(Vol.62 No.5 1998)
Increased Cellulose Production from Sucrose with Reduced
Levan Accumulation by an Acetobacter Strain Harboring a Recombinant Plasmid
Naoto TONOUCHI,・ Hideshi YANASE,＊ Yukiko KOJIMA, Takayasu TSUCHIDA,
Fumihiro YOSHINAGA, and Sueharu HORINOUCHI＊＊ 833
Effects of Difructose Anhydride III on Calcium Absorption
in Small and Large Intestines of Rats
Takuya SUZUKI, Hiroshi HARA, Takanori KASAI, and Fusao TOMITA 837
Physiological Role of L-Ascorbic Acid in Rats Exposed to
Cigarette Smoke
Tadao KURATA, Emiko SUZUKI,＊ Mizuki HAYASHI, and Mami KAMINAO 842
Correlation between the Drinkability of Beer and Gastric
Emptying
Yoko NAGAO,1 Hozue KODAMA,1 Toshihiko YONEZAWA,4 Ayako TAGUCHI,2 Seiji
FUJINO,2 Koh NAKAHARA,2 Ken HARUMA,3 and Tohru FUSHIKI1,＊ 846
Isolation, Characterization, and Sugar Chain Structure
of EndoPG Ia, Ib, and Ic from Stereum purpureum
Yuji HASUI, Yohko FUKUI, Junko KIKUCHI, Nozomu KATO, Kazuo MIYAIRI,
and Toshikatsu OKUNO＊ 852
Antifungal Cyclodepsipeptides, W493 A and B, from Fusarium
sp.: Isolation and Structural Determination
Kenichi NIHEI, Hiroshi ITOH, Kimiko HASHIMOTO,＊ Kazuo MIYAIRI, and
Toshikatsu OKUNO・ 858
Antioxidative and Prooxidative Actions of Xylose-Lysine
Maillard Reaction Products
Gow-Chin YEN・ and Mei-Lin LIU 864
Cloning and Characterization of the Azurin iso-1 Gene,
Concerned with the Electron Transport Chain Involved in Methylamine/Methanol
Oxidation in the Obligate Methylotroph Methylomonas sp. Strain J
Katsuhiko TAGUCHI,1,2・ Toshiaki KUDO,2 and Jiro TOBARI1 870
Isolation of Agrobacterium sp. Strain KNK712 That Produces
N-Carbamyl-D-Amino Acid Amidohydrolase, Cloning of the Gene for this Enzyme,
and Properties of the Enzyme
Hirokazu NANBA, Yasuhiro IKENAKA, Yukio YAMADA, Kazuyoshi YAJIMA, Masayuki
TAKANO, and Satomi TAKAHASHI 875
Screening, Characterization, and Cloning of the Gene for
N-Carbamyl-D-Amino acid Amidohydrolase from Thermotolerant Soil Bacteria
Yasuhiro IKENAKA, Hirokazu NANBA, Yukio YAMADA, Kazuyoshi YAJIMA, Masayuki
TAKANO, and Satomi TAKAHASHI 882
Sequence Analysis by Cloning of the Structural Gene of
Gassericin A, a Hydrophobic Bacteriocin Produced by Lactobacillus gasseri
LA39
Yasushi KAWAI, Tadao SAITO, Masakatsu SUZUKI, and Takatoshi ITOH 887
Identification of Deoxy-D-Fructosyl Phosphatidylethanolamine
as a Non-enzymic Glycation Product of Phosphatidylethanolamine and its
Occurrence in Human Blood Plasma and Red Blood Cells
Sittiwat LERTSIRI, Mayumi SHIRAISHI, and Teruo MIYAZAWA・ 893
Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA
Carboxyltransferase in Culture of Streptomyces fradiae
Du Bok CHOI,＊ Yongsoo PARK,＊＊ and Mitsuyasu OKABE＊＊,・ 902
Cysteine Protease Inhibitors Produced by the Industrial
Koji Mold, Aspergillus oryzae O-1018
Takao YAMADA,・ Jun HIRATAKE,＊ Mototsune AIKAWA, Tetsuyoshi SUIZU,
Yoshiyuki SAITO, Akitsugu KAWATO, Koji SUGINAMI, and Jun′ichi ODA＊ 907
Synthesis of a Lectin in Both Mycelia and Fruit Bodies
of the Ascomycete Mushroom Aleuria aurantia
Shigeru OGAWA, Eijiro NAKAJIMA, Hideyuki NAGAO, Masanari OHTOSHI, Akikazu
ANDO, and Yoshiho NAGATA・ 915
Liberation of Phospholipids from Z-Disks of Chicken Skeletal
Muscle Myofibrils by 0.1 mM Calcium Ions: Weakening Mechanism for Z-Disks
during Post-mortem Aging of Meat
Ken-ichiro SHIMADA, Dong Hyun AHN, and Koui TAKAHASHI・ 919
Deterioration of Connectin/Titin and Nebulin Filaments
by an Excess of Protease Inhibitors
Ryuichi TATSUMI, Akihito HATTORI, and Koui TAKAHASHI・ 927
Improvement of the Off-flavor of Soy Protein Isolate by
Removing Oil-body Associated Proteins and Polar Lipids
Masahiko SAMOTO,・ Chiaki MIYAZAKI, Jiroh KANAMORI, Takeshi AKASAKA,
and Yukio KAWAMURA＊,・ 935
Inhibitory Effects of Lipid Oxidation on the Activity
of Plasma Lecithin-Cholesterol Acyltransferase
Shin KAMIYAMA,・ Tokuhisa YAMATO, and Yuji FURUKAWA 941
Complexes of Serine Acetyltransferase and Isozymes of
Cysteine Synthase in Spinach Leaves
Xia ZHU,1 Takayuki YAMAGUCHI,1,2 and Masahiro MASADA1,・ 947
Direct Formation of Human Interleukin-11 by Cis-Acting
System of Plant Virus Protease in Escherichia coli
Tohru TAKAHASHI, Michiko NAKANISHI, Yoshio YAO, Ichiro UYEDA,1 and
Nobufusa SERIZAWA・ 953
Chemical Modifications of Momordin-a and Luffin-a, Ribosome-Inactivating
Proteins from the Seeds of Momordica charantia and Luffa cylindrica: Involvement
of His140, Tyr165, and Lys231 in the Protein-Synthesis Inhibitory Activity
Yuji MINAMI,・ M. Rafiqul ISLAM, and Gunki FUNATSU 959
Low Sucrase Activity in the Small Intestine of a Senescence-accelerated
Strain of Mouse, SAMP1
Makoto TAKENOSHITA, Mitsuharu YABUNE, Hiromi KATSURA, Ryoichi YAMAJI,
Hiroshi INUI, Kazutaka MIYATAKE, and Yoshihisa NAKANO 965
Antimutagenicity of Flavones and Flavonols to Heterocyclic
Amines by Specific and Strong Inhibition of the Cytochrome P450 1A Family
Kazuki KANAZAWA,・ Takatoshi YAMASHITA, Hitoshi ASHIDA, and Gen-ichi
DANNO 970
Rice Bifunctional α-Amylase/Subtilisin Inhibitor: Characterization,
Localization, and Changes in Developing and Germinating Seeds
Hiroshi YAMAGATA・, Kiyoshi KUNIMATSU, Hiroshi KAMASAKA, Takeshi KURAMOTO,
and Teruo IWASAKI＊ 978
Salicylic Acid Induces a Cytosolic Ca2＋ Elevation in
Yeast Izumi C. MORI,a Hidetoshi IIDA,b Frederick I. TSUJI,c Minoru
ISOBE,a
Nobuyuki UOZUMI,a,d and Shoshi MUTOa,d,・ 986
Note
Reduction of Stale Aldehyde of Beer with Membrane Fraction of Acetic Acid
Bacteria
Yukihiro NOMURA, Masanori YAMAMOTO, and Hidehiko KUMAGAI・ 990
Note
Effect of Magnetite on the Hematocrit Value in Exsanguinated Rats
Toru HAMAYA, Atsutane OHTA, Toshiaki KONO, Rikizo AONO,＊ and Koki
HORIKOSHI＊＊ 993
Note
Benzaldehyde O-Alkyloximes as New Plant Growth Regulators
Hiromichi YOSHIKAWA and Keiko DOI 996
Note
4-Hydroxy-3-nitrophenylacetic and Sinapic Acids as Antibacterial Compounds
from Mustard Seeds
Shoko TESAKI, Soichi TANABE, Haruhiro ONO,＊ Eri FUKUSHI,＊＊ Jun KAWABATA,＊＊
and Michiko WATANABE・998
Note
Effectiveness of Polyclonal and Monoclonal Antibodies Prepared for an Immunoassay
of the Etofenprox Insecticide
Shiro MIYAKE, Akiko HAYASHI,＊,・ Takako KUMETA,＊＊ Koji KITAJIMA,＊＊
Hiroshi KITA,・・ and Hideo OHKAWA＊,・・・1001
Note
Affinity of Antioxidative Polyphenols for Lipid Bilayers Evaluated with
a Liposome System
Tsutomu NAKAYAMA,＊ Koji ONO, and Kei HASHIMOTO＊＊ 1005
Note
Effects of Vitamin B6 Deficiency on Cytokine Levels and Lymphocytes in
Mice・
Shoko DOKE,＊,＊＊＊ Naoki INAGAKI,＊＊ Takashi HAYAKAWA,＊＊＊ and
Haruhito TSUGE＊＊＊,・・ 1008
Note
Manauealide C and Anhydrodebromoaplysiatoxin, Toxic Constituents of the
Hawaiian Red Alga, Gracilaria coronopifolia
Hiroshi NAGAI,・ Yukiko KAN, Tsuyoshi FUJITA, Bryan SAKAMOTO,＊ and
Yoshitsugi HOKAMA＊ 1011
Note
Antimicrobial Activity of a Compound Isolated from an Oil-Macerated Garlic
Extract
Hisae YOSHIDA, Nami IWATA, Hirotaka KATSUZAKI,＊ Rie NAGANAWA, Keiko
ISHIKAWA, Hiroyuki FUKUDA, Tsuchiyoshi FUJINO,・ and Atsushi SUZUKI 1014
Note
Molecular Cloning and Expression Patterns of Cu/Zn-Superoxide Dismutases
in Developing Soybean Seeds
Masaomi ARAHIRA, Van Hai NONG,・ Kazunari KADOKURA, Keitarou KIMURA,
Kyoko UDAKA,・・ and Chikafusa FUKAZAWA・・・ 1018
Note
Nisin Z Production by Lactococcus lactis IO-1 Using Xylose as a Carbon
Source
Noppawan CHINACHOTI, Hiromi MATSUSAKI,・・ Kenji SONOMOTO, and Ayaaki
ISHIZAKI・ 1022
Note
Insecticidal and Antifungal Activities of Aminorhodanine Derivatives
Yoshihiko INAMORI,・ Yukiko OKAMOTO, Yoko TAKEGAWA, Hiroshi TSUJIBO,
Yoshikazu SAKAGAMI,＊ Yuko KUMEDA,＊ Mitsunobu SHIBATA,＊＊ and Atsushi
NUMATA 1025
Note
Thermostable Neutral Protease Resembling Thermolysin Derived from Bacillus
brevis MIB001
Yukio TAKII,1,2 Yoshimi URATA,1 and Noriko UENO11028
Note
Interaction between the Carboxyl-terminal Heparin-binding Domain of Fibronectin
and (－)-Epigallocatechin Gallate
Masaki SAZUKA, Mamoru ISEMURA, and Satoko ISEMURA＊ 1031
Short Communication
