(Vol.61 No.5 1997)
Studies on Biosynthesis of Brassinosteroids
Akira Sakurai and ShozoFujioka 757
Effect of Dietary n-3/n-6 Fatty Acid Ratio on the Total
Count, Fatty Acid Composition, and Histamine and Leukotriene Concentrations
of Mast Cells in Tunica Mucosa
Bronchiorum of Type I Allergic Guinea Pig
Masanori Kuwamori, Masahiro Wada, Toshichika Takita, Tadahiro Tadokoro,
Akio Maekawa, and Satoshi Innami 763
Oxygen Sensitivity of NifA Protein of Azospirillum lipoferum
FS as Suggested by Gene Cloning and Expression in Escherichia coli
Toru Shigematsu, Akio Inoue, Makoto Hidaka, Haruhiko Masaki, and Takeshi
Uozumi 768
In Vitro and in Vivo Anti-platelet Effects of Enzymatic
Hydrolysates of Collagen and Collagen-related Peptides
Isao Nonaka, Shin-ichiro Katsuda,Takashi Ohmori, Tamotsu Shigehisa,
Tatsuyoshi Nakagami, and Susumu Maruyama 772
Growth Inhibition, Morphological Change, and Ectoenzyme
Release of LLC-PK1 Cells by Phosphatidylinositol-specific Phospholipase
C of Bacillus thuringiensis
Chisako Itami, Yukio Kimura, Ryo Taguchi, Hiroh Ikezawa, and Toshikatsu
Nakabayashi 776
Characterization of the Protein and Glycan Moieties in
Different Forms of Bovine Lactoferrin
Xiu-Yun Ye, Toshihide Nishimura, and Shigeru Yoshida 782
Thermal Gelation Profile Changes in Reconstituted Actomyosin
due to Storage under a High Salt Concentration and Low Temperature
Hiroyuki Tanji, Yoshihide Ikeuchi, Masatoshi Yoshizawa, and Atsushi
Suzuki 787
Characterization of 30-kDa Fragments Derived from -Conglycinin
Degradation Process during Germination and Seedling Growth of Soybean
Misako Kawai, Shun'ichi Suzuki, Minao Asano, Tetsuya Miwa, and Hiroshiro
Shibai 794
Efficient Expression of Mono- and Diacylglycerol Lipase
Gene from Penicillium camembertii U-150 in Aspergillus oryzae under the
Control of Its Own Promoter
Shotaro Yamaguchi, Kazuyuki Takeuchi, Tamio Mase, and Akira Matsuura,
800
Synthesis of 24a-Substituted Milbemycin A4 Derivatives
and Their Acaricidal Activity against Tetranychus urticae
Yoshihisa Tsukamoto, Hisaki Kajino, Kazuo Sato, Keiji Tanaka, and Toshiaki
Yanai 806
Purification of Amylases and Other Enzymes by a Forced-affinity
Chromatography Method
Mikihiko Kobayashi, Yutaka Sasaki, and Shoichi Kobayashi 813
Lipid Peroxidation in Linoleic Acid Micelles Caused by
H2O2 in the Presence of Myoglobin
Tsutomu Nakayama, Youhei Chiba, and Kei Hashimoto 817
Incorporation of Farnesyl Pyrophosphate Derivertives into
Abscisic Acid and Its Biosynthetic Intermediates in Cercospora cruenta
Hirotaka Yamamoto and Takayuki Oritani 821
Enzymatic Synthesis of Mannosyl-cyclodextrin by -Mannosidase
from Jack Bean
Kenichi Hamayasu, Koji Hara, Koki Fujita, Yukio Kondo, Hitoshi Hashimoto,
Toshiko Tanimoto, Kyoko Koizumi, Hirofumi Nakano, and Sumio Kitahata 825
High-level Expression of the Chemically Synthesized Gene
for Microbial Transglutaminase from Streptoverticillium in Escherichia
coli
Misako Kawai, Shino Takehana, and Hiroshi Takagi 830
Effects of Dietary Sesaminol and Sesamin on Eicosanoid
Production and Immunoglobulin Level in Rats Given Ethanol
Michiko Nonaka, Kanae Yamashita, Yoshie Iizuka, Mitsuo Namiki, and
Michihiro Sugano 836
High Level Expression of XMP Aminase in Escherichia coli
and Its Application for the Industrial Production of 5'-Guanylic Acid
Tatsuro Fujio, Tatsunari Nishi, Seiga Ito, and Akihiko Maruyama 840
Sequential Binding of Staphylococcal -Hemolysin to Human
Erythrocytes and Complex Formation of the Hemolysin on the Cell Surface
Jun Kaneko, Toshiko Ozawa, Toshio Tomita, and Yoshiyuki Kamio 846
Protoplast Formation from Schizophyllum commune by a Culture
Filtrate of Bacillus circulans KA-304 Grown on a Cell-wall Preparation
of S. commune as a Carbon Source
Katsushige Mizuno, Osamu Kimura, and Takashi Tachiki 852
Purification and Characterization of a Dipeptidyl Carboxypeptidase
from Pseudomonas sp. WO24
Wataru Ogasawara, Nobuhide Abe, Takashi Hagio, Hirofumi Okada, and
Yasushi Morikawa 858
Effects of Ethylene and Gibberellins on the Elongation
of Rice Seedlings (Oryza sativa L.)
