(Vol.61 No.4 1997)
Molecular Biology of the Pore-forming
Cytolysins from Staphylococcus aureus, ƒ¿- and ƒÁ-Hemolysins and Leukocidin
Toshio Tomita and Yoshiyuki Kamio 565
Cytotoxicity of Cholestane 3ƒÀ,5ƒ¿,6ƒÀ-Triol
on Cultured Intestinal Epithelial Crypt Cells (IEC-6)
Kimiko Ohtani, Tomoyo Terada, Masaharu Kamei, and Isao Matsui-Yuasa
573
The Phylogeny of Species of the
Genus Issatchenkia KUDRIAVZEV (Saccharomycetaceae) Based on the Partial
Sequences of 18S and 26S Ribosomal RNAs
Yuzo Yamada, Jun-ichi Yano, Tomoko Suzuki, and Kozaburo Mikata 577
Hydrolysis of Xylan by Aspergillus
niger Immobilized on Non-woven Fabrics
Hiroharu Tokuda, Takashi Urata, and Kotoyoshi Nakanishi 583
Chemical and Functional Properties
of Mutastein, an Inhibitor of Insoluble Glucan Synthesis by Streptococcus
sobrinus
Osamu Hayashida, Keiji Hasumi, and Akira Endo 588
Purification, Characterization,
and cDNA Cloning of a Novel ƒ¿-Galactosidase from Mortierella vinacea
Hajime Shibuya, Hideyuki Kobayashi, Taku Sato, Wong-Sin Kim, Shigeki
Yoshida, Satoshi Kaneko, Kunihiro Kasamo, and Isao Kusakabe 592
Purification and Properties of
ƒÀ-Fructofuranosidase from Clostridium perfringens
Makoto Ishimoto and Atsuko Nakamura 599
Optimization of the Culture Medium
for Growth and the Kinetics of Lactate Fermentation by Pediococcus sp.
ISK-1
Etmy Herawati and Ayaaki Ishizaki 604
Microbial Extracellular Glycolipid
Induction of Differentiation and Inhibition of the Protein Kinase C Activity
of Human Promyelocytic Leukemia Cell Line HL60
Hiroko Isoda, Dai Kitamoto, Hiroshi Shinmoto, Masatoshi Matsumura,
and Tadaatsu Nakahara 609
Purification and Some Properties of
Endo-1,4-ƒÀ-D-xylanase from a Fresh-water Mollusc, Pomacea insularus (de
Ordigny)
Izumi Yamaura, Takahiro Koga, Toshihiko Matsumoto, and Tetsuo Kato
615
Non-radioactive Adenosine 5'-Phosphosulfate
Sulfotransferase Assay by Coupling with Sulfite Reductase and O-Acetylserine(thiol)lyase
Takeshi Ara and Jiro Sekiya 621
Some Properties and Physiological
Roles of Phosphoenolpyruvate Carboxylase in@Rhodopseudomonas sp. No. 7
Grown on Ethanol under Anaerobic-light Conditions
Megumi Sadaie, Takanari Nagano, Tomoaki Suzuki, Hirofumi Shinoyama,
and Takaaki Fujii 625
Changes in PAF (Platelet-activating
Factor) Production during Cell Cycle of Yeast Saccharomyces cerevisiae
Reiko Nakayama, Hiroaki Udagawa, and Hidehiko Kumagai 631
Purification
and Characterization of Extracellular Poly(ƒÀ-D-1,4-mannuronide) Lyase
from Dendryphiella salina IFO 32139
Tomoko Shimokawa, Shigeki Yoshida, Toshio Takeuchi, Katsumi Murata,
Hideyuki Kobayashi, and Isao Kusakabe 636
Structural Characteristics
of the Disulfide-reduced Ovotransferrin N-Lobe Analyzed by Protein Fragmentation
Kimihiko Mizutani, Honami Yamashita, Hideo Oe, and Masaaki Hirose 641
New Antioxidative Indophenol-reducing
Phenol Compounds Isolated from the Mortierella sp. Fungus
Akira Hirota, Yasujiro Morimitsu, and Hiroshi Hojo 647
Inhibition of Aldose Reductase
and Sorbitol Accumulation by Astilbin and Taxifolin Dihydroflavonols in
Engelhardtia chrysolepis
Hiroyuki Haraguchi, Isao Ohmi, Ayumi Fukuda, Yukiyoshi Tamura, Kenji
Mizutani, Osamu Tanaka, and Wen-Hua Chou 651
Cloning and Sequence Analysis
of cDNA Encoding Endopolygalacturonase I from Stereum purpureum
Kazuo Miyairi, Mineo Senda, Masaki Watanabe, Yuji Hasui, and Toshikatsu
Okuno 655
Total Synthesis
of (+)-Phrymarolin I from (+)-Malic Acid
Momotoshi Okazaki, Fumito Ishibashi, Yoshihiro Shuto, and Eiji Taniguchi
660
Quantitative Study
of Yeast Growth in the Presence of Added Ethanol and Methanol Using a Calorimetric
Approach Oana-Arina Antoce, Vasile Antoce,
Katsutada Takahashi, and Fumiki Yoshizako 664
Secretory Production
of Erythropoietin and the Extracellular Domain of the Erythropoietin Receptor
by Bacillus brevis : Affinity Purification and Characterization
Masaya Nagao, Katsunori Inoue, Sung Kwon Moon, Seiji Masuda, Hiroaki
Takagi, Shigezo Udaka, and Ryuzo Sasaki 670
Purification
and Characterization of Glycoprotein from the Skin Mucus of the Rainbow
Trout, Salmo gairdneri
Toshihisa Sumi, Yoichiro Hama, Daisuke Maruyama, Makio Asakawa, and
Hiroki Nakagawa 675
Purification and
Characterization of the Chitinase (ChiA) from Enterobacter sp. G-1
Jae Kweon Park, Kenji Morita, Ikuo Fukumoto, Yukikazu Yamasaki, Tsuyoshi
Nakagawa, Makoto Kawamukai, and Hideyuki Matsuda 684
The Amino Acid Sequence of Mitogenic
Lectin-B from the Roots of Pokeweed (Phytolacca americana)
Ken-ichi Yamaguchi, Noriko Yurino, Mitsutaka Kino, Masatsune Ishiguro,
and Gunki Funatsu 690
Synthesis by an ƒ¿-Glucosidase
of Glycosyl-trehaloses with an Isomaltosyl Residue
Masashi Kurimoto, Tomoyuki Nishimoto, Tetsuya Nakada, Hiroto Chaen,
Shigeharu Fukuda, and Yoshio Tsujisaka 699
Characterization of IKI1
and IKI3 Genes Conferring pGKL Killer Sensitivity on Saccharomyces cerevisiae
Hirohiko Yajima, Masao Tokunaga, Akiko Nakayama-Murayama, and Fumio
Hishinuma 704
A Novel Metalloproteinase, Almelysin,
from a Marine Bacterium, Alteromonas sp. No. 3696: Purification and Characterization
Masahiro Shibata, Saori Takahashi, Ryoichi Sato, and Kohei Oda 710
Effect of Tween 20 on the Oxidative
Stability of Sodium Linoleate and Sodium Docosahexaenoate
Kazuo Miyashita, Nobuko Inukai, and Toru Ota 716
Increase of the Protease
Activity of Aqualysin I, a Thermostable Serine Protease, by Replacing Asn219
near the Catalytic Residue Ser222
Shie-Jea Lin, Dong-Wook Kim, Yoko Ryugo, Takayoshi Wakagi, and Hiroshi
Matsuzawa 718
Finding of UDP-Glucose: Nicotinic
Acid-N-glucosyltransferase Activity in Cultured Tobacco Cells and Its Properties
Hiroshi Taguchi, Kazuyoshi Sasatani, Hiroshi Nishitani, and Katsuzumi
Okumura 720
Stimulating Effect of
Xanthene Dyes on Immunoglobulin Produced in vitro by Rat Spleen Lymphocytes
Yuichiro Kuramoto, Koji Yamada, Beong Ou Lim, and Michihiro Sugano
723
A New Filter Assay in the Study
of Lysyl-tRNA Synthetase from Bacillus stearothermophilus
Teisuke Takita Satoshi Hashimoto, Kuniyo Inouye, and Ben'ichiro Tonomura
726
Zinc Resistance of Methylobacterium
Species
Takashi Kunito, Satoshi Shibata, Satoshi Matsumoto, and Hiroshi Oyaizu
729
Purification and
Properties of a Cysteine Proteinase Occurring in Dormant Wheat Seeds
Masaharu Kuroda, Toshihiro Kiyosaki, Soichi Arai, and Keiko Abe 732
Screening of Bacterial
Cellulose-producing Acetobacter Strains Suitable for Sucrose as a Carbon
Source
Akira Seto, Yukiko Kojima, Naoto Tonouchi, Takayasu Tsuchida, and Fumihiro
Yoshinaga 735
Spectroscopic Analysis of the
Cytoagglutinating Activity of Abrin-b Isolated from Abrus precatorius Seeds
against Leukemic Cells
Hideki Ohba, Tetsuya Toyokawa, Seiji Yasuda, Tomoaki Hoshino, Kyogo
Itoh, and Nobuyuki Yamasaki 737
Spasmolytic Activity of
Aurapten Analogs
Yasumasa Yamada, Masako Okamoto, Hiroe Kikuzaki, and Nobuji Nakatani
740
Total Synthesis of (+)-Paulownin
Momotoshi Okazaki, Fumito Ishibashi, Yoshihiro Shuto, and Eiji Taniguchi