Features of Tri101, the Trichothecene 3-O-Acetyltransferase Gene, Related
to the Self-defense Mechanism in Fusarium graminearum
Makoto KIMURA,＊ Yoshinori SHINGU, Katsuyoshi YONEYAMA,・ and Isamu
YAMAGUCHI 1033
Short Cmmunication
cDNA Cloning, Expression, and Characterization of the Human Bifunctional
ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase Enzyme
Ken YANAGISAWA1, Yoichi SAKAKIBARA1, Masahito SUIKO1, Yasunari TAKAMI2,
Tatsuo NAKAYAMA2, Hiroshi NAKAJIMA3, Katsuhiko TAKAYANAGI3, Yasuhiro NATORI4,
and Ming-Cheh LIU1,・ 1037
-1-
Increased Cellulose Production from Sucrose with Reduced
Levan Accumulation by an Acetobacter Strain Harboring a Recombinant Plasmid
Naoto TONOUCHI,・ Hideshi YANASE,＊ Yukiko KOJIMA, Takayasu TSUCHIDA, Fumihiro YOSHINAGA, and Sueharu HORINOUCHI＊＊
Bio-Polymer Research Co., Ltd., 3-2-1 Sakato, Takatsu-ku, Kawasaki,
Kanagawa 213-0012, Japan
＊Department of Biotechnology, Faculty of Engineering, Tottori University,
Tottori 680-0945, Japan
＊＊Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan
Received August 8, 1997
Cellulose production from sucrose by Acetobacter strains is accompanied
by the accumulation of a water-soluble polysaccharide, called levan. To
improve cellulose productivity, a levansucrase-deficient mutant, LD-2,
was derived from Acetobacter strain 757 and used as a host for the construction
of recombinant strains. An LD-2 mutant harboring a plasmid containing the
sucrase gene, sucZE3, from Zymomonas mobilis together with zliS, a gene
that encodes a secretion-activating factor under the control of the Escherichia
coli lac promoter, had sucrase activity and produced much cellulose and
little levan in a medium containing sucrose. In addition, a mutant levansucrase
gene, mutant sacB, from Bacillus subtilis, which encodes a protein with
little levan-forming activity, was generated by site-directed mutagenesis
and introduced into the LD-2 mutant. This introduction also resulted in
the higher cellulose productivity and little levan.
Acetobacter; cellulose; sucrose; levan; levansucrase
-2-
Effects of Difructose Anhydride III on Calcium Absorption
in Small and Large Intestines of Rats
Takuya SUZUKI, Hiroshi HARA, Takanori KASAI, and Fusao TOMITA
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060--8589, Japan
Received September 1, 1997
Difructose anhydride III (DFA III; di-D-fructo-furanose 1,2′:2,3′ dianhydride)
was prepared from inulin with Arthrobacter sp. H65-7 inulin fructotransferase
(depolymerizing) (inulase II; EC 2.4.1.93). DFA III is not hydrolyzed by
enzymes in the small intestine, but is metabolized by microorganisms in
the large intestine. We investigated the effects of DFA III on calcium
absorption in two experiments. In the in vivo experiment, we examined the
effects of DFA III, fructooligosaccharides, and raffinose on calcium absorption
in male Sprague-Dawley rats 5 weeks old at start of the experiment and
given feed containing 3％ of one of these oligosaccharides for two weeks.
The apparent calcium absorption was significantly higher in rats fed any
of these oligosaccharides than in control rats, and the increase with DFA
III was the greatest. Absorption in both the small and large intestines
was affected. In rats fed DFA III, the cecal wall thickened and soluble
calcium and the amounts of some organic acids were higher than in the control
groups. In an in vitro experiment with everted jejunal and ileal sacs of
rats, calcium absorption was higher when DFA III was present in the mucosal
fluid at all concentrations tested (up to 200 mM). In the jejunal sacs,
the increase in calcium absorption depended on the DFA III concentration.
In the ileal sacs, the absorption was maximum at 50 mM DFA III and did
not increase further at higher concentrations. These results indicate that
intact DFA III stimulates calcium absorption in the small intestine, and
that cecal fermentation of DFA III may contribute to the increase in calcium
absorption by the large intestine.
calcium; difructose anhydride III; oligosaccharide; rats; rat intestine
-3-
Physiological Role of L-Ascorbic Acid in Rats Exposed to
Cigarette Smoke
Tadao KURATA, Emiko SUZUKI,＊ Mizuki HAYASHI, and Mami KAMINAO
Institute of Environmental Science for Human Life, and ＊Department of Human Biological Studies, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
Received September 8, 1997
This study clarifies the effect of exposure to cigarette smoke on L-ascorbic
acid (AsA) metabolism and on the activities of drug-metabolizing enzymes.
Male Wistar rats were used. The test rats (group T) were exposed to sidestream
smoke from cigarette for 2 h every day for 25 days. During the experimental
period, the excreted amount of AsA in the urine from group T was higher
than that from the control group (group C). At the end of the experimental
period, the AsA content of the plasma and tissues, the liver cytochrome
P-450 content and the activities of drug-metabolizing enzymes in group
T were each higher than those in group C.
L-ascorbic acid; cigarette smoke; cytochrome P-450; drug-metabolizing enzyme;
rat
-4-
Correlation between the Drinkability of Beer and Gastric
Emptying
Yoko NAGAO,1 Hozue KODAMA,1 Toshihiko YONEZAWA,4 Ayako TAGUCHI,2 Seiji FUJINO,2 Koh NAKAHARA,2 Ken HARUMA,3 and Tohru FUSHIKI1,＊
＾{1}Laboratory of Nutrition Chemistry, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
＾{2}Research Laboratory for New Product, Kirin Brewery Co. Ltd., 1-17-1
Namamugi, Tsurumi-ku, Yokohama 230-0052, Japan
＾{3}First Department of Internal Medicine, Hiroshima University School
of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-0037, Japan
＾{4}Kirin-Seagram Ltd., 970 Shibanuta, Gotemba 412-0003, Japan
Received September 11, 1997
＊ To whom correspondence should be addressed: Tohru FUSHIKI, Ph.D., Laboratory
of Nutrition Chemistry, Division of Applied Life Sciences, Graduate School
of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
A subjective evaluation of beer drinkability and the degree of stomach
fullness were found to correlate with the relaxed cross-sectional area
of the pylorus antrum measured by real-time ultrasonography. Five kinds
of beer with a different malt/adjuncts ratio and degree of attenuation
were used. Each beer was given to 9 healthy volunteers at the rate of 3
ml/kg/15 min, and they each recorded the degree of stomach fullness, desire
to drink and tastiness every 30 min. With increasing volume drunk, the
degree of tastiness and desire to drink were lowered, and the degree of
stomach fullness raised. The relaxed cross-sectional area of the pylorus
antrum measured by an ultrasonic image analyzer every 30 min was highly
correlated with the degree of stomach fullness, tastiness of the beer,
and desire to drink (p＝0.0021, <0.0001, 0.001). The beer giving the
lowest degree of stomach fullness was appraised to be tasty and highly
drinkable. These findings suggest that the rate of gastric emptying is
one of the factors determining the drinkability of beer, and that measurement
of the relaxed cross-sectional area of the pylorus antrum is useful to
evaluate stomach fullness during beer drinking.
beer drinkability; gastric emptying; ultrasonography; pylorus antrum cross
section; subjective evaluation
-5-
Isolation, Characterization, and Sugar Chain Structure
of EndoPG Ia, Ib, and Ic from Stereum purpureum
Yuji HASUI, Yohko FUKUI, Junko KIKUCHI, Nozomu KATO, Kazuo MIYAIRI, and Toshikatsu OKUNO＊
Department of Bioresources Science, Faculty of Agriculture, Hirosaki University, Hirosaki, Aomori 036--8561, Japan
Received September 11, 1997
Three endopolygalacturonases (endoPG Ia, Ib, and Ic) were isolated from
the culture filtrate of Stereum purpureum, the causative fungus of apple
silver-leaf disease. Their properties, including specific activities, optimum
pHs, thermal stabilities, and kinetic parameters (Km and Vmax) were compared.
