chiba University, Matsudo, Japan
Received October 13, 1997
We have reported a simple and rapid method to discriminate species in the
genus Pleurotus by analysis of restriction-fragment-length polymorphism
of whole-cell DNA, and found that several restriction enzymes gave DNA
bands useful in such discrimination, but other enzymes tested did not.
In the present study, we report the reason why there were useful and useless
enzymes; the effective enzymes digested rDNA into small fragments that
did not interfere with the detection of DNA bands useful for discrimination.
The origin of these discriminative DNA bands was found to be mitochondrial
DNA when the banding profiles of whole-cell DNA, mitochondrial DNA, and
nuclear DNA were compared. Consequently, our method could be used for rapid
and simple identification of mitochondrial DNA type in the genus Pleurotus.
The results were used to study mitochondrial inheritance, and we found
that only the nucleus but not the mitochondria migrated during the mating
of Pleurotus cornucopiae with P. citrinopileatus.
mitochondrial DNA type; RFLP; mitochondrial inheritance; Pleurotus spp.
-28-
Hypercholesterolemic Effect in Rats of a Dietary Addition
of the Nitric Oxide Synthase Inhibitor, L-NωNitroarginine, by Less Synthesis
of Bile Acids
Abdelkrim KHEDARA, Tsuyoshi GOTO, Jun KAYASHITA,＊ and Norishisa KATO・
Department of Applied Biochemistry, Faculty of Applied Biological Science,
Hiroshima University, Higashi-Hiroshima 739, Japan
＊Development, Health Care, Kissei Pharmaceutical Company, Matsumoto 399,
Japan
Received October 16, 1997
We have previously reported that feeding rats with a diet containing 0.02％
L-Nωnitroarginine (L-NNA), a specific inhibitor of nitric oxide synthase,
induced hypercholesterolemia. This present study was conducted to examine
the underlying mechanism for hypercholesterolemia in rats. In experiment
1, feeding a diet containing 0.02％ L-NNA for 5 wk elevated the concentration
of serum cholesterol and reduced the excretion of fecal bile acids, but
did not affect the excretion of fecal neutral sterols. Reduced activity
of hepatic cholesterol 7 α-hydroxylase, the rate-limiting enzyme for the
biosynthesis of bile acids from cholesterol, was observed in the rats receiving
L-NNA. In experiment 2, rats were fed for 5 wk on a diet with or without
0.02％ L-NNA that was or was not supplemented with 4％ L-arginine. The
L-NNA treatment elevated the serum concentrations of total cholesterol,
free cholesterol and esterified cholesterol, and reduced the activity of
hepatic cholesterol 7 α-hydroxylase, serum nitrate (a metabolite of NO)
and the ratio of HDL-cholesterol versus serum total cholesterol. These
alterations were suppressed by supplementing the L-NNA-containing diet
with L-arginine. The results suggest that lower NO production by L-NNA
caused hypercholesterolemia by a mechanism involving impaired bile acid
synthesis.
nitric oxide; cholesterol; bile acids; L-arginine; rats
-29-
In Search of Circular Permuted Variants of Escherichia
coli Dihydrofolate Reductase
Masahiro IWAKURA
National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Received October 21, 1997
A circularized form of a Cys-free mutant of Escherichia coli dihydrofolate
reductase (DHFR) was used to search for a proteolytic site that gave new
N- and C-termini on circularized DHFR with enzyme activity. Of the six
site-specific proteolytic enzymes tested, three proteases, Achromobacter
protease I (lysine-specific endopeptidase), asparaginylendopeptidase, and
Staphylococcus aureus V8 protease, cleaved a single site of the circularized
DHFR to form circular permuted variants. Twenty-four possible sites for
cleavage were found formation of eight circular permuted variants was suggested
by results of N-terminal sequence analysis of the linearized proteins isolated
by gel filtration in the presence of 5 M guanidine hydrochloride. Mapping
of the predicted cleavage sites on the DHFR molecule suggested that they
were not all at a specific loop and, therefore, there are many possible
circular permuted variants.
circular permutation; dihydrofolate reductase; Escherichia coli; N-terminal
analysis; partial digestion
-30-
Purification and Characterization of Trehalose Phosphorylase
from Catellatospora ferruginea
Kazuo AISAKA,・ Tomomi MASUDA, Tadashi CHIKAMUNE, and Kazuyo KAMITORI・・
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida-shi, Tokyo 194, Japan
Received November 7, 1997
Trehalose phosphorylase was purified from the cell extracts of Catellatospora
ferruginea. The enzyme had an apparent molecular weight of 400,000 by gel
filtration and 98,000 by SDS-PAGE, suggesting that the enzyme was a tetramer.