743
Synthesis of ƒ¿-Clausenan
Hidetaka Tsukasa 746
Variety of Intracellular
Products among Recombinants Obtained by Interspecific Protoplast Fusion
in Streptomycetes
Masanori Okanishi, Yumiko Yamaura, and Takaki Furuta 748
Short Communication Asymmetric
Total Syntheses of (+)-Coronafacic Acid and (+)-Coronatine
Hiroaki Toshima, Shinji Nara, and Akitami Ichihara 752
Short Communication Recognition
of Osteopontin by Rat Bone Marrow Derived Osteoblastic Primary Cells
Tomoko Yabe, Atsuko Nemoto, and Toshimasa Uemura 754
-1-
Review
Molecular Biology of the Pore-forming
Cytolysins from Staphylococcus aureus, ƒ¿- and ƒÁ-Hemolysins and Leukocidin
Toshio Tomita and Yoshiyuki Kamio*
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Amamiya-machi 1-1, Tsutsumi-dori, Aoba-ku, Sendai 981, Japan
@Staphylococcus aureus is an important opportunistic pathogen. It produces
a variety of extracellular proteins, which may play important roles in
infections by this bacterium. Staphyloccal ƒ¿-toxin is a pore-forming 33-kDa
polypeptide. In the first part of this article, we will refer to the roles
of cell membranes in the pore formation by ƒ¿-toxin as well as the molecular
dissection of ƒ¿-toxin for understanding its pore-forming nature. Staphylococcal
ƒÁ-hemolysin and leukocidin are bi-component cytolysins, which have different
cell specificities towards erythrocytes and leukocytes, respectively. We
have found that these bi-component cytolysins share a common component.
In the second part of this article, we will refer to the current status
of knowledge of molecular cloning of the genes coding for ƒÁ-hemolysin
and leukocidin, molecular domains of the toxins which decide the cell specificities,
and mode of action of these bi-component toxins.
Key words: Staphylococcus aureus; pore-forming toxins; ƒ¿-hemolysin; ƒÁ-hemolysin;
leukocidin
-2-
Cytotoxicity of Cholestane 3ƒÀ,5ƒ¿,6ƒÀ-Triol
on Cultured Intestinal Epithelial Crypt Cells (IEC-6)
Kimiko Ohtani,õ Tomoyo Terada, Masaharu Kamei,* and Isao Matsui-Yuasa*
Faculty of Human Life Science, Osaka City University, 3-3-138 Sugimoto,
Sumiyoshi-ku, Osaka 558, Japan
* Osaka City Institute of Public Health and Environmental Science, 8-34
Higashiuemachi, Tennoji-ku, Osaka 543, Japan
Received April 22, 1996
@The effects of cholestane 3ƒÀ,5ƒ¿,6ƒÀ-triol on intestinal epithelial
crypt cells were investigated using the IEC-6 cell line. Cholestane 3ƒÀ,5ƒ¿,6ƒÀ-triol
decreased SH groups (glutathione and protein SH) in the cell, and showed
cytotoxicity in a time-dependent manner. Although the concentration of
cholestane 3ƒÀ,5ƒ¿,6ƒÀ-triol used in this study (100 ƒÊM) was very high
compared with that in plasma of experimental animals, cholestane 3ƒÀ,5ƒ¿,6ƒÀ-triol
did not show any cytotoxicity on IEC-6 cells without fetal calf serum (FCS).
The level of cytotoxicity was dependent upon the concentration of FCS in
the culture medium. Unknown components in FSC (not VLDL or LDL) were suggested
to be associated with the cytotoxicity of cholestane 3ƒÀ,5ƒ¿,6ƒÀ-triol.
Moreover, the fact that even heat-treated FCS (100‹C for 30 min) still
mediated the cytotoxicity suggested the participation of non-protein components.
Key words: cholesterol oxide; cholestane 3ƒÀ,5ƒ¿,6ƒÀ-triol; IEC-6 cell;
intestinal epithelial crypt cell; cytotoxicity
-3-
The Phylogeny of Species of the
Genus Issatchenkia KUDRIAVZEV (Saccharomycetaceae) Based on the Partial
Sequences of 18S and 26S Ribosomal RNAs
Yuzo Yamada,õ Jun-ichi Yano, Tomoko Suzuki, and Kozaburo Mikata*
Laboratory of Applied Microbiology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422, Japan *Institute for Fermentation, 2-17-85 Juso-honmachi, Yodogawa-ku, Osaka 532, Japan
Received June 5, 1996
@The ten strains of Issatchenkia species were examined for their partial
base sequences of 18S and 26S rRNAs. In the 18S rRNA partial base sequences
(positions 1451-1618, 168 bases), the strains of the species of the genus
Issatchenkia were found to be not uniform phylogenetically. The calculated
base differences numbered 5-0. The strains of Issatchenkia species examined
had 3-1 base differences with the type strain of Pichia membranaefaciens.
Especially, the type strain of Issatchenkia orientalis, the type species
of the genus Issatchenkia was found to be closely related phylogenetically
to that of P. membranaefaciens. The calculated number of base differences
was only one. The base sequences on the fingerprint segment were comprised
of four bases (four kinds of AUAU, CCAU, AUAG, and ACAU), as found in P.
membranaefaciens (ACAA). In the 26S rRNA partial base sequences, the calculated
number of base differences was 8-0 (positions 1611-1835, 225 bases), and
the calculated percent similarities were 61-80 (positions 493-622, 130
bases), within the genus Issatchenkia. Discussion was made phylogenetically
and taxonomically, especially on the phylogenetic relationship between
the type species of the genera Issatchenkia and Pichia and on a circumscription
of the genus Issatchenkia.
Key words: Issatchenkia; Issatchenkia orientalis; partial base sequences
of 18S and 26S rRNAs; phylogeny; taxonomy
-4-
Hydrolysis of Xylan by Aspergillus
niger Immobilized on Non-woven Fabrics
Hiroharu Tokuda,õ Takashi Urata, and Kotoyoshi Nakanishi
Department of Brewing and Fermentation, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya, Tokyo 156, Japan
Received July 2, 1996
@Aspergillus niger, which produces xylan-degrading enzymes, was immobilized
on non-woven fabrics. The maximum xylan hydrolysis activity (15 U/cm3-support)
and the highest stability were obtained when the fungus was immobilized
on non-woven fabric made of silk. The enzymatic properties of the immobilized
preparation were similar to those of the free enzyme. Ten times repeated
batch hydrolysis of birch-wood xylan was done over a period of 450 h. Hydrolysis
of different xylan substrates such as oat spelts and rice bran by the immobilized
mycelia was also investigated.
Key words: immobilization; Aspergillus niger; xylan; hydrolysis; non-woven
fabrics
-5-
Chemical and Functional Properties
of Mutastein, an Inhibitor of Insoluble Glucan Synthesis by Streptococcus
sobrinus
Osamu Hayashida, Keiji Hasumi, and Akira Endo
Department of Applied Biological Science, Faculty of Agriculture, Tokyo Noko University, Fuchu-shi, Tokyo 183, Japan
Received July 15, 1996
@Mutastein, a potent inhibitor of insoluble glucan synthesis by Streptococcus
sobrinus, is a protein with a molecular weight of `2~106. Amino acid
and ELISA analyses suggested that mutastein is a mixture of heterogenous
polymers of ƒ¿-casein contained in the culture medium of the producing
strain, Aspergillus terreus M3328. Mutastein strongly inhibited the primer-dependent
insoluble glucan synthase of S. sobrinus B13. The primer-independent soluble
glucan synthase was not affected by mutastein while primer-dependent soluble
glucan synthase was slightly activated.