Their properties were very similar to one another except for the substrate
specificity and relative molecular mass. The sugar chains of endoPG Is
were released by hydrazinolysis, and one major sugar chain common to endoPG
Is was isolated. The pyridylamino sugar was characterized by a two-dimensional
mapping method using HPLC, and identified as a high mannose type N-linked
sugar chain, Manα1--6(Manα1--3)Manα1--6(Manα1--3) Manβ1--4GlcNAcβ1--4
GlcNAc (designated as M5.1). Observation of the course of Western blot
analysis for the proteins from the culture filtrate with endoPG I antibodies
showed that the fungus secreted three endoPG Is into the culture broth
during the growing period.
endopolygalacturonase Ia; endopolygalacturonase Ib; endopolygalacturonase
Ic; N-linked sugar chain; Stereum purpureum
-6-
Antifungal Cyclodepsipeptides, W493 A and B, from Fusarium
sp.: Isolation and Structural Determination
Kenichi NIHEI, Hiroshi ITOH, Kimiko HASHIMOTO,＊ Kazuo MIYAIRI, and Toshikatsu OKUNO・
Department of Biochemistry and Biotechnology, Faculty of Agriculture
and Life Science, Hirosaki University, Hirosaki 036-8561, Japan
＊Department of Applied Chemistry, Faculty of Science and Technology, Keio
University, Yokohama 8522, Japan
Received December 5, 1997
W493 A and B, which showed strong antifungal activity, were isolated from
a culture broth of Fusarium sp. The structure of W493 B was determined
to be that of a cyclodepsipeptide, cyclo(3S,4R-HMTA-D-allo-Thr-L-Ala-D-Ala-L-Gln-D-Tyr-L-Ile)
(1) by MS and NMR data, an amino acid analysis, and synthesis of the component.
HMTA represents 3-hydroxy-4-methyltetradecanoic acid. W493 A (2) had a
similar structure, except that L-Ile in 1 was replaced by L-Val. The absolute
configuration of each amino acid was determined by chiral HPLC, and the
sequence of the components was determined by HMBC experiments. The sequence
of the two alanines was determined to be a L-Ala-D-Ala by a chiral HPLC
analysis of the peptide fragment containing only one Ala residue. The absolute
configuration of HMTA obtained from the hydrolysis of W493 B was determined
to be 3S and 4R by comparing with four isomers prepared by enantioselective
synthesis via Sharpless asymmetric epoxidation.
cycloheptadepsipeptide; antifungal substance; Fusarium sp.; W493 A; W493
B
-7-
Antioxidative and Prooxidative Actions of Xylose-Lysine
Maillard Reaction Products
Gow-Chin YEN・ and Mei-Lin LIU
Departmant of Food Science, National Chung Hsing University, 250 Kuokuang Road, Taichung 402, Taiwan, Republic of China
Received September 12, 1997
Maillard reaction products were prepared by heating xylose and lysine at
pH 9.0 and 100°C for 3 h, and then fractionated by ethyl ether and ethanol
into acidic, neutral and basic low-molecular-weight, ethanol-soluble and
ethanol-insoluble fractions. The ethanol-soluble and -insoluble fractions
were the major fractions of the xylose-lysine Maillard reaction products
(XL MRPs), contributing 79.5％ and 20.1％, respectively. XL MRPs revealed
an inhibitory effect on linoleic acid peroxidation induced by the Fenton
reaction, but did not inhibit liposome peroxidation induced by Fe2＋, where
it had a prooxidative action. XL MRPs caused oxidative damage to deoxyribose
and 2′-deoxyguanosine (2′-dG) induced by the Fenton reaction. The ethanol-soluble
and -insoluble fractions also caused oxidative damages while the low-molecular-weight
fractions displayed an antioxidative effect in inhibiting the oxidative
damage to deoxyribose that was induced by the Fenton reaction. The prooxidative
action of the ethanol-soluble and -insoluble fractions resembled that of
the untractionated products in the 2′-dG assay. In these systems with
deoxyribose, 2′-dG, linoleic acid and liposomes, XL MRPs exhibited either
antioxidative or prooxidative properties, which might have been due to
competition between their reducing power and scavenging activity toward
the hydroxyl radical. However, the low-molecular-weight fractions did not
show any prooxidative activity in these oxidation systems.
Maillard reaction products; xylose; lysine; antioxidant; prooxidant
-8-
Cloning and Characterization of the Azurin iso-1 Gene,
Concerned with the Electron Transport Chain Involved in Methylamine/Methanol
Oxidation in the Obligate Methylotroph Methylomonas sp. Strain J
Katsuhiko TAGUCHI,1,2・ Toshiaki KUDO,2 and Jiro TOBARI1
1Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul′s)
University, Nishi-Ikebukuro, Toshima-ku, Tokyo 171-0021, Japan
2Laboratory of Microbiology, The Institute of Physical and Chemical Research
(RIKEN) 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Recived September 12, 1997
Two azurin-type blue copper proteins, which is concerned with the electron
transport chain involved in methylamine/methanol oxidation, have been found
in the obligate methylotroph Methylomonas sp. strain J. The azurin iso-1
gene was cloned and sequenced to analyze the role in the electron transport
chain. PCR products synthesized with primers based on the N- and C-terminal
amino acid sequences of azurin iso-1 were used as probes for cloning. One
complete open reading frame (the azurin iso-1 gene) and one partial orf
(orf1) were found in a cloned Eco105I-HindIII fragment, pMAZ3, with a total
of 1066 bp. The gene encoded 148 amino acid residues. The amino acid sequence
after Ala-21, deduced from the nucleotide sequence, was identical to that
of the azurin iso-1 protein. The gene was in a region separate from the
mau gene cluster in the chromosome. Escherichia coli expressed azurin iso-1.
The results of northern blotting analysis suggested that expression of
the azurin iso-1 gene is regulated by a complex regulatory network controlling
oxidation of methylamine or methanol in this strain; for example, copper
ions affected the expression of the azurin iso-1 gene.
obligate methylotroph; methylamine dehydrogenase; blue copper protein;
azurin
-9-
Isolation of Agrobacterium sp. Strain KNK712 That Produces
N-Carbamyl-D-Amino Acid Amidohydrolase, Cloning of the Gene for this Enzyme,
and Properties of the Enzyme
Hirokazu NANBA, Yasuhiro IKENAKA, Yukio YAMADA, Kazuyoshi YAJIMA, Masayuki TAKANO, and Satomi TAKAHASHI
Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
Received September 16, 1997
Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino acid
amidohydrolase (DCase) was isolated from soil. The bacterium had D-specific
hydantoinase activity also. Both enzymes are suitable for use in the production
of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was
cloned into Escherichia coli. The cloned DNA fragment contained one open
reading frame, predicted to encode a peptide of 304 amino acids, with a
calculated molecular weight of 34,285. The DCase gene was overexpressed
under the control of the lac promoter, and DCase accounted for 50％ of
the soluble protein in the cells. The enzyme was purified and some properties
were investigated. Both the optimum pH and the pH that gave greatest stability
were about pH 7.0. The optimum temperature was 65°C, and the enzyme was
stable at 55°C. The enzyme had strict specificity toward N-carbamyl-D-amino
acids, and was inhibited by thiol reagents, Cu2＋, Hg2＋, Ag＋, and ammonia.
N-carbamyl-D-amino acid amidohydrolase; D-amino acid
-10-
Screening, Characterization, and Cloning of the Gene for
N-Carbamyl-D-Amino acid Amidohydrolase from Thermotolerant Soil Bacteria
Yasuhiro IKENAKA, Hirokazu NANBA, Yukio YAMADA, Kazuyoshi YAJIMA, Masayuki TAKANO, and Satomi TAKAHASHI
Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
Received September 16, 1997
For the production of D-amino acids, thermotolerant bacteria producing
N-carbamyl-D-amino acid amidohydrolase were isolated from soil by enrichment
culture at 45°C with N-carbamyl-D-amino acids as the sole nitrogen source.