Koji Furukawa, Young-Yell Yang, Ichiro Honda, Tadashi Yanagisawa, Akira
Sakurai, Nobutaka Takahashi, and Yuji Kamiya 864
Acyl Amino Acid Derivatives as Novel Inhibitors of Influenza
Neuraminidase
Mitsuyo Kondoh, Toshiyuki Furutani, Masayuki Azuma, Hiroshi Ooshima,
and Jyoji Kato 870
Cloning and Sequencing of a cDNA Encoding -Glucosidase
from Sugar Beet
Hirokazu Matsui, Shunsuke Iwanami, Hiroyuki Ito, Haruhide Mori, Mamoru
Honma, and Seiya Chiba 875
Absolute Configurations of Some Hydroxy-fatty Acids Produced
by the Insect Genus Laccifer
Akio Ichikawa, Haruko Takahashi, Takashi Ooi, and Takenori Kusumi 881
Isolation and Partial Amino Acid Sequence of Bacteriocins
Produced by Lactobacillus acidophilus
Takatsugu Tahara and Kazuo Kanatani 884
Stimulating Effect of Ileal Pancreaticobiliary Secretion
on Ileal Apolipoprotein A-IV mRNA Expression in Fasted Rats
Kei Sonoyama, Reiko Fujiwara, and Yoritaka Aoyama 887
Biological Activity of Purpurogallin
Yoshihiko Inamori, Chikaaki Muro, Eiko Sajima, Motoharu Katagiri, Yukiko
Okamoto, Hajime Tanaka, Yoshikazu Sakagami, and Hiroshi Tsujibo 890
Preparation of Glutaraldehyde Cross-linked Complex from
Support
Toshiro Matsui, Tomoyuki Oki, Kiyoshi Matsumoto, and Yutaka Osajima
893
Occurrence of Cobalamin Coenzymes in the Photosynthetic
Green Alga, Chlorella vulgaris
Fumio Watanabe, Katsuo Abe, Shigeo Takenaka, Yoshiyuki Tamura, Isao
Maruyama, and Yoshihisa Nakano 896
Action of a Thermostable Trehalose Synthase from Thermus
aquaticus on Sucrose
Tomoyuki Nishimoto, Tetsuya Nakada, Hiroto Chaen, Shigeharu Fukuda,
Toshiyuki Sugimoto, Masashi Kurimoto, and Yoshio Tsujisaka 898
Selective Incorporation of Polyunsaturated Fatty Acids
into Organelle Phospholipids of Animal Cells
Hironori Masui, Reiko Urade, and Makoto Kito 900
Inhibition of Glucan Synthesis by Flavipin-crosslinked
Casein Polymers
Osamu Hayashida, Yamaji Nakano, Keiji Hasumi, and Akira Endo 903
Zinc Finger-like Motif Conserved in a Family of RNA Binding
Proteins
Yuichi Matsushima, Kiyoshi Matsumura, and Yasuo Kitagawa, 905
Expression of Gene Encoding Endo-1,4--glucanase in Suspension-cultured
Poplar (Poplus alba L.) Cells
Takumi Takeda, Fukumi Sakai, and Takahisa Hayashi 907
A Gene Homologous to the Streptomyces Chymotrypsin-like
Protease (SAM-P20) Gene Is Tandemly Located
Seiichi Taguchi, Takahiro Ogawa, Takeshi Endo, and Haruo Momose 909
New Dihydroquinolinone Toxic to Artemia salina Produced
by Penicillium sp. NTC-47
Hideo Hayashi, Tadashi Nakatani, Yoshiki Inoue, Mitsuru Nakayama, and
Hiroshi Nozaki 914
Peculiar Archaea Found in Japanese Paddy Soils
Yuko Kudo, Satoshi Shibata, Taro Miyaki, Toshihiro Aono, and Hiroshi
Oyaizu 917
Grandinal, a New Phloroglucinol Dimer from Eucalyptus
grandis
Inder Pal Singh, Rie Hayakawa, Hideo Etoh,, Midori Takasaki, and Takao
Konoshima 921
Occurrence of Free N-Glycans in Pea (Pisum sativum. L)
Seedlings
Yoshinobu Kimura, Shigeaki Takagi, and Tomonori Shiraishi 924
Structure and Activity of a New Form of the Glutamate
Transporter of the Nematode Caenorhabditis elegans
Tsuyoshi Kawano, Kyoko Takuwa, and Terumi Nakajima 927
-1-
Studies on Biosynthesis of Brassinosteroids
Akira Sakurai and Shozo Fujioka
The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan
@Biosynthesis of steroidal plant hormones, brassinosteroids, was studied
using the cell culture system of Catharanthus roseus. Feeding labeled compounds
of possible intermediates to the cultured cells, followed by analyzing