Key words: mutastein; glucan synthase; Streptococcus sobrinus
-6-
Purification, Characterization,
and cDNA Cloning of a Novel ƒ¿-Galactosidase from Mortierella vinacea
Hajime Shibuya, Hideyuki Kobayashi,*,õ Taku Sato, Wong-Sin Kim,** Shigeki Yoshida, Satoshi Kaneko,* Kunihiro Kasamo,* and Isao Kusakabe
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305,
Japan
* National Food Research Institute, Ministry of Agriculture, Forestry,
and Fisheries, Ibaraki 305, Japan
** Department of Biology Education, Wonkwang University, Iri-city, Chonbuk
570-749, Korea
Received July 29, 1996
@A novel ƒ¿-galactosidase, designated ƒ¿-galactosidase II, was isolated
from the culture filtrate of Mortierella vinacea. The molecular size of
the purified enzyme estimated by gel filtration was 60 kDa, which agreed
with that, 51-62 kDa, estimated by SDS-PAGE. The enzyme was thermolabile
at neutral pH, but the addition of BSA to the enzyme solution at the concentration
of 0.01% increased its stability considerably. The enzyme appears to be
novel because it showed a distinct substrate specificity from other microbial
ƒ¿-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan,
that is, the enzyme liberated not only side-chain ƒ¿-galactosyl residue
from 63-mono-ƒ¿-D-galactopyranosyl-ƒÀ-1,4-D-mannotetraose but also terminal
ƒ¿-galactosyl residue from 63-mono-ƒ¿-D-galactopyranosyl-ƒÀ-1,4-D-mannotriose.
In addition, the enzyme acted on galactomannans effectively. ƒ¿-Galactosidase
II cDNA was cloned and its nucleotides sequenced. The deduced amino acid
sequence showed that the mature enzyme consisted of 376 amino acid residues
with a molecular mass of 41,334 Da. The derived amino acid sequence of
the enzyme showed 31-49% sequence similarity with those of ƒ¿-galactosidases
from other origins.
Key words: ƒ¿-galactosidase; Mortierella vinacea; cDNA cloning; galactomanno-oligosaccharides;
specificity of ƒ¿-galactosidase
-7-
Purification and Properties of
ƒÀ-Fructofuranosidase from Clostridium perfringens
Makoto Ishimotoõ and Atsuko Nakamura
Tokyo Kaseigakuin University, Aiharamachi 2600, Machida-shi, Tokyo 194-02, Japan
Received July 29, 1996
@ƒÀ-Fructofuranosidase [EC 3.2.1.26] in Clostridium perfringens was induced
in the presence of sucrose and suppressed in the presence of glucose or
maltose. The enzyme seems to be present in protoplasm in a soluble state.
The ƒÀ-fructofuranosidase from C. perfringens cells grown on sucrose was
purified by ammonium sulfate precipitation, DEAE-cellulose chromatography,
Sephadex G-150 gel filtration, and hydroxylapatite chromatography to a
homogeneous state. The molecular weight was 37,000 by gel filtration using
Sephadex G-150 and by SDS-polyacrylamide gel electrophoresis. The amino
acid composition is not much different from those of other microorganisms,
but the Glx content was a little higher. The enzyme was inhibited by heavy
metals, such as Hg2+, Cu2+, and Ag+, as well as pCMB; the activity was
restored by incubating with mercaptoethanol. Fructose and amines including
Tris and aniline had inhibitory effects.
Key words: ƒÀ-fructofuranosidase; invertase; fructosidase; Clostridium
perfringens; spheroplast
-8-
Optimization of the Culture Medium
for Growth and the Kinetics of Lactate Fermentation by Pediococcus sp.
ISK-1
Etmy Herawati and Ayaaki Ishizaki
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812, Japan
Received August 8, 1996
@The growth of a newly isolated strain of Pediococcus sp., designated
ISK-1, was very slow and the concentration of cells in the medium remained
low. Fermentation with an initial 30 g/liter glucose required about 60
h. To stimulate fermentation, we attempted to optimize the medium by flask
culture and jar fermentation tests. Mevalonic acid and mieki (soy bean
hydrolyzate) stimulated fermentation and increased the rate of formation
of DL-lactate. Kinetic analysis of the fermentation showed that mevalonic
acid markedly increased the specific glucose consumption rate and the specific
lactate production rate. Mieki and mevalonic acid had a synergistic effect,
but the effect of mevalonic acid was different from that of mieki.
Key words: Pediococcus sp. ISK-1; lactate fermentation; kinetics studies;
mieki; mevalonic acid
-9-
Microbial Extracellular Glycolipid
Induction of Differentiation and Inhibition of the Protein Kinase C Activity
of Human Promyelocytic Leukemia Cell Line HL60
Hiroko Isoda, Dai Kitamoto,* Hiroshi Shinmoto,** Masatoshi Matsumura, and Tadaatsu Nakaharaõ
Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305, Japan
* National Chemical Laboratory for Industry Agency of Industrial Science
& Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan
** National Food Research Institute, Ministry of Agriculture, Forestry,
and Fisheries, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan
Received August 22, 1996
@The biological activities of 7 microbial extracellular glycolipids including
mannosylerythritol lipid (MEL)-A, MEL-B, polyol lipid (PL), rhamnolipid
(RL), sophorose lipid (SL), succinoyl trehalose lipid (STL)-1, and STL-3
were investigated. All glycolipids except for RL were found to induce cell
differentiation instead of cell proliferation in the human promyelocytic
leukemia cell line HL60. To identify the differentiation direction of the
induced cells, the leukocyte esterase activities were cytologically investigated,
and the results showed that MEL-A, MEL-B, and PL induced HL60 to differentiate
into granulocytes, while SL, STL-1, and STL-3 induced differentiation into
monocytes. The 6 effective glycolipids also increased nitroblue tetrazolium
(NBT) reducing ability, which is a common differentiation-associated characteristic
in monocytes and granulocytes. Furthermore, it was also observed that these
6 glycolipids inhibited the activity of phospholipid- and Ca2+-dependent
protein kinase. Additionally, the 6 effective glycolipids also induced
the human myelogenous leukemia cell line K562 and the human basophilic
leukemia cell line KU812 to differentiate into monocytes, granulocytes,
and megakaryocytes.
Key words: biosurfactant; glycolipid; leukemia; differentiation; PKC
-10-
Purification and Some Properties of
Endo-1,4-ƒÀ-D-xylanase from a Fresh-water Mollusc, Pomacea insularus (de
Ordigny)
Izumi Yamaura, Takahiro Koga, Toshihiko Matsumoto, and Tetsuo Kato
Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Kumamoto 860, Japan
Received August 30, 1996
@Endo-1,4-ƒÀ-D-xylanase (EC 3.2.1.8) was purified from viscera of a fresh-water
mollusc, Pomacea insularus (de Ordigny). The purified enzyme, with a molecular
weight of 47,000, gave a single protein band in sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The amino-terminal sequence
was Ala-Ala-Gly-Ala-Gly-Val-Thr-Ser-Glu-Lys-Asp-Arg-Leu-Arg-Arg-Ser-Asp-Lys-Thr-Val-His-Val-Asn-.
The enzyme was stable from pH about 4.5 to 9.5 and had its maximum activity
at pH about 5.5. The purified enzyme produced X2, X3, X4, and larger xylooligosaccharides
from birchwood xylan. The enzyme activity was greatly inhibited by Ag+,
Hg2+, Cu2+, N-bromosuccinimide, and p-chloromercuribenzoic acid. On the
other hand, the enzyme activity was greatly elevated by the addition of
chloride ion.