The enzyme activities and substrate specificities of these strains were
examined by the resting cells reaction. One of the enzymes, produced by
Pseudomonas sp. strain KNK003A, was purified and characterized, and the
amino acids of its N-terminal region were sequenced. A DNA fragment containing
the gene for a thermostable N-carbamyl-D-amino acid amidohydrolase was
then cloned into Escherichia coli. The gene encoded a peptide of 312 amino
acids, with a calculated molecular weight of 35,000. The similarity of
the deduced amino acid sequences of this enzyme and a related enzyme from
a mesophile, Agrobacterium sp. strain KNK712, was 60％. A database was
searched for similar sequences.
N-carbamyl-D-amino acid amidohydrolase; thermotolerant enzyme; D-amino
acid
-11-
Sequence Analysis by Cloning of the Structural Gene of
Gassericin A, a Hydrophobic Bacteriocin Produced by Lactobacillus gasseri
LA39
Yasushi KAWAI, Tadao SAITO, Masakatsu SUZUKI, and Takatoshi ITOH
Laboratory of Animal Products Chemistry, Biological Resource Science, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Received September 17, 1997
Gassericin A, a bacteriocin from Lactobacillus gasseri LA39, was purified
to homogeneity from the culturesupernatant mainly by reverse-phase chromatography.
The molecular weight of gassericin A was found to be 5,652 by mass analysis,
unlike the estimated 3,800 found by SDS-PAGE. However, when the purified
preparation was treated with lysylendopeptidase, it migrated as a single
band to 5,600 with bacteriocin activity on SDS-PAGE. N- and C-terminal
amino acids could not be identified. The internal amino acid could be identified
after gassericin A was hydrolyzed with lysylendopeptidase. The DNA of the
structural gene of gassericin A was sequenced by cloning of the gene from
chromosomal DNA with an oligonucleotide probe. The structural gene of gassericin
A was found on the chromosomal DNA as an open reading frame encoding a
protein composed of 91 amino acids. The amino acid sequence of mature gassericin
A was predicted to be 58 residues from the DNA sequence and results of
mass analysis. These results suggested that gassericin A has a closed circular
structure with N- and C-terminals linked. Gassericin A is a hydrophobic
class II bacteriocin; it was 98％ identical with acidocin B produced by
Lactobacillus acidophilus M46. bacteriocin; Lactobacillus gasseri; gassericin
A; acidocin B; lactic acid bacteria
-12-
Identification of Deoxy-D-Fructosyl Phosphatidylethanolamine
as a Non-enzymic Glycation Product of Phosphatidylethanolamine and its
Occurrence in Human Blood Plasma and Red Blood Cells
Sittiwat LERTSIRI, Mayumi SHIRAISHI, and Teruo MIYAZAWA・
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai 981-8555, Japan
Received September 22, 1997
The amino-carbonyl reaction (Maillard reaction), also known as glycation,
of egg-yolk phosphatidylethanolamine (PE) was induced by incubating PE
(50 mg/ml) with D-glucose (222 mM) in a methanol medium containing 2,6-di-tert-butyl-p-cresol
as an antioxidant at 37°C for 4 days. The resultant product, glycated
PE (・・PE), was then isolated from the reaction mixture by two-step normal
and reversed-phase high-performance liquid chromatography with UV diode
array detection and was characterized as having a 1:1:1 elemental ratio
of phosphorus:nitrogen:sugar. The Fourier transform-nuclear magnetic resonance
spectrum and infrared absorbance spectrum indicate the isolated ・・PE
to have been deoxy-D-fructosyl PE, which is an Amadori product of PE. The
fast atom bombardment-mass spectrometric data for the glycation product
of authentic dioleoyl PE (1,2-di-9-octadecenoyl-sn-glycero-3-phosphoethanolamine)
show that the molecular weight of ・・PE corresponds to that of glucose-conjugated
PE in the form of an Amadori product. This Amadori product formation was
also confirmed in PE/phosphate buffer dispersions and in phosphatidylcholine-PE
liposome/phosphate buffer suspensions in the presence of D-glucose at 37°C.
・・PE was degraded by phospholipase A2, C and D. Freshly spiked blood
plasma and red blood cells (RBC) from normal human volunteers contained
substantial levels of ・・PE, the concentration corresponding to at least
9 mol％ of PE. Remarkable formation of ・・PE, up to 15--45 mol％ of PE
in human blood plasma and RBC, was further confirmed by prolonged incubation
with 5--45 mM D-glucose. The ・・PE formation in RBC was found to be proportional
to the glycated hemoglobin formation.
amino-carbonyl reaction; glycation; Maillard reaction; Amadori product;
phosphatidylethanolamine
-13-
Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA
Carboxyltransferase in Culture of Streptomyces fradiae
Du Bok CHOI,＊ Yongsoo PARK,＊＊ and Mitsuyasu OKABE＊＊,・
＊United Graduate School of Agricultural Science, Gifu University, 1-1
Yanagido, Gifu 501-1193, Japan
＊＊Laboratory of Biotechnology, Department of Applied Biological Chemistry,
Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, 422-8529,
Japan
Received September 24, 1997
To investigate why more tylosin was produced when Streptomyces fradiae
T1558 was cultured in a rapeseed oil medium than in a glucose or starch
medium, we measured the activity of methylmalonyl-CoA carboxyltransferase
(EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme,
which catalyzes the formation of the precursor of tylosin, protylonolide,
was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which
was 2.5- and 1.3-fold that with the glucose or starch medium, respectively.
The intracellular propionic acid concentration was 1.2 g/g of dry weight,
which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively.
The addition of propionic acid increased tylosin production in batch culture:
when 0.2 g/l (final concentration) propionic acid was added to the glucose
medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the
amount without propionic acid. These findings suggest that in glucose medium,
intracellular propionic acid is a limiting factor because of the low activity
of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.
Streptomyces fradiae; tylosin; methylmalonyl-CoA carboxyltransferase; propoinic
acid
-14-
Cysteine Protease Inhibitors Produced by the Industrial
Koji Mold, Aspergillus oryzae O-1018
Takao YAMADA,・ Jun HIRATAKE,＊ Mototsune AIKAWA, Tetsuyoshi SUIZU, Yoshiyuki SAITO, Akitsugu KAWATO, Koji SUGINAMI, and Jun′ichi ODA＊
Research Institute, Gekkeikan Sake Co. Ltd., 24, Shimotoba Koyanagi-cho, Fushimi-ku, Kyoto 612--8385, Japan
＊Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011,
Japan Received October 15, 1997 Aspergillus oryzae O-1018 (FERM P-15834)
separated from industrial koji for brewing sake was found to produce five
papain-inhibitory compounds in the culture supernatant. The five isolated
inhibitors were named CPI-1 to CPI-5, and their structures were elucidated
by spectroscopic analyses and chemical degradation. We determined the structures
of CPI-2, CPI-3 and CPI-4 as 4-amino-1-[[N-||[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl]||||amino]butane,
5-amino-1-[[N-[(2S, 3S)-3-trans-carbox||||yoxiran-2-carbonyl]-L-isoleucyl]amino]pentane
and N8-||||[N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleu||cyl]spermidine,
respectively. We also confirmed by a degradation experiment that CPI-1
consisted of L-trans-epoxysuccinic acid, L-tyrosine and spermidine, and
that CPI-5 was composed of L-trans-epoxysuccinic acid, L-phenylalanine
and spermidine. Although CPI-4 was identified as kojistatin A,1) the other
CPIs seemed to be novel compounds. All CPIs were cysteine protease-specific
inhibitors with appreciable selectivity toward cathepsin B and L. The inhibition
potency of CPIs against cysteine proteases was as high as or higher than
that of E-64. In particular, CPI-2, -3 and -4 were ten times more effective
than E-64 toward cathepsin B and L, and CPI-1 and -5 were about 100 times
more inhibitory than E-64 toward cathepsin L.
Aspergillus oryzae; koji mold; cysteine protease inhibitor; trans-epoxysuccinyl
derivative
-15-
Synthesis of a Lectin in Both Mycelia and Fruit Bodies
of the Ascomycete Mushroom Aleuria aurantia
Shigeru OGAWA, Eijiro NAKAJIMA, Hideyuki NAGAO, Masanari OHTOSHI, Akikazu ANDO, and Yoshiho NAGATA・
Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University, Matsudo 271-0092, Japan
Received October 16, 1997
In several organisms that form fruit bodies, the synthesis of lectins is
developmentally regulated. Aleuria aurantia is an ascomycete that forms
a fruit body known as orange peel mushroom. To find whether the mycelia
of this organism synthesize a lectin, mycelial isolates were obtained from
a wild fruit body by spore germination and regeneration from a fragment
of the fruit body. The isolates were identified as A. aurantia by analysis
of their DNA. The mycelial isolates synthesized a lectin with the same
properties as those of fruit-body lectin in terms of subunit molecular
mass, immunochemical reactivity, binding specificity for L-fucose, and
N-terminal amino acid sequence. Vegetatively growing mycelia synthesized
as much lectin as the fruit body, so such synthesis was not developmentally
regulated, unlike some other organisms that form fruit bodies.
ascomycete mushroom Aleuria aurantia; Aleuria aurantia lectin; lectin synthesis
in mycelia
-16-
Liberation of Phospholipids from Z-Disks of Chicken Skeletal
Muscle Myofibrils by 0.1 mM Calcium Ions: Weakening Mechanism for Z-Disks
during Post-mortem Aging of Meat
Ken-ichiro SHIMADA, Dong Hyun AHN, and Koui TAKAHASHI・
Meat Science Laboratory, Department of Animal Science, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
Received October 17, 1997
Weakening of the Z-disks of skeletal muscle myofibrils contributes to the
tenderization of meat during post-mortem aging. To elucidate the weakening
mechanism, we compared Z-disks weakened by post-mortem aging of chicken
breast muscle with those of myofibrils treated with a solution containing
0.1 mM CaCl2 and 1 μM calpastatin domain I. In both cases, the Z-disks
were weakened with a corresponding liberation of their constituent phospholipids
(PLs). The liberation of PLs specific to 0.1 mM calcium ions was minimal
at pH 6.5 and maximal at 35°C together with the Z-disk weakening. Binding
of calcium ions to PLs in the Z-disks was determined by 45Ca-autoradiography.