the metabolites by gas chromatography-mass spectrometry disclosed the pathways
from a plant sterol, campesterol, to brassinolide. There are two pathways,
named the early C6-oxidation pathway and late C6-oxidation pathway, both
of which would be operating in a wide variety of plants. Recent findings
of brassinosteroid-deficient mutants of Arabidopsis and the garden pea
by several groups, and the possible blocked steps of the mutants in the
biosynthetic pathways are also introduced
Key words: biosynthesis; brassinosteroids; plant cell cultures; Catharanthus
roseus; brassinosteroid mutants
-2-
Effect of Dietary n-3/n-6 Fatty Acid Ratio on the Total
Count, Fatty Acid Composition, and Histamine and Leukotriene Concentrations
of Mast Cells in Tunica Mucosa
Bronchiorum of Type I Allergic Guinea Pig
Masanori Kuwamori,* Masahiro Wada,* Toshichika Takita,* Tadahiro Tadokoro,**
Akio Maekawa,** and Satoshi Innami *,
* Department of Nutrition, and ** Department of Agricultural Chemistry, Faculty of Agriculture, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156, Japan
Received July 9, 1996
@To search for the most effective dietary n-3/n-6 ratio to suppress the
type I allergic response, we performed basic experiments that applied parameters
associated with the type I allergy. Guinea pigs fed on diets containing
lipids with the n-3/n-6 ratio at different levels and the polyunsaturated
fatty acid/saturated fatty acid ratio of a fixed level were sensitized
with ovalbumin and reared for two weeks.
@The lowest or critical level of the n-3/n-6 ratio which produced a significant
difference in the parameters was as follows: about 2.0 for the response
of mast cells and eosinophils; 0.5 and 1.0, respectively, for the uptake
of n-3 and n-6 polyunsaturated fatty acids and decreased histamine production;
and 0.2 for decreased leukotriene B4 and total leukotrienes 4, and increased
leukotrienes 5/leukotrienes 4.
@The critical level of the n-3/n-6 ratio thus differed widely according
to the parameter. Overall, the upper limit for the dietary n-3/n-6 ratio
to suppress antigen-induced type I allergic responses is suggested to be
around 1.0.
Key words: n-3/n-6 fatty acid ratio; mast cells; eosinophils; leukotriene;
guinea pig
-3-
Oxygen Sensitivity of NifA Protein of Azospirillum lipoferum
FS as Suggested by Gene Cloning and Expression in Escherichia coli
Toru Shigematsu, Akio Inoue, Makoto Hidaka, Haruhiko Masaki, and Takeshi Uozumi
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan
Received September 3, 1996
@We cloned and sequenced a 2.8-kb Sal I fragment of Azospirillum lipoferum
FS as a homologue of the Klebsiella oxytoca nifA gene. The amino acid sequence
deduced from an open reading frame of 1872 bases showed 91% identity to
that of the A. brasilense NifA, and the putative central 54 interaction
domain was conserved as well as the C-terminal DNA-binding domain. The
NifA function on the nifH promoter was examined in Escherichia coli using
a combination of a nifA driver plasmid and a nifH-lacZ reporter plasmid,
in which the transcriptional activation of the nifH promoter by the NifA
was evaluated with the -galactosidase activity. The A. lipoferum NifA
activated the nifH promoter solely under microaerobic conditions, while
the K. oxytoca NifA activated it irrespective of the oxygen condition.
These observations suggest that oxygen sensitivity is an intrinsic property
of the A. lipoferum NifA.