Key words: fresh-water mollusc; Pomacea insularus ; ƒÀ-1,4-xylanase; N-terminal
sequence; xylooligosaccharides
-11-
Non-radioactive Adenosine 5'-Phosphosulfate
Sulfotransferase Assay by Coupling with Sulfite Reductase and O-Acetylserine(thiol)lyase
Takeshi Araõ and Jiro Sekiya
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan
Received September 17, 1996
@Adenosine 5'-phosphosulfate (APS) sulfotransferase is thought to be an
enzyme that transfers the sulfo-group of APS to a carrier compound with
a thiol group in the assimilatory sulfate reduction pathway of higher plants.
We developed a rapid, non-radioactive assay for APS sulfotransferase. Sulfite
released by APS sulfotransferase reaction in the presence of excess dithiothreitol
was further converted to cysteine by coupling with yeast sulfite reductase
and cabbage O-acetylserine(thiol)lyase. The cysteine thus formed was measured
colorimetrically. By this method, 5 to 300 nmol of sulfite could be assessed.
When the method was applied to APS sulfotransferase, the enzyme activity
was APS-dependent with the partially purified enzyme. We could also detect
APS sulfotransferase activity in some higher plants by this method.
Key words: adenosine 5'-phosphosulfate; adenosine 5'-phosphosulfate sulfotransferase;
enzyme assay; higher plants
-12-
Some Properties and Physiological
Roles of Phosphoenolpyruvate Carboxylase in Rhodopseudomonas sp. No. 7
Grown on Ethanol under Anaerobic-light Conditions
Megumi Sadaie, Takanari Nagano, Tomoaki Suzuki, Hirofumi Shinoyama, and Takaaki Fujiiõ
Department of Bioproduction Science, Faculty of Horticulture, Chiba University, 648 Matsudo, Matsudo-shi, Chiba 271, Japan
Received September 20, 1996
@Phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) and phosphoenolpyruvate
kinase (EC 4.1.1.49 <ATP>, PEPK) were detected in cells of Rhodopseudomonas
sp. No. 7 grown photoanaerobically on ethanol and acetate. The activity
of PEPC was about 3 times higher in the ethanol-grown cells than the acetate-grown
cells. PEPC was purified as an electrophoretically homogeneous and stable
protein (Mr, about 400,000; subunit Mr, about 102,000). The enzyme absolutely
required Mn2+ or Mg2+ for the appearance of its activity. Acetyl CoA (40
ƒÊM) reduced the Km against phosphoenolpyruvate and HCO-(/)3 to about 1/20
(0.23 mM) and 1/2 (1.7 mM), respectively. At that time, the V(/)max increased
to about 30 times (90 ƒÊmol/min/mg protein). In the presence of acetyl
CoA the circular dichroism spectrum of the enzyme showed the decrease of
an ƒ¿-helix structure. The enzyme was not activated by fructose-1,6-bisphosphate.
The enzyme was inhibited by aspartate (Ki, 0.208 mM). Besides, the enzyme
was inhibited by nucleotides (ATP and GTP). The enzyme activity was activated
to about 15 times by 3.25 M ethanol.
Key words: acetyl CoA; ethanol assimilation; photosynthetic bacteria; phosphoenolpyruvate
carboxylase; Rhodopseudomonas sp.
-13-
Changes in PAF (Platelet-activating
Factor) Production during Cell Cycle of Yeast Saccharomyces cerevisiae
õ
Reiko Nakayama,õõ Hiroaki Udagawa,* and Hidehiko Kumagai *
Department of Food Science, Kyoto Women's University, Imakumano, Kitahiyoshi-cho,
Higashiyama-ku, Kyoto 605, Japan
* Department of Food Science and Technology, Faculty of Agriculture, Kyoto
University, Kitashirakawa, Sakyou-ku, Kyoto 606, Japan
Received September 20, 1996
@Yeast Saccharomyces cerevisiae cells were cultured synchronously and
the change of platelet-activating factor (PAF) production during the cell
cycle was investigated at each phase of the cycle. The basal PAF contents
of diploid AKU4103 cells in G1 and M phases were higher than those of cells
in S phase. Both diploid and haploid strains showed the same level of PAF
production in response to the calcium ionophore A23187. A23187-stimulated
PAF productions of cells in G1 and M phases were about 20 times higher
than that of cells in S phase. The contents of PAF precursor in G1 and
M phases cells of AKU4103 were higher than those in S phase cells, and
the ratio of A23187-stimulated PAF to the precursor was highest in G1 phase
cells. We also examined the change in a PAF-synthesizing enzyme, acetyltransferase,
activity during the cell cycle using a microsomal fraction. Specific activity
was the highest at G1 phase, and total activity was higher at M phase.
The enzyme activities of cells in S phase of strains AKU4103 and RAY-3Aa
were one-third and one-tenth of those in G1 phase of corresponding cells,
respectively. These results suggest that PAF production was higher at G1
and M phases and lower at S phase, and changes in PAF productivity during
cell cycle were related to the precursor contents and the synthesizing
enzyme activities in those cells. These data suggest that PAF may control
the cell cycle phase in budding yeast.
Key words: PAF (platelet-activating factor); yeast; Saccharomyces cerevisiae;
acetyltransferase; cell cycle
-14-
Purification
and Characterization of Extracellular Poly(ƒÀ-D-1,4-mannuronide) Lyase
from Dendryphiella salina IFO 32139
Tomoko Shimokawa, Shigeki Yoshida,õ Toshio Takeuchi,* Katsumi Murata,* Hideyuki Kobayashi,** and Isao Kusakabe
Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoodai,
Tsukuba-shi, Ibaraki 305, Japan
* Research and Development Department, Kibun Food Chemifa Co., Ltd., 2-1-1
Irifune, Chuo-ku, Tokyo 104, Japan
** National Food Research Institute, Ministry of Agriculture, Forestry,
and Fisheries, Ibaraki 305, Japan
Received September 26, 1996
@An extracellular endo poly(ƒÀ-D-1,4-mannuronide) lyase of Dendryphiella
salina IF 32139 was purified to homogeneity by Q Sepharose FF and Sephacryl
S-200 HR column chromatographies. The purified enzyme had a molecular weight
of 35,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and an isoelectric point of 3.65 by isoelectric focusing. The optimum pH
and temperature for enzyme activity were pH 5.0 and 45‹C, respectively.
The enzyme was stable from pH 4 to 10 and at temperature below 40‹C. Some
divalent cations, Ca2+, Mn2+, and Zn2+, increased the enzyme activity.
Hg2+ and NBS strongly inhibited the activity. This enzyme susceptibly degraded
poly-M, produced a wide range of 4,5-unsaturated oligomannuronic acids,
and further degraded these unsaturated oligomannuronic acids to produce
the unsaturated monomer and dimer as final products.
Key words: poly(ƒÀ-d-1,4-mannuronide) lyase; alginate lyase; alginate;
Dendryphiella salina
-15-
Structural Characteristics
of the Disulfide-reduced Ovotransferrin N-Lobe Analyzed by Protein Fragmentation
Kimihiko Mizutani, Honami Yamashita, Hideo Oe,õ and Masaaki Hirose õõ
The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan
Received September 26, 1996
@The isolated N-lobe (1-332) and C-lobe (342-686) of ovotransferrin were
each found to form an opaque gel by incubation with 70 mM 2-mercaptoethanol
at 37‹C for 24 h, suggesting that a protein conformational change due
to extensive disulfide reduction was the initial mechanism prior to subsequent
intermolecular protein aggregation forming gel networks. To investigate
the conformational state of an extensively disulfide-reduced protein, the
structural characteristics of a two-disulfide protein form that had been
shown to be produced by extensive disulfide reduction of the N-lobe [H.
Yamashita, T. Nakatsuka, and M. Hirose, J. Biol. Chem., 270, 29806-29812
(1995)] were analyzed, as a model protein, by the protein fragmentation
approach. When the two-disulfide form was proteolyzed with chymotrypsin,
a protease-resistant 8-kDa fragment was produced. This fragment was purified
by ion-exchange column chromatography, and chemical analyses and matrix-assisted
laser desorption ionization time-of-flight mass spectrometry clearly showed
that the 8-kDa fragment comprised Ala1-Tyr72 of ovotransferrin. The fragment
was found by amino acid analysis to retain the two intra-chain disulfide
bonds intact. Far-UV CD spectrum analyses revealed that the 8-kDa fragment
retained a native-like folded conformation. These data are consistent with
the view that the N-terminal segment, Ala1-Tyr72, assumed a local native-like
conformation in the two-disulfide form of the ovotransferrin N-lobe.