Acidic PLs were strongly radioactive and neutral PLs were appreciably radioactive.
It is very probable that acidic PLs would bind electrostatically to α-actinin
under physiological conditions, and that this interaction would be broken
by the binding of calcium ions at 0.1 mM to PLs, resulting in the partial
liberation of PLs from Z-disks. We conclude, therefore, that the liberation
of PLs by the binding of 0.1 mM calcium ions was the main cause for Z-disk
weakening during the post-mortem aging of chicken.
post-mortem aging; Z-disk weakening; phospholipid liberation; calcium ion
-17-
Deterioration of Connectin/Titin and Nebulin Filaments
by an Excess of Protease Inhibitors
Ryuichi TATSUMI, Akihito HATTORI, and Koui TAKAHASHI・
Meat Science Laboratory, Department of Animal Science, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8589, Japan
Received October 22, 1997
We studied the effect of protease inhibitors at a high concentration on
connectin and nebulin filaments in myofibrils. Calpastatin domain I at
0.1 mM bound to connectin and nebulin filaments, and deteriorated their
physico-chemical properties; the calcium-binding ability of connectin and
nebulin filaments was suppressed, the susceptibility of both filaments
to trypsin was markedly decreased, and the resting tension of mechanically
skinned fibers was increased by 2.5 times that of the control at a sarcomere
length of 3.6 μm. This indicates that the connectin filaments were made
more rigid. The same phenomenon was observed from the treatment of skinned
fibers with 1 mM leupeptin whose resting tension was increased to 2 times
the control value. Microscopically, both protease inhibitors induced dense
aggregation and disappearance of the regular striation of myofibrils due
to their non-specific binding to many myofibrillar proteins. The use of
excess calpastatin domain I and leupeptin should therefore be avoided in
physiological and biochemical studies on connectin and nebulin filaments,
as well as on myofibrils. calcium-binding; calpastatin domain I; connectin/titin;
nebulin; leupeptin
-18-
Improvement of the Off-flavor of Soy Protein Isolate by
Removing Oil-body Associated Proteins and Polar Lipids
Masahiko SAMOTO,・ Chiaki MIYAZAKI, Jiroh KANAMORI, Takeshi AKASAKA, and Yukio KAWAMURA＊,・
Central Research Institute, Fuji Oil Co. Ltd., 4-3 Kinunodai, Yawara,
Tsukuba-gun, Ibaraki 300-2436, Japan
＊Protein Science Laboratory, National Food Research Institute, 2-1-2 Kannondai,
Tsukuba, Ibaraki 305-0856, Japan
Received October 20, 1997
The precipitate formed by ultracentrifuging a defatted soybean extract
at 200,000×・・ for 50 min at pH 7.5 was composed of particles of 100--200
nm in diameter and enriched with 34-kDa, 24-kDa and 18-kDa proteins. An
SDS-PAGE analysis showed these proteins to migrate to a position identical
to that of oil-body-associated proteins (OBAPs; Herman, Planta, 172, 336--345,
1987).1) They were recovered in the precipitate of soy protein with 30--40％
saturated ammonium sulfate in the presence of 10 mM 2-ME. The lipid composition
of the precipitate by a TLC analysis showed that most of the polar lipids
in the soybean extract had been condensed in the fraction, suggesting the
association between OBAP and the polar lipids. Removal of OBAP and the
polar lipids from the soybean extract by conventional centrifugation (10,000×・・
for 10 min) in the presence of 30 mM Na2SO4 and 30 mM CaCl2 at pH 2.8 was
achieved with concomitant improvement of the volatile off-flavor. A soy
protein isolate (SPI) prepared from such a soybean extract contained far
fewer volatile off-flavor compounds than normal SPI did.
soy protein isolate; lipid; off-flavor
-19-
Inhibitory Effects of Lipid Oxidation on the Activity
of Plasma Lecithin-Cholesterol Acyltransferase
Shin KAMIYAMA,・ Tokuhisa YAMATO, and Yuji FURUKAWA
Laboratory of Nutrition, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai 981-8555, Japan
Received October 20, 1997
We investigated the effects of free radical generation on the esterification
of cholesterol by lecithin-cholesterol acyltransferase (LCAT). A water-soluble
free radical initiator, 2,2′-azobis-amidinopropane dihydrochloride (AAPH),
inhibited the activity of plasma LCAT as a function of the incubation time
after its addition. When a small amount of oxidized HDL was added to plasma,
LCAT activity was dose-dependently inhibited. To identify the effects of
HDL oxidation on LCAT activity, a purified enzyme and cofactor in a vesicle
solution (an artificial substrate) were used. i) LCAT activity was inhibited
by the oxidation of substrate vesicles, this inhibition being related to
the degree of oxidation. ii) This inhibition was observed even if apolipoprotein
A-I was not oxidized. iii) Oxidized phosphatidylcholine, but not oxidized
cholesterol, in the vesicles affected LCAT activity. iv) The addition of
0--40％ of oxidized vesicles to normal substrate vesicles resulted in the
activity of LCAT being inhibited in a dose-dependent manner. These results
suggest that the esterification of cholesterol by LCAT may be affected
by the oxidation of substrate phosphatidylcholine via free radical generation
in the plasma.
lecithin-cholesterol acyltransferase activity; oxidized low-density lipoprotein;
high-density lipoprotein; cholesterol reverse-transport
-20-
Complexes of Serine Acetyltransferase and Isozymes of
Cysteine Synthase in Spinach Leaves
Xia ZHU,1 Takayuki YAMAGUCHI,1,2 and Masahiro MASADA1,・
1Department of Bioresources Chemistry, Faculty of Horticulture, Chiba
University, Matsudo 648, Chiba 271-8510, Japan
2Engineering Department, Kazami Co., Ltd., Sukedo 1-26, Ashikaga City,
Tochigi 326-0044, Japan
Received October 22, 1997
Polyclonal antibodies against cysteine synthase (CSase; EC 4.2.99.8) isozymes
1, 2, and 3 were used for the detection of complexes of these isozymes
with serine acetyltransferase (SATase; EC 2.3.1.30). SATase was partially
purified and found to complex with these isozymes by western blotting and
immunotitration. When the complexes were treated with a high concentration
of O-acetyl-L-serine, they did not dissociate. However, some complexes
with CSase 1 or 3 dissociated when left for 24 h at 4°C. Results of western
blotting on SDS-PAGE showed that CSase 2 complexed with SATase. CSases
1, 2, and 3 all could complex with SATase, but the tightness of the bond
differed.
enzyme complexes; cysteine biosynthesis; serine acetyltransferase; cysteine
synthase; O-acetylserine(thiol)-lyase
-21-
Direct Formation of Human Interleukin-11 by Cis-Acting
System of Plant Virus Protease in Escherichia coli
Tohru TAKAHASHI, Michiko NAKANISHI, Yoshio YAO, Ichiro UYEDA,1 and Nobufusa SERIZAWA・
Biomedical Research Laboratories, Sankyo Co. Ltd., 2-58 Hiromachi 1-chome,
Shinagawa-ku, Tokyo 140-8710, Japan
1Laboratory of Plant Virology and Mycology, Department of Agrobiology and
Bioresources, Faculty of Agriculture Hokkaido University, Kita 9, Nishi
9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan
Received October 30, 1997
To produce a large amount of recombinant proteins in Escherichia coli,
we constructed a unique cis-acting expression system using a plant virus
protease. This new expression system could directly produce recombinant
proteins, that had a biologically active form. A gene of nuclear inclusion-a
(NIa), which had a specific amino acid sequence, was fused with a foreign
protein gene at the same protein reading frame. One of the NIa-specific
cleavage amino acid sequences, Gln-Ala, was also contained at the protein-protein
junction. In the case of human interleukin-11 (hIL-11), a 23-kDa specific
signal band was obtained from recombinant bacteria. N-terminal sequencing
of the 23-kDa protein showed that NIa specifically cleaved the fusion protein
at Gln-Ala, producing Ala-hIL-11. Furthermore, we could produce the mature
rhIL-11 by extending the culture time. This 23-kDa protein had the same
biological activity as hIL-11 in a mouse plasmacytoma, T1165. Combined
with fermentation control, we produced mature rhIL-11 in E. coli.