The enzyme was specific for trehalose in phosphorolysis and specific for
β-D-glucose 1-phosphate in synthesis. In addition to D-glucose, D-xylose
and D-fucose were also possible sugar acceptors during synthesis. Phosphate
ions were a key to the activity and stability of the enzyme, controlling
the equilibrium of the reversible reaction and the heat stability of the
enzyme. The enzyme was strongly inhibited by p-chloromercuribenzoate and
pyridoxal phosphate. The enzyme was inactivated by heat or by storage frozen
with ammonium chloride and lithium chloride.
trehalose phosphorylase; Catellatospora ferruqinea; disaccharide; phosphorylase
-31-
Physiological Properties of a Neutralo-sensitive Mutant
Derived from Facultative Alkaliphilic Bacillus sp. C-125
Shinya MOROTOMI,1,2 Makio KITADA,1,・ Ron USAMI,2 Koki HORIKOSHI,2 and Toshiaki KUDO1
1Microbiology Laboratory, The Institute of Physical and Chemical Research
(Riken), 2-1, Hirosawa, Wako-shi, Saitama 351-01, Japan
2Bioengineering Laboratory, Department of Applied Chemistry, Toyo University,
Kawagoe, Saitama 350, Japan
Received April 7, 1997
A neutralo-sensitive mutant (M-12) was isolated from the facultative alkaliphilic
Bacillus sp. C-125. This mutant strain was able to grow to the same extent
as did the parent strain above pH 8, but did not grow below pH 7.5. The
same extent of oxygen uptake was shown by the cells of the parent and mutant
strains at pH 10.3. On the other hand, the oxygen uptake rate was about
one-fifth of that of the parent strain at pH 7. NADH-dependent oxygen uptake
by everted vesicles of the mutant was lower than that of the parent strain
at pH 7--7.5, while the rate at pH 8--9 was almost identical in both strains.
The activity at pH 7 of cytochrome c oxidase of right-side-out membrane
vesicles of the mutant strain was lower than that of the parent strain
at pH 7, while both samples had almost the same enzymatic activity at pH
8.5. These results suggest that poor respiratory activities of the mutant
strain at pH 7 are the reason why this mutant strain was unable to grow
at neutral pH.
facultative alkaliphile; neutralo-sensitive mutant; oxygen uptake; cytochrome
c oxidase
-32-
A Spin Trap, N-tert-Butyl-α-phenylnitrone Extends the
Life Span of Mice
Kieko SAITO,＊,・ Hisashi YOSHIOKA,＊＊ and Richard G. CUTLER＊,・・
＊Gerontology Research Center, National Institute on Aging, NIH, 4940
Eastern Avenue Baltimore MD 21224, USA
＊＊Institute for Environmental Sciences, University of Shizuoka, 52-1
Yada Shizuoka 422, Japan
Received August 22, 1997
To characterize the pharmacological effects of N-tert-butyl-α-phenylnirone
(PBN) on life span, we administered PBN in drinking water to 24.5-month-old
mice, and the survivors were counted. Their water consumption and body
weights were measured as biological markers. PBN-treated animals as compared
with control animals had prolonged mean and maximum life spans. Their water
consumption decreased but no significant change was found in their body
weights, indicating that the metabolism was improved. Results showed that
PBN indeed affects physiological functions and extends life span. We propose
that nitric oxide release from PBN may be involved in altering the aging
process.
N-tert-butyl-α-phenylnitrone (PBN); oxidative stress: life span; reactive
oxygen species; nitric oxide
-33-
Purification and Properties of Acetylacetoin Synthase
from Bacillus sp. YUF-4
Sadaharu UI,・ Takeshi HOSAKA, Kimiya MIZUTANI, and Akio MIMURA
Department of Applied Chemistry ＆ Biotechnology, Faculty of Engineering, Yamanashi University, Kofu, Yamanashi 400, Japan
Received August 25, 1997
In Bacillus sp. YUF-4, acetylacetoin synthase was induced by acetoin, while
glucose inhibited the induction. The enzyme was purified 111-fold by 6
purification steps, and a further purification followed, by HPLC using
a TSK gel, Phenyl-5PW RP. The resulting enzyme gave a single band with
a molecular mass of 62 kDa by SDS-PAGE and 220 kDa by gel filtration. Some
enzymic characteristics were studied.