Key words: ovotransferrin fragment; thiol-dependent gelation; disulfide-reduced
ovotransferrin; ovotransferrin gelation
-16-
New Antioxidative Indophenol-reducing
Phenol Compounds Isolated from the Mortierella sp. Fungus
Akira Hirota, Yasujiro Morimitsu, and Hiroshi Hojo
Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422, Japan
Received September 30, 1996
@Four indophenol-reducing compounds, USF-406A, B, C, and D, were isolated
from a culture filtrate of the fungus, Mortierella sp. USF-406 strain by
chemical screening. Their structures were elucidated by spectroscopic evidence
and chemical degradation. Two were new phenol compounds, N-(4,6-dihydroxy-2,3,5-trimethylbenzoyl)-glycine
and its methyl ester, and the others were their known derivatives. Biosynthetic
studies, using 13C-labelled compounds and 13C-NMR spectroscopy, showed
they were derived from the same tetraketide precursor. Their antioxidative
activities were measured, and they acted as electron donors and also as
radical scavengers. The production of these phenol compounds with antioxidative
activity might have be related to the production of unsaturated fatty acids
by the same fungus.
Key words: indophenol-reducing compound; antioxidant; radical scavenging
activity; Mortierella sp.
-17-
Inhibition of Aldose Reductase
and Sorbitol Accumulation by Astilbin and Taxifolin Dihydroflavonols in
Engelhardtia chrysolepis
Hiroyuki Haraguchi,õ Isao Ohmi, Ayumi Fukuda, Yukiyoshi Tamura,* Kenji Mizutani,* Osamu Tanaka,* and Wen-Hua Chou**
Faculty of Engineering, Fukuyama University, Gakuen-cho, Fukuyama 729-02,
Japan
* Biology and Chemistry Laboratory, Maruzen Pharmaceutical Co., Ltd., Sanada
Takanishi-cho, Fukuyama 729-01, Japan
** South China Institute of Botany, The Academy of Science of China, Guangzhou,
China
Received October 1, 1996
@Dihydroflavonol taxifolin and its glycoside, astilbin, from Engelhardtia
chrysolepis inhibited rat lens and recombinant human aldose reductase.
Taxifolin also inhibited sorbitol accumulation in human red blood cells.
Furthermore, this dihydroflavonol aglycone maintained the clarity of rat
lens incubated with a high concentration of glucose. These dihydroflavonols
may be effective for preventing osmotic stress in hyperglycemia.
Key words: astilbin; taxifolin; aldose reductase; sorbitol accumulation;
Engelhardtia chrysolepis
-18-
Cloning and Sequence Analysis
of cDNA Encoding Endopolygalacturonase I from Stereum purpureum
Kazuo Miyairi, Mineo Senda,* Masaki Watanabe, Yuji Hasui, and Toshikatsu Okuno
Department of Bioresources Science, Faculty of Agriculture, Hirosaki University, and * Hirosaki University Gene Research Center, Hirosaki-shi, Aomori 036, Japan
Received October 7, 1996
@Endopolygalacturonase (endoPG) I was obtained from Stereum purpureum
by an improved easier purification procedure. It was found that EndoPG
I consisted of three glycosilated proteins with the same isoelectric point
and different molecular masses, 42, 45, and 48 kDa, respectively. However,
the enzymatic deglycosilation product of endoPG I gave a single band at
the position corresponding to 39 kDa on SDS-PAGE. Furthermore, the N-terminal
amino acid sequences of three endoPGs were identical one another up to
20 residues. A cDNA library was constructed and positive cDNA clones encoding
endoPG I were isolated by using antibody raised against the purified endoPG
I. Nucleotide sequence analysis of the cDNA disclosed a 1212-bp open reading
frame that encoded 403 amino acid residues. The N-terminal amino acid sequence
(residues 1-20) of endoPG I coincided with the deduced amino acid sequence
starting from the 25th residue. Therefore, the sequence of the first 24
residues represented a signal peptide and the remaining sequence, consisting
of 379 residues, was the mature protein with molecular mass of 39.1 kDa.
The deduced sequence of endoPG I showed 30-45% similarity in comparison
with those of bacterial and fungal endoPGs, and the sequence of putative
active site residues reported for the endoPGs was highly conserved in the
sequence of endoPG I.
Key words: endopolygalacturonase; cDNA cloning; deduced amino acid sequence;
Stereum purpureum
-19-
Total Synthesis of (+)-Phrymarolin
I from (+)-Malic Acid
Momotoshi Okazaki, Fumito Ishibashi,* Yoshihiro Shuto, and Eiji Taniguchi **
Faculty of Agriculture, Ehime University, Matsuyama 790, Japan
* Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan
** Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan
Received October 14, 1996
@(+)-Phrymarolin I was stereoselectively synthesized from (R)-(+)-3-hydroxybutanolide
that had been prepared from (+)-malic acid. The procedure is more efficient
than our previous synthesis in terms of fewer reaction steps and the easier
availability of the starting material.
Key words: stereoselective synthesis; (+)-phrymarolin I; furofuran lignan;
(R)-(+)-3-hydroxybutanolide
-20-
Quantitative Study
of Yeast Growth in the Presence of Added Ethanol and Methanol Using a Calorimetric
Approach Oana-Arina Antoce,*,õ Vasile Antoce,*
Katsutada Takahashi,*,õõ and Fumiki Yoshizako**
* Laboratory of Biophysical Chemistry, College of Agriculture, Osaka
Prefecture University, Sakai, 1-1 Gakuen-cho, Osaka 593, Japan
** Laboratory of Biological Materials and Bioengineering, Research Center
for Advanced Science and Technology, Osaka Prefecture University, Sakai,
1-2 Gakuen-cho, Osaka 593, Japan
Received October 14, 1996
@Using a calorimeter with 24 sample units the heat evolved during incubation
of yeast cultures at 30‹C was detected in the form of growth thermograms.
Ethanol and methanol added to the culture medium produced changes in the
growth thermograms that could be analyzed to calculate the 50% inhibitory
concentration (Ki) and minimum inhibitory concentration (MIC). Correlation
of the heat evolution curves with the number of cells and the turbidity
of the culture was found to be very good. It was found that addition of
ethanol and methanol up to 7.65% had clear effects of inhibition on growth
of all yeast strains studied, reducing the growth rate constant and delaying
growth. However, the amounts of ethanol produced from the nutrients available
in the culture vial was only little affected by the initial addition of
up to 7.65% (v/v) of ethanol or methanol in the medium.
Key words: ethanol inhibition; methanol inhibition; yeast growth; calorimetric
measurement
-21-
Secretory Production
of Erythropoietin and the Extracellular Domain of the Erythropoietin Receptor
by Bacillus brevis : Affinity Purification and Characterization
Masaya Nagao, Katsunori Inoue,* Sung Kwon Moon, Seiji Masuda, Hiroaki Takagi,** Shigezo Udaka,*** and Ryuzo Sasaki
Department of Food Science and Technology, Faculty of Agriculture, Kyoto
University, Kyoto 606-01, Japan
* Medic Co., Ltd., Shiga 520-23, Japan
** Research Laboratory, Higeta Shoyu Co., Ltd., Chiba 288, Japan
*** Department of Brewing and Fermentation, Tokyo University of Agriculture,
Setagaya, Tokyo 156, Japan
Received October 14, 1996
@Bacillus brevis secretes a large amount of cell wall proteins into the
culture medium. For construction of Bacillus brevis expression-secretion
vectors of human erythropoietin (EPO) and the extracellular domain of mouse
erythropoietin receptor (sEPOR), cDNA for each mature form was inserted
into a plasmid containing the promoter region and the signal-peptide encoding
region of a cell wall protein. Culture supernatants of transformants were
affinity purified using a monoclonal antibody-fixed gel for EPO and an
EPO-fixed gel for sEPOR. The affinity purification efficiently removed
unwanted proteins, giving samples with sufficiently high purity to analyze
amino acid sequences of N-terminal regions and biological activities. Combination
of this secretory production and affinity purification may facilitate isolation
of a large amount of pure EPO and sEPOR, and is useful for further understanding
the molecular mechanism of interaction between EPO and EPOR.