gene expression; clover yellow vein virus; potyvirus; nuclear inclusion-a;
interleukin-11
-22-
Chemical Modifications of Momordin-a and Luffin-a, Ribosome-Inactivating
Proteins from the Seeds of Momordica charantia and Luffa cylindrica: Involvement
of His140, Tyr165, and Lys231 in the Protein-Synthesis Inhibitory Activity
Yuji MINAMI,・ M. Rafiqul ISLAM, and Gunki FUNATSU
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Received November 4, 1997
Effects of chemical modifications on the protein-synthesis inhibitory (PSI)
activities of momordin-a and luffin-a were investigated. Treatment with
a 50-fold excess of diethylpyrocarbonate at pH 6.5 modified one histidine
residue in momordin-a and luffin-a and reduced their PSI activities to
10％ and 8.3％, respectively. Modifications with a 20-fold excess of KI3
at pH 7.0 at 0°C greatly reduced their PSI activities to 10％ by iodination
of nearly one tyrosine residue. The PSI activity of momordin-a was rapidly
reduced to 6.4％ by the modification of one lysine residue with trinitrobenzensulfonic
acid as in the case of luffin-a reported previously. By analyses of the
tryptic peptides from the modified momordin-a and luffin-a, the modified
residues were identified as His140, Tyr165, and Lys231. Furthermore, the
amounts of three modified momordin-a binding to rat liver ribosomes were
reduced to about half or less than half of that of native momordin-a. From
these results, it was suggested that His140, Tyr165, and Lys231 are highly
exposed on the surface of momordin-a and luffin-a molecules and are involved
in their PSI activities, probably by binding to ribosomes.
ribosome-inactivating protein; protein-synthesis inhibitor; chemical modification;
bitter gourd seed protein; sponge gourd seed protein
-23-
Low Sucrase Activity in the Small Intestine of a Senescence-accelerated
Strain of Mouse, SAMP1
Makoto TAKENOSHITA, Mitsuharu YABUNE, Hiromi KATSURA, Ryoichi YAMAJI, Hiroshi INUI, Kazutaka MIYATAKE, and Yoshihisa NAKANO
Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
Received November 4, 1997
The small intestinal sucrase activity in a senescence-accelerated strain
of mouse, SAMP1, was significantly lower than that in other strains, including
its control strain, SAMR1. In contrast, the activity of isomaltase, which
usually associates with sucrase to form a complex enzyme (SI complex),
in SAMP1 was comparable to that in other strains. Thus, the ratio of the
sucrase to isomaltase activities (S/I ratio) in SAMP1 was very low (about
0.15), compared with that in other strains (around 0.7). The S/I ratio
in SAMP1 was abnormally low, even at a young age, indicating that senescence
did not result in the low sucrase activity. Western blot analysis suggests
that a large part of the isomaltase subunit occurred alone without the
association of the sucrase subunit in this strain. In contrast, Northern
blot analysis shows that the level of mRNA for the SI complex in SAMP1
was comparable to that in SAMR1. When the pancreatico-biliary ducts were
ligated in SAMP1 to reduce the level of pancreatic proteases, a remarkable
increase was observed in the sucrase activity, whereas the isomaltase activity
was increased to a much smaller extent. This marked increase in sucrase
activity resulted in the S/I ratio increasing to 0.84 18 h after the ligation.
These results suggest the sucrase subunit of the SI complex to be abnormally
unstable against pancreatic proteases in SAMP1.
sucrase; isomaltase; senescence-accelerated mouse; pancreatic protease;
small intestine
-24-
Antimutagenicity of Flavones and Flavonols to Heterocyclic
Amines by Specific and Strong Inhibition of the Cytochrome P450 1A Family
Kazuki KANAZAWA,・ Takatoshi YAMASHITA, Hitoshi ASHIDA, and Gen-ichi DANNO
Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokkodai, Nada-ku, Kobe 657-8501, Japan
Received November 10, 1997
We found the mechanism in flavonoids that can strongly suppress the mutagenicity
of one of the food-derived and carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole
(Trp-P-2). The antimutagenicity was evaluated by IC50 value, the amount
required for 50％ inhibition of the mutagenicity of 0.1 nmol Trp-P-2, with
Salmonella typhimurium TA98 strain in the presence of S9 mix. The flavones
and flavonols were two orders stronger as antimutagens than such antimutagenic
phytochemicals as chlorophylls and catechins. We had previously found flavonoids
to be a desmutagen to neutralize Trp-P-2 before or during attack of DNA,
because they had no effect on either the ultimate mutagenic form of Trp-P-2
(N-hydroxy-Trp-P-2) or the mutated cells. The desmutagenicity of the flavonoids
did not depend on the hydroxy number or position that should be associated
with antioxidative potency, and was also unaffected by the solubility of
Trp-P-2 in the assay solution. The inhibitory effect of the flavonoids
on the metabolic activation of Trp-P-2 to N-hydroxy-Trp-P-2 was almost
in parallel with the antimutagenic IC50 value, when determined with a Saccharomyces
cerevisiae AH22 cell simultaneously expressing both rat cytochrome P450
1A1 and yeast reductase. The Ki values of flavones and flavonols for the
enzyme were less than 1 μM, while the Km value of Trp-P-2 was 25 μM.
The antimutagenicity of the flavones and flavonols was thus concluded to
be due to inhibition of the activation process of Trp-P-2 by P450 1A1 to
the ultimate carcinogenic form. They were also able to act as antimutagens
toward other indirect mutagens that were activated by P450 1A1.
antimutagenicity; flavonoid; cytochrome P450 inhibition; cancer prevention;
heterocyclic amine
-25-
Rice Bifunctional α-Amylase/Subtilisin Inhibitor: Characterization,
Localization, and Changes in Developing and Germinating Seeds
Hiroshi YAMAGATA・, Kiyoshi KUNIMATSU, Hiroshi KAMASAKA, Takeshi KURAMOTO, and Teruo IWASAKI＊
Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Nada, Kobe 657-8501, Japan
Received November 17, 1997
A bifunctional α-amylase/subtilisin inhibitor (RASI) was purified to electrophoretic
homogeneity from rice (Oryza sativa L.) bran. Its molecular mass was 21
kDa by SDS-PAGE and its isoelectric point was 9.05. Purified RASI inhibited
subtilisin Carlsberg strongly and inhibited α-amylase from germinating
rice seeds weakly. It inhibited rice α-amylase more than barley α-amylase,
and the inhibition of rice α-amylase was greater at higher pHs. RASI did
not inhibit trypsin, chymotrypsin, cucumisin, or mammalian α-amylase.
The RASI was in the outermost part of the rice grain and its subcellular
site seemed to be aleurone particles in aleurone cells. SDS-PAGE and western
blotting showed that RASI was synthesized in the late milky stage in developing
seeds, and it remained fairly constant during the first 7 days of germination.
Oryza sativa L.; rice seed; α-amylase; subtilisin; bifunctional inhibitor
-26-
Salicylic Acid Induces a Cytosolic Ca2＋ Elevation in
Yeast
Izumi C. MORI,a Hidetoshi IIDA,b Frederick I. TSUJI,c Minoru ISOBE,a Nobuyuki UOZUMI,a,d and Shoshi MUTOa,d,・
aGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa,
Nagoya 464-8601, Japan
bDepartment of Biology, Tokyo Gakugei University, Nukuikita-machi, Koganei-shi,
Tokyo 184-0015, Japan
cMarine Biology Research Division, Scripps Institution of Oceanography,
University of California, San Diego, La Jolla CA92093, USA
dNagoya University Bioscience Center, Nagoya University, Chikusa, Nagoya
464-8601, Japan
Received December 8, 1997
Cytosolic free calcium ion concentration ([Ca2＋]cyt) after a salicylic
acid (SA)-stimulus was monitored in cells of the yeast Saccharomyces cerevisiae
expressing apoaequorin, which constitutes a Ca2＋-sensitive luminescent
protein, aequorin, when combined with coelenterazine. SA induced a transient
[Ca2＋]cyt elevation that was dependent on the concentration of SA and
pH of the SA solution. The SA-induced [Ca2＋]cyt elevation was not reduced
in Ca2＋-deficient medium, suggesting that Ca2＋ was mobilized from an
intracellular Ca2＋ store(s). Benzoic acid, butyric acid and sorbic acid
did not induced a [Ca2＋]cyt elevation.
aequorin; cytosolic Ca2＋; salicylic acid; Saccharomyces cerevisiae
-27-
Note
Reduction of Stale Aldehyde of Beer with Membrane Fraction of Acetic Acid
Bacteria
Yukihiro NOMURA, Masanori YAMAMOTO, and Hidehiko KUMAGAI・
Somatech Center, House Foods Corporation, 1-5-7 Mikuriya-sakaemachi, Higashiosaka-shi, Osaka 577-8520, Japan
Received August 8, 1997
The oxidation of trans-2-nonenal, the main compound causing staleness in
beer, with a membrane fraction of acetic acid bacteria was investigated.
For use of the fraction, it was necessary to inactivate membrane-bound
alcohol dehydrogenase (ADH) first to avoid oxidation of alcohol, while
maintaining membrane-bound aldehyde dehydrogenase (ALDH) activity. ADH
was completely inactivated by treatment of the membrane fraction at 60°C
for 15 min, without any loss of ALDH activity. ALDH activity was not affected
by heat treatment even in the presence of 10％ alcohol. Results of HPLC
showed that a fraction incubated at 60°C for 15 min oxidized 50 ppb trans-2-nonenal
added to a commercial beer sample without affecting the alcohol content.