acetylacetoin; acetylacetoin synthase; acetoin; diacetyl; 2,3-butanediol
cycle
-34-
Need for Aromatic Residue at Position 115 for Proteolytic
Activity Found by Site-directed Mutagenesis of Tryptophan 115 in Thermolysin
Kuniyo INOUYE,＊・ Nozomi MAZDA,＊＊ and Motoki KUBO＊＊・・
＊Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
＊＊Department of Chemistry and Biochemistry, Numazu College of Technology,
Numazu, Shizuoka 410-8501, Japan
Received August 27, 1997
In thermolysin, tryptophan 115 seems to be at the S2 subsite. Trp-115 was
replaced with tyrosine, phenylalanine, leucine, and valine during site-directed
mutagenesis in order to evaluate the role of Trp-115 in the proteolytic
activity of thermolysin. The mutant enzymes with Tyr-115 or Phe-115 had
as much proteolytic activity as the wild-type enzyme, but the other two
mutant enzymes had no activity. We found earlier that the substitution
of Trp-115 with alanine, glutamic acid, lysine, and glutamine causes the
enzyme to lose all activity, so an aromatic amino acid at position 115
seems to be essential for thermolysin.
Bacillus stearothermophilus; metalloproteinase; site-directed mutagenesis;
thermolysin; tryptophan
-35-
Xylooligosaccharide Production by Aspergillus oryzae I3
Immobilized on a Nonwoven Fabric
Hiroharu TOKUDA, Keigo SATO, and Kotoyoshi NAKANISHI
Department of Brewing and Fermentation, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156, Japan
Received September 12, 1997
Immobilized mycelia were screened for xylooligosaccharide production from
xylan, and 20 strains of Aspergillus oryzae were selected. For its high
activity and operational stability of xylooligosaccharides formation, immobilized
A. oryzae I3 was selected for further examination. Batch production of
xylooligosaccharides from xylan by the immobilized mycelia was repeated
a total of 4 times.
immobilized mycelia; xylan hydrolysis; operational stability; nonwoven
fabrics; xylooligosaccharide
-36-
Okaramine G, a New Okaramine Congener from Penicillium
simplicissimum ATCC 90288
Hideo HAYASHI・ and Atsushi SAKAGUCHI
Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
Received September 16, 1997
Okaramine G (1), a new okaramine congener, was isolated from Penicillium
simplicissimum ATCC 90288. The structure of 1 was determined by a spectroscopic
investigation. Okaramine G exhibited insecticidal activity against silkworms.
Penicillium simplicissimum; okaramines; insecticidal compound; insecticide
-37-
Purification of Pomacea canaliculata α-Fucosidase Isoforms
with Different Thermostabilities
Koji HIRATA, Yoichi ASO,・ Shoji YASUDA, and Masatsune ISHIGURO
Laboratory of Protein Chemistry and Engineering, Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka 812-8581, Japan
Received September 26, 1997
Two isoforms, F1 (pI 4.7) and F2 (pI 4.9), of α-fucosidase were purified
from the viscera of Pomacea canaliculata. The thermostability of F1 was
higher than that of F2. Both were 260-kDa proteins containing polypeptides
(55 and 52 kDa) and sugar. There were small differences between F1 and
F2 in the optimum conditions for the enzyme reaction and pH stability.
α-fucosidase; apple snail; isoform; Pomacea canaliculata
-38-
Autoxidation Reaction Mechanism for L-Ascorbic Acid-related
Compounds in Methanol without Metal Ion Catalysis
Noriko MIYAKE and Tadao KURATA
Institute of Environmental Science for Human Life, Ochanomizu University, Bunkyo-ku, Tokyo 112, Japan
Received October 13, 1997
The autoxidation mechanism for L-ascorbic acid (ASA)-related compounds
such as D-arabo-ascorbic acid (＝erythorbic acid; ERA) and triose reductone
(TR) in methanol without metal ion catalysis was studied. The oxidation
reaction of these ASA-related compounds seems to proceed via the C(2) oxygen
adduct of ERA (or TR) by a similar reaction mechanism to that of ASA.
L-ascorbic acid; autoxidation; D-erythorbic acid; triose reductone; C(2)
oxygen adduct
-39-
Bacterial Reduction of 9-Fluorenone to 9-Hydroxyfluorene
Tomomi INOUE, Tetsuya TASAKA, Reiji MARUYAMA, Yasuteru SUMITA, Shin ONO, and Masami INOUE・
Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930, Japan
Received October 17, 1997
A wild type of the Gram-positive bacterium, Bacillus brevis, reduced polycyclic
aromatic compounds such as 9-fluorenone to the corresponding alcohol, 9-hydroxyfluorene,
at 30°C in an anaerobic atmosphere in a 97％ yield by extraction with
an organic solvent. The products could be also continuously isolated by
dialysis from a flowing reaction solution. reduction; Bacillus brevis;
9-fluorenone; 9-hydroxyfluorene; dialysis
-40-
Theanine-induced Reduction of Brain Serotonin Concentration
in Rats
Hidehiko YOKOGOSHI,・ Mikiko MOCHIZUKI, and Kotomi SAITOH
Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Received October 17, 1997
Following the administration of theanine, the brain tryptophan content
significantly increased or tended to increase, but the contents of serotonin
and 5-hydroxyindole acetic acid (5HIAA) decreased. The use of inhibitors
of serotonin metabolism enable us to speculate that theanine reduced serotonin
synthesis and also increased serotonin degradation in the brain.