Key words: erythropoietin; soluble erythropoietin receptor; Bacillus brevis;
secretory production; affinity purification
-22-
Purification
and Characterization of Glycoprotein from the Skin Mucus of the Rainbow
Trout, Salmo gairdneri
Toshihisa Sumi, Yoichiro Hama, Daisuke Maruyama, Makio Asakawa,* and Hiroki Nakagawaõ
Department of Applied Biological Sciences, Faculty of Agriculture, Saga
University, Honjyo-machi, Saga 840, Japan
* Laboratory of Food Science, Faculty of Education, Kumamoto University,
3-Kurokami, Kumamoto 860, Japan
Received October 21, 1996
@Mucus glycoprotein (RGP) was purified and characterized from the skin
mucus of rainbow trout, Salmo gairdneri. RGP was found to contain 30.1%
NeuAc, 26.0% GalNAc, 5.0% Gal, and 26.0% amino acids. The protein moiety
of RGP is very rich in Thr (32.4 mol%). Neither NeuGc nor KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic
acid) was found in RGP. Alkaline borohydride treatment of RGP yielded a
major disaccharide alditol, NeuAcƒ¿2¨6GalNAc-ol and more than 4 minor
oligosaccharide alditols including NeuAc¨(GalNAcƒ¿1¨)GalNAc-ol. It was
evident that an average RGP molecule has approximately 500 NeuAc-containing
oligosaccharide chains, which are attached to the Thr and Ser residues
of the protein moiety and spaced at an average of 3 amino acids apart.
Key words: sialoglycoprotein; mucus glycoprotein; mucus; rainbow trout;
mucin
-23-
Purification and
Characterization of the Chitinase (ChiA) from Enterobacter sp. G-1
Jae Kweon Park, Kenji Morita, Ikuo Fukumoto, Yukikazu Yamasaki,** Tsuyoshi Nakagawa,* Makoto Kawamukai,õ and Hideyuki Matsuda
Department of Biochemistry and Biotechnology, Faculty of Life and Environmental
Science, and
* Research Institute of Molecular Genetics, Shimane University, 1060 Nishikawatsu,
Matsue, Shimane 690, Japan,
** Institute of Industrial Science and Technology in Shimane Prefecture,
Matsue 690, Japan
Received October 23, 1996
@Enterobacter sp. G-1 which produces chitinolytic and chitosanolytic enzymes,
was previously isolated in our laboratory. One major chitinase, designated
ChiA, was purified 42.9-fold from a culture filtrate of Enterobacter sp.
G-1. To purify the chitinase, ammonium sulfate fractionation, DEAE-Sephadex
A-50 column chromatography, and gel filtration on Sephadex G-100 column
chromatography were used. The ChiA protein had a molecular weight of 60,000
estimated by SDS polyacrylamide gel electrophoresis and an isoelectric
point of 6.6. The optimal pH and optimal temperature of ChiA against colloidal
chitin were pH 7.0, and 40‹C, respectively. The purified ChiA degraded
colloidal chitin mainly to GlcNAc2 with a small amount of GlcNAc3 and GlcNAc4.
ChiA hydrolyzed flaked chitin, colloidal chitin, and ethylenglycol chitin,
but did not hydrolyze carboxymethyl cellulose (CMC), nor >90% deacetylated
flaked chitosan. The chitinase activity was 42% inhibited by 10 mM EDTA,
but was not inhibited by Ca(>12+ (<50 mM) or NaCl (<400 mM). The
purified ChiA hydrolyzed colloidal chitin and chitin-related compounds
in an endo splitting manner.
Key words: Enterobacter sp. G-1; chitinase; chitin; activity staining;
purification
-24-
The Amino Acid Sequence of Mitogenic
Lectin-B from the Roots of Pokeweed (Phytolacca americana)
Ken-ichi Yamaguchi, Noriko Yurino, Mitsutaka Kino, Masatsune Ishiguro, and Gunki Funatsu
Laboratory of Protein Chemistry and Engineering, Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka 812-81, Japan
Received October 23, 1996
@The complete amino acid sequence of pokeweed lectin-B (PL-B) has been
analyzed by first sequencing seven lysylendopeptidase peptides derived
from the reduced and S-pyridylethylated PL-B and then connecting them by
analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated
PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides
linked to Asn96 and Asn139, and has a molecular mass of 34,493 Da. PL-B
is composed of seven repetitive chitin-binding domains having 48-79% sequence
homology with each other. Twelve amino acid residues including eight cysteine
residues in these domains are absolutely conserved in all other chitin-binding
domains of plant lectins and class I chitinases. Also, it was strongly
suggested that the extremely high hemagglutinating and mitogenic activities
of PL-B may be ascribed to its seven-domain structure.
Key words: pokeweed mitogen; lectin; chitin-binding protein; amino acid
sequence
-25-
Synthesis by an ƒ¿-Glucosidase
of Glycosyl-trehaloses with an Isomaltosyl Residue
Masashi Kurimoto,õ Tomoyuki Nishimoto, Tetsuya Nakada, Hiroto Chaen, Shigeharu Fukuda, and Yoshio Tsujisaka*
Hayashibara Biochemical Laboratories, Inc., 7-7 Amase-minamimachi, Okayama
700, Japan
* Hayashibara Institute Corp., 1-2-3 Shimoishii, Okayama 700, Japan
Received October 24, 1996
@Glycosyl-trehaloses with an isomaltosyl residue were synthesized by ƒ¿-glucosidase
from Aspergillus niger by using maltotetraose as a glucosyl donor and trehalose
as the acceptor. The one trisaccharide and two tetrasaccharides formed
were isolated by successive column chromatography. The results of an enzymatic
digestion, methylation analysis, and 13C-NMR studies indicated that these
oligosaccharides were ƒ¿-isomaltosyl ƒ¿-glucoside, ƒ¿-isomaltotriosyl ƒ¿-glucoside
and ƒ¿-isomaltosyl ƒ¿-isomaltoside. These oligosaccharides were not fermented
to an acid by Streptococcus mutans, and they effectively inhibited water-insoluble
glucan synthesis from sucrose by glucosyltransferase. In an in vitro utilization
test with human intestinal bacteria, these oligosaccharides were predominantly
utilized by Bifidobacteria. Key words: trehalose; isomaltosyl residue;
ƒ¿-glucosidase
-26-
Characterization of IKI1
and IKI3 Genes Conferring pGKL Killer Sensitivity on Saccharomyces cerevisiaeõ
Hirohiko Yajima,*,õõ Masao Tokunaga,*,**,õõõ Akiko Nakayama-Murayama,* and Fumio Hishinuma*
* Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi,
Tokyo 194, Japan
** Laboratory of Applied Microbiology, Faculty of Agriculture, Kagoshima
University, 1-21-24 Korimoto, Kagoshima 890, Japan
Received October 31, 1996
@The Saccharomyces cerevisiae iki mutants show an insensitive phenotype
to the pGKL killer toxin, and we have cloned some IKI genes by complementation
of this phenotype [Kishida et al., Biosci. Biotech. Biochem., 60, 798-801
(1996)]. Here, we identified and characterized the IKI1 and IKI3 genes.
DNA sequencing of the genes showed that both have 100% identity with hypothetical
genes identified by the yeast genome project, YHR187w (481,911-480,985
in chromosome VIII) for IKI1, and YLR384c (888,852-892,898 in chromosome
XII) for IKI3. Both are novel genes with no significant identity with other
known genes and they do not belong to any homology domain group, gene family,
or superfamily. The disruption of IKI1 is not lethal, but growth of the
disruptant was slower than that of the wild type at all temperatures examined.
The disruptant was the killer-insensitive phenotype. The sequence of the
IKI1 gene predicted a hydrophilic protein with a molecular mass of 35 kDa
(309 amino acids). A 35-kDa protein band was also detected by immunoblotting
the 25,000~g pellet fraction of the wild type yeast cell lysate. Disruption
of the IKI3 gene is also non-lethal and it has the killer-insensitive phenotype.
Iki3p may contain a transmembrane domain near the NH2-terminal region (97-113
residues in a total of 1349 amino acids).
Key words: linear plasmid pGKL1; killer toxin; iki mutants; IKI1; IKI3
-27-
A Novel Metalloproteinase, Almelysin,
from a Marine Bacterium, Alteromonas sp. No. 3696: Purification and Characterization
Masahiro Shibata, Saori Takahashi, Ryoichi Sato,* and Kohei Odaõ
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute
of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan
* Central Research Institute, Maruha, Wadai, Tsukuba 300-24, Japan
Received November 5, 1996
@We have discovered a novel metalloproteinase, which has high activity
at low temperatures, from the culture supernatant of a marine bacterium.