Our sensory panel found that the stale odor of trans-2-nonenal disappeared
when the membrane fraction was so treated, with the resulting odor better
than that of beer with trans-2-nonenal added but not treated with the fraction.
acetic acid bacteria; membrane fraction; stale aldehyde; trans-2-nonenal
-28-
Note
Effect of Magnetite on the Hematocrit Value in Exsanguinated Rats
Toru HAMAYA, Atsutane OHTA, Toshiaki KONO, Rikizo AONO,＊ and Koki HORIKOSHI＊＊
Bio Science Laboratories, Meiji Seika Kaisha Ltd., 5-3-1 Chiyoda, Sakado-shi,
Saitama 350-0289, Japan
＊Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta,
Midori-ku, Yokohama-shi, Kanagawa 226-8501, Japan
＊＊Department of Chemistry, Faculty of Engineering, Toyo University, Kujirai,
Kawagoe-shi, Saitama 350-8585, Japan
Received August 15, 1997
Magnetite prepared by an enzyme-dependent reaction gradually released iron
ion into the acidic-to-neutral buffer solution. A preparatory experiment
was performed to examine the efficiency of magnetite as an iron supplement.
Feeding exsanguinated rats with being magnetite resulted in the hematocrit
value being recovered without any serious adverse effect on the digestive
organs.
magnetite; iron supplement; adverse effect
-29-
Note
Benzaldehyde O-Alkyloximes as New Plant Growth Regulators
Hiromichi YOSHIKAWA and Keiko DOI
Department of Environmental Chemistry, Kyushu Kyoritsu University, Yahatanishi-ku, Kita-kyushu, Fukuoka 807-8585, Japan
Received August 25, 1997
||Fourty-two kinds of benzaldehyde O-alkyloximes de||||rived from benzaldehydes
were prepared and their biologi||cal activities were investigated. Introduction
of a fluorine or bromine atom to the benzene ring of the oximes enhanced
their phytotoxic activity. The O-alkyloximes with a fluorine atom at the
3 or 4 position of the benzene ring were more active than the other oximes
in the GA3-induced α-amylase induction inhibition test. In the transpiration
test, 4-bromobenzaldehyde O-carboxylmethyloxime was the most active. The
O-alkyloximes exhibited weak abscisic acid-like activity by inhibiting
not only the germination, root growth and transpiration of higher plants
but also GA3-induced α-amylase induction in embryoless barley seeds.
benzaldehyde O-alkyloximes; growth inhibitor; α-amylase induction; transpiration
-30-
Note
4-Hydroxy-3-nitrophenylacetic and Sinapic Acids as Antibacterial Compounds
from Mustard Seeds
Shoko TESAKI, Soichi TANABE, Haruhiro ONO,＊ Eri FUKUSHI,＊＊ Jun KAWABATA,＊＊ and Michiko WATANABE・
Food Science Laboratory, Faculty of Education, Tokyo Gakugei University,
Koganei-shi, Tokyo 184-8501, Japan
＊Skylark Food Science Institute, Chiba-shi, Chiba 261-0023, Japan
＊＊Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589,
Japan
Received October 3, 1997
A methanol extract from yellow mustard seeds had antibacterial activity
against Escherichia coli, Salmonella enteritidis, and Staphylococcus aureus.
Two compounds with such activity were isolated from the extract. By instrumental
analysis, the compounds were identified as 4-hydroxy-3-nitrophenylacetic
and sinapic acids. Examination of the structure-activity relationship showed
that the hydroxyl and nitro groups of the first compound were involved
in the activity against all three species. The two methoxyl groups and
the hydroxyl group in sinapic acid were effective against E. coli and all
of the substituents of the benzene ring were effective against S. enteritidis.
The presence of the propenoic group of the second compound was effective
against S. aureus.
antibacterial activity; sinapic acid; 4-hydroxy-3-nitrophenylacetic acid
-31-
Note
Effectiveness of Polyclonal and Monoclonal Antibodies Prepared for an Immunoassay
of the Etofenprox Insecticide
Shiro MIYAKE, Akiko HAYASHI,＊,・ Takako KUMETA,＊＊ Koji KITAJIMA,＊＊ Hiroshi KITA,・・ and Hideo OHKAWA＊,・・・
Central Research Institute Iatron Laboratories Inc., Mitoaza Mitodai
1460-6, Takomachi, Katori-gun, Chiba 289-2247, Japan
＊Department of Biological and Environmental Science, Faculty of Agriculture,
Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-0013, Japan
＊＊Institute of Life Science Mitsui-Toatsu Chemicals Inc., 1144 Togo,
Mobara-shi, Chiba 297-0017, Japan
Received October 13, 1997
Two polyclonal antibodies and three monoclonal antibodies specific to the
etofenprox insecticide were prepared from rabbits and mice, respectively.
The monoclonal antibodies were more reactive with etofenprox than the polyclonal
antibodies by C-ELISA. Monoclonal antibody cl 205-65 was found to be the
most tolerant to methanol, most highly reactive and most specific to etofenprox
among the antibodies tested.
polyclonal antibody; monoclonal antibody; immunoassay; etofenprox; insecticide
-32-
Note
Affinity of Antioxidative Polyphenols for Lipid Bilayers Evaluated with
a Liposome System
Tsutomu NAKAYAMA,＊ Koji ONO, and Kei HASHIMOTO＊＊
School of Food and Nutritional Sciences, University of Shizuoka, 52-1
Yada, Shizuoka 422-8526, Japan
＊＊Faculty of Agriculture, Utsunomiya University, Mine-cho, Utsunomiya
321-8505, Japan
Received October 15, 1997
We developed a method to measure the amounts of antioxidative polyphenols
and ubiquinones incorporated into the lipid bilayers of liposomes to estimate
their affinities for cell membranes. Results were expressed in terms of
an ``affinity factor′′, calculated by division of the amount of compound
incorporated by the amount added to the liposomal solution. The results
reflected dose-dependence of the biological activities of the compound
found in earlier in vitro experiments with mammalian and bacterial cells.
antioxidant; liposome; model membrane; polyphenol; ubiquinone
-33-
Note
Effects of Vitamin B6 Deficiency on Cytokine Levels and Lymphocytes in
Mice・
Shoko DOKE,＊,＊＊＊ Naoki INAGAKI,＊＊ Takashi HAYAKAWA,＊＊＊ and Haruhito TSUGE＊＊＊,・・
＊Gifu City Women′s College, 2693 Nagarafukumitsu, Gifu 502-0817, Japan
＊＊Faculty of Pharmacy, Gifu Pharmaceutical University, 5-6-1 Mitahorahigashi,
Gifu 502-8585, Japan
＊＊＊The United Graduate School of Agricultural Science, Gifu University,
1-1 Yanagido, Gifu 501-1193, Japan
Received October 20, 1997
The effects of vitamin B6 (B6) deficiency on cytokine levels and proportions
of lymphocyte subsets in BALB/c mice were investigated. The proportion
of lymphocytes from the thymus and spleen of mice given no B6, that were
CD4＋CD8－ T cells, was larger than in mice given B6, and the ratio of
CD8＋ to CD4＋ T cells in the thymus of mice given no B6 was lower. The
concentrations of interleukin-5 and -10 in spleen cells stimulated in vitro
with concanavalin A were significantly higher in the mice with B6 deficiency,
as was their plasma corticosterone concentrations. These results suggested
that B6 is necessary to maintain cytokine levels and lymphoid function
in the thymus and spleen of mice.
vitamin B6 deficiency; cytokine; lymphocyte
-34-
Note
Manauealide C and Anhydrodebromoaplysiatoxin, Toxic Constituents of the
Hawaiian Red Alga, Gracilaria coronopifolia
Hiroshi NAGAI,・ Yukiko KAN, Tsuyoshi FUJITA, Bryan SAKAMOTO,＊ and Yoshitsugi HOKAMA＊
Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto, Mishima-gun,
Osaka 618-8503, Japan
＊John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu,
HI 96922, U.S.A.
Received October 29, 1997
Manauealide C (1) and anhydrodebromoaplysiatoxin (4), toxic constituents
of the Hawaiian red alga, Gracilaria coronopifolia which has been concerned
with food poisoning cases, were studied. The absolute structure of manauealide
C was determined as 1 by chemical conversion and spectroscopic methods.
The first complete assignment of 13C chemical shifts for anhydrodebromoaplysiatoxin
(4) was established. The biological activity of 4 was also investigated.
Gracilaria coronopifolia; manauealide C; anhydrodebromoaplysiatoxin; absolute
configuration; marine algal toxin
-35-
Note
Antimicrobial Activity of a Compound Isolated from an Oil-Macerated Garlic
Extract
Hisae YOSHIDA, Nami IWATA, Hirotaka KATSUZAKI,＊ Rie NAGANAWA, Keiko ISHIKAWA, Hiroyuki FUKUDA, Tsuchiyoshi FUJINO,・ and Atsushi SUZUKI
Biodevelopment Division, Central Institute, Nagoya Seiraku Co., Ltd.,
310 Nakasuna-cho, Tempaku-ku, Nagoya, Aichi 468-8588, Japan
＊Faculty of Bioresources, Mie University, 1515 Kamihama-cho, Tsu, Mie
514-0008, Japan
Received November 10, 1997
A compound showing antimicrobial activity was isolated from an oil-macerated
garlic extract by silica gel column chromatography and preparative TLC.