theanine; tryptophan; brain serotonin; neurotransmitter; rat
-41-
Optical Resolution by Preferential Crystallization of
(RS)-2-Amino-3-(2-carboxyethylthio)propanoic Acid
Tadashi SHIRAIWA,・ Motoki KUBO, Mitsuhiro WATANABE, Hidemasa NAKATANI, Masanori OHKUBO, and Hidemoto KUROKAWA
Faculty of Engineering and High Technology Research Center, Kansai University, Yamate-cho, Suita, Osaka 564, Japan
Received October 20, 1997
Electrophilic additions of DL- and L-Cys to propenoic||acid afforded (RS)-
and (R)-2-amino-3-(2-carboxy||||ethylthio)propanoic acids [(RS)- and (R)-ACE],
respec||tively. (RS)-ACE was found to exist as a conglomerate based on
its melting point, solubility, and infrared spectrum. (RS)-ACE was optically
resolved by preferential crystallization to yield (R)- and (S)-ACE. The
obtained (R)- and (S)-ACE were efficiently recrystallized from water, taking
account of the solubility of (RS)-ACE, to give them in optically pure form.
optically active 2-amino-3-(2-carboxyethylthio)propanoic acid; conglomerate;
optical resolution; preferential crystallization
-42-
Binding of Paratropomyosin to β-Connectin from Chicken
Skeletal Muscle
Minoru YAMANOUE, Sha FEI,＊ and Takahide OKAYAMA
Department of Biofunctional Chemistry, Faculty of Agriculture, and ＊Graduate School of Science and Technology, Kobe University, Kobe, Hyogo 657, Japan
Received October 21, 1997
We found that paratropomyosin bound to β-connectin, in examining binding
of paratropomyosin at the junction of A- and I-bands of sarcomeres. The
turbidity of a mixture of β-connectin and paratropomyosin was greater
with more paratropomyosin added, but high concentrations of Ca2＋ suppress
this increase. These results suggest that paratropomyosin is released from
connectin filaments at the A-I junction region by increased concentrations
of calcium ions in postmortem skeletal muscles.
paratropomyosin; β-connectin; rigor-linkage; meat tenderization; postmortem
ageing
-43-
Complete Amino Acid Sequence of Chitinase-A from Leaves
of Pokeweed (Phytolacca americana)
Takeshi YAMAGAMI,・ Miho TANIGAWA, Masatsune ISHIGURO, and Gunki FUNATSU
Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Received November 18, 1997
The complete amino acid sequence of pokeweed leaf chitinase-A was determined.
First all 11 tryptic peptides from the reduced and S-carboxymethylated
form of the enzyme were sequenced. Then the same form of the enzyme was
cleaved with cyanogen bromide, giving three fragments. The fragments were
digested with chymotrypsin or Staphylococcus aureus V8 protease. Last,
the 11 tryptic peptides were put in order. Of seven cysteine residues,
six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and
Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of
208 amino acid residues and had a molecular weight of 22,391. It consisted
of only one polypeptide chain without a chitin-binding domain. The length
of the chain was almost the same as that of the catalytic domains of class
IL chitinases. These findings suggested that this enzyme is a new kind
of class IIL chitinase, although its sequence resembles that of catalytic
domains of class IL chitinases more than that of the class IIL chitinases
reported so far. Discussion on the involvement of specific tryptophan residue
in the active site of PLC-A is also given based on the sequence similarity
with rye seed chitinase-c.
chitinase; amino acid sequence; Phytolacca americana; pokeweed
-44-
Structure-related Emission Spectrometric Analysis of the
Chemiluminescence of Catechins, Theaflavins and Anthocyanins
Teruo MIYAZAWA・ and Kiyotaka NAKAGAWA
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai 981-8555, Japan
Received November 14, 1997
This study for the first time achieved an emission spectrometric analysis
of the chemiluminescence of flavonoids in the presence of hydrogen peroxide,
acetaldehyde and horseradish peroxidase, and revealed that the maximum
emission wavelengths (Emax) strictly differ among catechins (Emax 630 nm),
theaflavins (Emax 690 nm) and anthocyanins (Emax 675 nm) according to their
chemical structures. This technique enabled the direct incorporation of
dietary tea catechin into rat intestinal mucosal cells to be spectrometrically
confirmed.
catechins; theaflavins; anthocyanins; chemiluminescence; emission spectrometry