The strain was identified as Alteromonas sp. No. 3696. The metalloproteinase,
named almelysin, was purified to homogeneity from the cultured supernatant
at 10‹C by two column chromatographies. About 20 mg of purified almelysin
was obtained from 18.4 liters of the culture supernatant. The molecular
mass of almelysin was estimated to be 28 kDa by SDS-PAGE and the isoelectric
point was 4.3. The optimum pH for activity of almelysin was pH 8.5-9.0
and 6.5 using casein and (7-methoxycoumarin-4-yl)acetyl(MOCAc)-Pro-Leu-Gly-Leu-(N
3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)[A2pr(Dnp)]-Ala-Arg-NH2 as
substrates, respectively. Almelysin was stable between pH 7.5-8.0 and below
40‹C. The optimum temperature for the activity was observed to be 40‹C
using both casein and MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 as substrates.
The activity of almelysin was inhibited by such metallo chelators as EDTA
and o-phenanthroline, while talopeptin, phosphoramidon, and SMPI, typical
metalloproteinase inhibitors, had no effect. Almelysin primarily cleaved
the Ala14-Leu15 bond and Phe24-Phe25 bond, and secondarily the Tyr16-Leu17
bond in oxidized insulin B-chain. However, almelysin could not cleave the
His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds, which were cleaved by other
metalloproteinases. These results indicate that the substrate specificity
of almelysin is different from other metalloproteinases. Interestingly,
Alteromonas sp. No. 3696 strain produced another proteinase as well as
almelysin at 25‹C.
Key words: metalloproteinase; marine bacteria; Alteromonas sp.; purification;
characterization
-28-
Note
Effect of Tween 20 on the Oxidative
Stability of Sodium Linoleate and Sodium Docosahexaenoate
Kazuo Miyashita,õ Nobuko Inukai, and Toru Ota
Faculty of Fisheries, Hokkaido University, 3-1-1 Minatocho, Hakodate 041, Japan
Received July 18, 1996
@Sodium linoleate (LA) and sodium docosahexaenoate (DHA) were oxidized
in an aqueous phase with or without Tween 20. The oxidative stability of
LA with Tween 20 was slightly less than that of LA without Tween 20. In
contrast, when DHA was dispersed in a buffer containing Tween 20, the stability
of DHA was markedly increased, suggesting a specific effect of Tween 20
on protecting DHA against oxidation in aqueous micelles. Key words: Tween
20; oxidative stability; sodium salt; DHA; aqueous solution
-29-
Note
Increase of the Protease
Activity of Aqualysin I, a Thermostable Serine Protease, by Replacing Asn219
near the Catalytic Residue Ser222
Shie-Jea Lin, Dong-Wook Kim, Yoko Ryugo, Takayoshi Wakagi, and Hiroshi Matsuzawa
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received September 24, 1996
@Functional role of Asn219 of aqualysin I, a thermostable serine protease
from Thermus aquaticus, was investigated by using site-directed mutagenesis.
Replacement of Asn219 with serine increased the catalytic efficiency (k(/)cat/Km)
for synthetic peptide substrates about twice as much as that of the wild
type, while threonine replacement caused a slight decrease in the efficiency.
Such replacements resulted in a significant change of k(/)cat rather than
Km, indicating that the side chain in the vicinity of the catalytic residue
Ser222 affects the catalytic rate constant.
Key words: aqualysin I; site-directed mutagenesis; catalytic efficiency;
protein engineering; heat-stable protease
-30-
Note
Finding of UDP-Glucose: Nicotinic
Acid-N-glucosyltransferase Activity in Cultured Tobacco Cells and Its Properties
Hiroshi Taguchi,õ Kazuyoshi Sasatani, Hiroshi Nishitani,* and Katsuzumi Okumura
Laboratory of Biological Chemistry, Faculty of Bioresources, Mie University,
Tsu, Mie 514, Japan
* R & D Center, Terumo Corporation, 1500 Inokuchi, Nakai-machi, Ashigarakami-gun,
Kanagawa 259-01, Japan
Received October 3, 1996
@We have already identified N-(ƒÀ-D-glucopyranosyl)nicotinic acid, which
was isolated from cultured tobacco cells, as a novel niacin metabolite.
The enzyme activity for the biosynthesis of this compound, UDP-glucose:
nicotinic acid-N-glucosyltransferase, was found in cell-free extracts from
cultured cells of tobacco (Nicotiana tabacum XD-6) and properties were
investigated in this experiment. The general catalytic properties were
as follows: optimum temperature was 25‹C, optimum pH was 6.1, the Km for
NiA and UDP-glucose were calculated at 220 ƒÊM and 4.3 mM, respectively.
The enzyme activity was lowered by heavy metal ions, isonicotinic acid,
benzoic acid, and 6-hydroxynicotinic acid. The roles of niacin metabolites
in cultured tobacco cells are discussed in relation to the enzyme.
Key words: UDP-glucose: nicotinic acid-N-glucosyltransferase; N-(ƒÀ-d-glucopyranosyl)nicotinic
acid; nicotinic acid; UDP-glucose; cultured tobacco cells
-31-
Note
Stimulating Effect of
Xanthene Dyes on Immunoglobulin Produced in vitro by Rat Spleen Lymphocytes
Yuichiro Kuramoto,õ Koji Yamada, Beong Ou Lim, and Michihiro Sugano
Laboratory of Food Science, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University 46-09, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-81, Japan
Received October 7, 1996
@The effects of food additives on immunoglobulin produced in rat splenic
lymphocytes were examined. The xanthene dye, Rose Bengal, enhanced IgE
production, while inhibiting the production of IgG and IgM, at 50 ƒÊM.
Among the xanthene dyes, Rose Bengal having 4 iodine and 4 chlorine atoms
exerted the highest Ig production-regulating activity in splenocytes, and
dihalogenated fluorescein, a diiodo compound, exerted similar activity,
while the dichloro and dibromo compounds did not. These results suggest
that halogen atoms, especially the iodine atom, in xanthene dyes play an
important role in regulation of Ig production.
Key words: xanthene dye; IgE; spleen lymphocytes
-32-
Note
A New Filter Assay in the Study
of Lysyl-tRNA Synthetase from Bacillus stearothermophilusõ
Teisuke Takita,õõ Satoshi Hashimoto, Kuniyo Inouye, and Ben'ichiro Tonomuraõõõ
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606-01, Japan
Received October 7, 1996
@A glass microfiber filter was found useful for detection of the complex
of lysyl-tRNA synthetase from Bacillus stearothermophilus with lysyladenylate.
Detected c.p.m. values of the 3H-labeled complex trapped by the glass microfiber
filter was about 2-fold higher than those by nitrocellulose filter and
about 3-fold higher than those by DEAE-cellulose filter because of its
higher counting efficiency. The use of the glass fiber filter is more suitable
for the lysyl-tRNA synthetase assay than the use of either nitrocellulose
or DEAE-cellulose filters, which were used for other aminoacyl-tRNA synthetases.
Effects of pH on the filter assay of lysyl-tRNA synthetase activity using
the glass microfiber filter were also examined.
Key words: aminoacyl-tRNA synthetase; filter assay; lysyladenylate; lysyl-tRNA
synthetase
-33-
Note
Zinc Resistance of Methylobacterium
Species õ
Takashi Kunito, Satoshi Shibata, Satoshi Matsumoto, and Hiroshi Oyaizu
Department of Applied Biological Chemistry, Graduate School of Agriculture and Agricultural Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received October 7, 1996
@Zinc (Zn)-resistant Methylobacterium strains were isolated from soils
collected in Japan. Although the soils used had never been polluted with
Zn, Zn-resistant Methylobacterium-like red colonies were detected at 102
to 103 per gram of dry soil. The 16S ribosomal DNA sequence of the representative
eleven strains were most similar to Methylobacterium radiotolerans. Furthermore,
all of Methylobacterium strains from culture collections showed moderate
or high Zn resistance. Thus, Zn resistance might be an inherent characteristic
in Methylobacterium species.