On basis of the results of NMR and MS analyses, it was identified as Z-4,5,9-trithiadeca-1,6-diene-9-oxide
(Z-10-devinylajoene; Z-10-DA). Z-10-DA exhibited a broad spectrum of antimicrobial
activity against such microorganisms as gram-positive and gram-negative
bacteria and yeasts. The antimicrobial activity of Z-10-DA was comparable
to that of Z-ajoene, but was superior to that of E-ajoene. Z-10-DA and
Z-ajoene are different in respect of substitution of the allyl group by
the methyl group flanking a sulfinyl group. This result suggests that substitution
by the methyl group would also be effective for the inhibition of microbial
growth.
ajoene; antimicrobial effect; garlic; oil-macerated garlic extract; Allium
sativum
-36-
Note
Molecular Cloning and Expression Patterns of Cu/Zn-Superoxide Dismutases
in Developing Soybean Seeds
Masaomi ARAHIRA, Van Hai NONG,・ Kazunari KADOKURA, Keitarou KIMURA, Kyoko UDAKA,・・ and Chikafusa FUKAZAWA・・・
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba, Ibaraki 305-8642, Japan
Received
Assuming that the amount of superoxide radicals generated in vivo correlates
with the production of ergastic substances such as storage proteins, the
coordinated response of detoxication enzymes such as superoxide dismutases
is largely exploited to understand the self-defense systems of plant.
Here we examined expression of the genes for superoxide dismutases during
seed development of soybean. The cDNAs encoding a cytosolic copper/zinc
form and an iron form of the above enzyme have been cloned and then employed
as probes, separately. Northern blotting results suggested that both superoxide
dismutase mRNAs are expressed at the maximum level, preceding a developmental
stage when mRNA encoding glycinin, soybean 11S-storage protein, at the
maximum.
superoxide dismutase; cDNA cloning; mRNA expression; soybean; seed development
-37-
Note
Nisin Z Production by Lactococcus lactis IO-1 Using Xylose as a Carbon
Source
Noppawan CHINACHOTI, Hiromi MATSUSAKI,・・ Kenji SONOMOTO, and Ayaaki ISHIZAKI・
Laboratory of Microbial Technology, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
Received November 21, 1997
Lactococcus lactis IO-1 was able to use xylose as a carbon source for nisin
Z production; the yield based on sugar consumption was about 20％ superior
to that made with glucose under the same fermentation conditions. The optimal
conditions for nisin Z production were with 4％ xylose at pH 6.0 and 37°C.
Addition of 0.1 M CaCl2 increased nisin Z production specifically, but
not cell growth, acid production, or xylose consumption, and resulted in
the maximum nisin Z activity of about 1.5 times that without CaCl2. xylose
fermentation; Lactococcus lactis; nisin Z production; calcium chloride
-38-
Note
Insecticidal and Antifungal Activities of Aminorhodanine Derivatives
Yoshihiko INAMORI,・ Yukiko OKAMOTO, Yoko TAKEGAWA, Hiroshi TSUJIBO, Yoshikazu SAKAGAMI,＊ Yuko KUMEDA,＊ Mitsunobu SHIBATA,＊＊ and Atsushi NUMATA
Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki-shi,
Osaka 569-0000, Japan
＊Osaka Prefectural Institute of Public Health, Nakamichi, Higashinari-ku,
Osaka 537-0000, Japan
＊＊Fumakilla Ltd., Ono-cho, Saiki-gun, Hiroshima 739-0000, Japan
Received November 26, 1997
Aminorhodanine (1) showed strong insecticidal activity against Culex pipiens
pallens and Musca domestica, with respective LD50 values of 0.21 μg/insect
and 0.87 μg/insect. Compound 1 had antifungal activity against Aspergillus
niger ATCC-16404, Trichophyton mentagrophytes IFO-32412, Candida albicans
ATCC-10231, Hansenula anomala OPS-308 and Penicillium expansum IFO-8800.
In particular, 1 had potent antifungal activity against Aspergillus niger
ATCC-16404, its minimal inhibitory concentration (MIC) being 6.25 μg/ml.
Both activities of 1 were much higher than those of rhodanine (4), suggesting
that the introduction of an amino group into N-3 of 4 plays an important
role in the biological activity of rhodanine-related compounds. On the
other hand, N-acetylaminorhodanine (2) and N-benzoylaminorhodanine (3)
did not show either activity, suggesting that the free amino group at N-3
of 1 is closely related to the inhibitory activity of rhodanine derivatives.
insecticidal activity; antifungal activity; aminorhodanine; pathogenic
fungus; rhodanine
-39-
Note
Thermostable Neutral Protease Resembling Thermolysin Derived from Bacillus
brevis MIB001
Yukio TAKII,1,2 Yoshimi URATA,1 and Noriko UENO1
1Department of Food Science and Nutrition, School of Human Environmental Sciences and 2Interdisciplinary Research Institute for Biosciences, Mukogawa Women′s University, Nishinomiya, Hyogo 663-8558, Japan
Received November 26, 1997
A microbe producing a protease with strong thermostability that was released
extracellularly was isolated from soil. The isolate, MIB001, grew at from
15 to 51°C and pH 5.1--8.8 and was tentatively identified as a strain
of Bacillus brevis. Rabbit antisera raised against a pure preparation of
the protease did not cross-react with thermolysin or neutral metalloprotease
from Bacillus stearothermophilus KP1236. Bacillus brevis; Bacillus thermoproteolyticus;
Bacillus stearothermophilus; neutral protease; thermolysin
-40-
Note
Interaction between the Carboxyl-terminal Heparin-binding Domain of Fibronectin
and (－)-Epigallocatechin Gallate
Masaki SAZUKA, Mamoru ISEMURA, and Satoko ISEMURA＊
School of Food and Nutritional Sciences, University of Shizuoka, Yada,
Shizuoka 422-8526, Japan
＊Nippon Dental University Junior Collage at Niigata, Niigata, 951-8151,
Japan
Received
We have previously reported that (－)-epigallocatechin gallate (EGCg) inhibited
lung carcinoma cell adhesion to fibronectin (FN) and demonstrated its interaction
with FN. In the present work, we studied the interaction between thermolysin
fragments of FN and EGCg. An amino acid sequence analysis of the fragment
bound by EGCg-agarose provided its identification as a carboxyl-terminal
heparin-binding domain. Thus, the inhibition of cancer cell adhesion to
FN by EGCg is not caused by its direct binding to the cell-binding domain
containing an Arg-Gly-Asp-sequence.
fibronectin; (－)-epigallocatechin gallate; (＋)-catechin; heparin-binding
domain; thermolysin
-41-
Short Communication
Features of Tri101, the Trichothecene 3-O-Acetyltransferase Gene, Related
to the Self-defense Mechanism in Fusarium graminearum
Makoto KIMURA,＊ Yoshinori SHINGU, Katsuyoshi YONEYAMA,・ and Isamu YAMAGUCHI
Microbial Toxicology Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Received December 22, 1997
A structural gene of Tri101, which encodes trichothecene 3-O-acetyltransferase,
was isolated as a 3-kb XhoI-XbaI fragment from the trichothecene producer
Fusarium graminearum strain F15. The gene contained no introns, and the
coding region was 0.7-kb downstream of a putative UTP-ammonia ligase gene
which obviously is not related to the biosynthesis of trichothecenes. Tri101
was expressed when T-2 toxin was added, but this induction was not dependent
on the expression level of Tri6, a transcription activator gene in the
trichothecene biosynthetic and regulatory gene cluster.
Fusarium mycotoxin; trichothecene biosynthesis; resistance gene; cosmid
clones; transcription regulatory gene
-42-
Short Communication
cDNA Cloning, Expression, and Characterization of the Human Bifunctional
ATP Sulfurylase/Adenosine 5′-Phosphosulfate Kinase Enzyme
Ken YANAGISAWA1, Yoichi SAKAKIBARA1, Masahito SUIKO1, Yasunari TAKAMI2, Tatsuo NAKAYAMA2, Hiroshi NAKAJIMA3, Katsuhiko TAKAYANAGI3, Yasuhiro NATORI4, and Ming-Cheh LIU1,・
1Department of Biochemistry, University of Texas Health Center, Tyler,
TX 75710, U.S.A.
2Department of Biochemistry, Miyazaki Medical College, Miyazaki 889-16,
Japan
3Department of Biochemistry, Unitika R ＆ D Center, Uji, Kyoto 611, Japan
4Research Institute, International Medical Center of Japan, Shinjuku, Tokyo
162, Japan
Received January 20, 1998
A cDNA encoding the human bifunctional ATP sulfurylase/adenosine 5′-phosphosulfate
(APS) kinase was cloned and sequenced. The enzyme contains an APS kinase
domain in its N-terminal portion and an ATP sulfurylase domain in its C-terminal
portion. Recombinant full-length enzyme and its constituent APS kinase
and ATP sulfurylase domains were individually expressed, purified, and
shown to have their respective enzymatic activities.
ATP sulfurylase; APS kinase; bifunctional enzyme