Key words: zinc resistance; heavy metal; Methylobacterium; methylotroph;
16S rDNA
-34-
Note
Purification and
Properties of a Cysteine Proteinase Occurring in Dormant Wheat Seeds
Masaharu Kuroda,* Toshihiro Kiyosaki, Soichi Arai, and Keiko Abe *
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Received October 9, 1996
@A cysteine proteinase was purified from dormant seeds of wheat (Triticum
aestivum, cultivar Norin 61) and its molecular mass was estimated to be
about 23 kDa by gel filtration. The Km of this proteinase at pH 5.5 was
calculated as 2 ƒÊM for Z-Phe-Arg-MCA, and its activity was inhibited by
cysteine proteinase inhibitors including antipain, E-64, and leupeptin.
Oryzacystatin-I, a proteinaceous cysteine proteinase inhibitor in rice
seeds, also inhibited its activity.
Key words: cysteine proteinase; Triticum aestivum; wheat; dormant seed
-35-
Note
Screening of Bacterial
Cellulose-producing Acetobacter Strains Suitable for Sucrose as a Carbon
Source
Akira Seto, Yukiko Kojima, Naoto Tonouchi, Takayasu Tsuchida, and Fumihiro Yoshinaga
Bio-Polymer Research Co., Ltd., 3-2-1 Sakato, Takatsu-ku, Kawasaki 213, Japan
Received October 15, 1996
@For potential application in the economic production of bacterial cellulose,
cellulose-producing Acetobacter strains capable of using sucrose as a carbon
source were screened for using corn steep liquor as a nitrogen source.
A total of 1500 strains were isolated and four were selected through static
and shaking flask cultures. Much higher cellulose accumulation was observed
in a jar fermentor using these strains in sucrose-CSL medium than seen
using the previously isolated strain, BPR2001, in fructose-CSL medium.
It was found that these strains also had high cellulose production from
glucose. The pH of the cultures of the isolated strains did not decrease,
and it was suggested that these strains have low glucose-oxidizing ability.
Key words: Acetobacter; cellulose; sucrose; screening; glucose
-36-
Note
Spectroscopic Analysis of the
Cytoagglutinating Activity of Abrin-b Isolated from Abrus precatorius Seeds
against Leukemic Cells
Hideki Ohba,õ Tetsuya Toyokawa, Seiji Yasuda, Tomoaki Hoshino,* Kyogo Itoh,* and Nobuyuki Yamasaki**
Kyushu National Industrial Research Institute, Shuku-machi, Tosu, Saga
841, Japan
* Department of Immunology, Kurume University School of Medicine, Kurume
830, Japan
** Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University,
Fukuoka 812, Japan
Received October 21, 1996
@The cytoagglutinating activity of abrin-b, a toxic lectin isolated from
Abrus precatorius seeds, against cultured cell strains derived from accute
lymphoblast leukemia (ALL) was investigated by visible (VIS) spectroscopy.
Upon addition of abrin-b, the turbidity at 600nm of cell suspension decreased
and this change could be recorded as the cytoagglutination curve. From
this curve, the cytoagglutination velocity (CV) and cytoagglutination intensity
(CI) of each cell strain was measured. Each cell strain showed the respective
CV and CI values and the cell strains derived from the T cell line were
strongly agglutinated by abrin-b compared with those derived from the B
cell line. Further, it has become apparent that the cytoagglutinating activity
increased with an increase in the order of the differentiation of cell
strains.
Key words: spectroscopic analysis; cytoagglutinating activity; abrin-b;
accute lymphoblast leukemia
-37-
Note
Spasmolytic Activity of
Aurapten Analogs
Yasumasa Yamada,õ Masako Okamoto, Hiroe Kikuzaki,* and Nobuji Nakatani *
Department of Food Science and Nutrition, Faculty of Human Life and
Science, Doshisha Women's College, Kamigyo-ku, Kyoto 602, Japan
* Department of Food and Nutrition, Faculty of Human Life Science, Osaka
City University, Sumiyoshi-ku, Osaka 558, Japan
Received October 24, 1996
@Seven coumaric compounds analogous to aurapten were synthesized. Their
spasmolytic activity against Ba2+, acetylcholine and histamine was evaluated
to investigate their structure-activity relationship. The results of the
bioassay demonstrated the important roles of the cis type of double bond
at C-2' and the epoxide between C-6' and 7'.
Key words: spasmolytic activity; aurapten; acetylcholine; histamine; barium
ion
-38-
Note
Total Synthesis of (+)-Paulownin
Momotoshi Okazaki, Fumito Ishibashi,* Yoshihiro Shuto, and Eiji Taniguchi **
Faculty of Agriculture, Ehime University, Matsuyama 790, Japan
* Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan
** Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan
Received November 5, 1996
@(+)-Paulownin, a furofuran lignan from Paulownia tomentosa, was stereoselectively
synthesized from (R)-(+)-3-hydroxybutanolide in 12 steps with a yield of
4.4%.
Key words: stereoselective synthesis; (+)-paulownin; furofuran lignan;
(R)-(+)-3-hydroxybutanolide
-39-
Note
Synthesis of ƒ¿-Clausenan
Hidetaka Tsukasa
Toyotama Koryo Co. Ltd., 1-14-5 Nihonbashi Kakigaracho, Chuo-ku, Tokyo 103, Japan
Received November 15, 1996
@ƒ¿-Clausenan [3-methyl-2-(3-methyl-1,3-butadienyl)furan], which is found
in Clausena willdenovii, was synthesized by starting from methyl 3-(3-methyl-2-furanyl)propionate
in a 34% overall yield in 4 steps.
Key words: ƒ¿-clausenan; Clausena willdenovii; methyl 3-formylpropionate;
methyl 3-(3-methyl-2-furanyl)propionate; methyl 4-oxo-5-methyl-6-formylhexanoate
-40-
Note
Variety of Intracellular
Products among Recombinants Obtained by Interspecific Protoplast Fusion
in Streptomycetes
Masanori Okanishi, Yumiko Yamaura, and Takaki Furuta
Faculty of Agriculture, Tamagawa University, Tamagawagakuen, Machida-shi, Tokyo 194, Japan
Received November 21, 1996
@This paper describes with the multiformity of intracellular prod ucts
among the recombinants obtained by interspecific protoplast fusion between
Streptomyces griseus and S. griseoruber and between S. griseus and S. rochei.
The products were extracted from the mycelium with methanol and analyzed
by thin-layer chromatography using three detection methods. Detection with
anisaldehyde-sulfuric acid reagent and Ehrlich's reagent showed that 29-37%
of the recombinants produced new products different from those of the parental
species, while about 50% of the recombinants did not produce any detectable
substances. In a bioautogram against Bacillus subtilis, about 50% of the
recombinants showed clear antibiotic activity in their methanol extracts,
while the parental species did not. The distribution of the products (parental,
mixed, new, or not expressed types) among the recombinants is discussed
compared with that of taxonomic allelo-characters.
Key words: interspecific hybrids; intracellular products; protoplast fusion;
secondary metabolites; streptomycetes
-41-
Short Communication Asymmetric
Total Syntheses of (+)-Coronafacic Acid and (+)-Coronatine
Hiroaki Toshima, Shinji Nara, and Akitami Ichihara
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Received December 6, 1996
@An asymmetric total synthesis of (+)-coronafacic acid, starting from
(R)-(+)-4-acetoxy-2-cyclopen-1-one as a chiral source, was accomplished.
Construction of the 1-hydrindanone framework was carried out by using intramolecular
1,6-conjugate addition as the key step. Coupling between (+)-coronafacic
acid and protected coronamic acid, and subsequent deprotection provided
(+)-coronatine. This is the first asymmetric total synthesis of (+)-coronatine.
Key words: coronafacic acid; coronatine; coronamic acid; asymmetric synthesis;
1,6-conjugate addition
-42-
Short Communication Recognition
of Osteopontin by Rat Bone Marrow Derived Osteoblastic Primary Cells
Tomoko Yabe, Atsuko Nemoto, and Toshimasa Uemuraõ
Bionic Design Group, National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba Research Center, Ibaraki 305, Japan
Received December 11, 1996
@To study the role of osteopontin, we did cell adhesion and ALP assays
of rat bone marrow osteoblastic cells (RBMO) on collagen Type I and osteopontin
surfaces. The RBMO proved to adhere much more strongly to the osteopontin
and to have higher ALP activity on the osteopontin, which suggests that
pre-osteoblasts differentiate into osteoblasts that form bone by recognizing
osteopontins.
Key words: osteopontin; osteoblast