Key words: nitrogen fixation; nifA; Azospirillum lipoferum; nifH promoter;
transcriptional activation
-4-
In Vitro and in Vivo Anti-platelet Effects of Enzymatic
Hydrolysates of Collagen and Collagen-related Peptides
Isao Nonaka, Shin-ichiro Katsuda,Takashi Ohmori, Tamotsu Shigehisa, Tatsuyoshi Nakagami, and Susumu Maruyama*
Department of Health and Dietary Science, Research and Development Center,
Nippon Meat Packers, Inc., 3-3 Midorigahara, Tsukuba-shi, Ibaraki 300-26,
Japan
* National Institute of Bioscience and Human Technology, Agency of Industrial
Science and Technology, 1-1 Higashi, Tsukuba-shi, Ibaraki 305, Japan
Received September 13, 1996
@Collagen-related peptides, Gly-Pro-Arg and its analogues, were examined
for their inhibitory effects on platelet aggregation induced by the addition
of ADP. Human platelet aggregation was suppressed by more than 50% with
each of Gly-Pro-Arg and such Gly-Pro-Arg-containing peptides as Gly-Pro-Arg-Gly,
Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Pro-Pro, and Gly-Pro-Arg-Pro-Pro-Pro at
a concentration of 0.3 mM. The inhibitory effects of these peptides were
about 10 times higher in human PRP than in rat PRP.
@Other Gly-Pro-Arg analogues such as Sar-Pro-Arg, Gly-Pro-Lys, Gly-Ala-Arg,
and Ala-Gly-Pro-Arg had no inhibitory effect at a concentration from 0.1
to 0.8 mM even in human PRP. Intravenous and oral administrations of Gly-Pro-Arg
and enzymatic hydrolysates of collagen suppressed the decrease in platelet
count for endotoxin-induced DIC in rats. Collagen itself has been regarded
as a potent inducer of platelet aggregation, but these findings suggest
that collagen-related peptides and enzymatic hydrolysates of collagen prevent
platelet aggregation.
Key words: collagen; collagenase; collagen-related synthetic peptide; fibrinogen;
thrombin
-5-
Growth Inhibition, Morphological Change, and Ectoenzyme
Release of LLC-PK1 Cells by Phosphatidylinositol-specific Phospholipase
C of Bacillus thuringiensis
Chisako Itami, Yukio Kimura, Ryo Taguchi,* Hiroh Ikezawa,* and Toshikatsu Nakabayashi
Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Mukogawa
Women's University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663, Japan
* Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences,
Nagoya city University, Mizuho-ku, Nagoya, Aichi 467, Japan
Received September 30, 1996
@Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus
thuringiensis added to a culture of LLC-PK1 cells inhibited cell growth
by 40%. In contrast with normal cells, the cells cultured in the presence
of PI-PLC showed needle-like appendages which seemed to have been formed
due to portions of the cell remaining adhered to the culture dish as the
cell shrank. When LLC-PK1 cells were treated with PI-PLC, significant amounts
of alkaline phosphatase and alkaline phosphodiesterase I were released
specifically from the apical surface of the LLC-PK1 cells. Furthermore,
PI-PLC treatment caused a delay of enzyme production and dome formation.
These data indicate that glycosyl-phosphatidylinositol (GPI)-anchored proteins
on the surface of LLC-PK1 cells are important in cell growth and differentiation.
Also, the combined use of LLC-PK1 cells and PI-PLC of B. thuringiensis
is effective for investigating the function of GPI-anchor proteins.
Key words: phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis;
ectoenzyme release; growth inhibition; morphological change; LLC-PK1 cells
-6-
Characterization of the Protein and Glycan Moieties in
Different Forms of Bovine Lactoferrin
Xiu-Yun Ye, Toshihide Nishimura, and Shigeru Yoshida
Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739, Japan
Received October 11, 1996
@The BrCN cleavage of lactoferrin-a or -b (LF-a or LF-b) led to the observation
of four fragments by SDS-PAGE, whose molecular masses were 77, 58, 52,
and 30 kDas, or 74, 54, 47, and 30 kDas, respectively. N-Terminal amino
acid sequence analyses show that the sequences of 58, 52, and 30 kDa fragments
(residues 64-471, 130-471, and 472-689) of LF-a coincide with those of
the 54, 47, and 30 kDa fragments of LF-b, respectively. All these fragments,
which were positive by PAS staining, were not stained after being treated
with glycopeptidase F. This treatment changed the 58 and 52 kDa fragments
of LF-a to the 54 and 47 kDa fragments, respectively, whose molecular masses
were the same as those of the treated fragments of LF-b. The 58 and 52
kDa fragments of LF-a bound to the lectin, Ricinus communis agglutinin,
while the 54 and 47 kDa fragments of LF-b hardly bound to it.
Key words: lactoferrin; glycoprotein; glycopeptide; glycan; milk
-7-
Thermal Gelation Profile Changes in Reconstituted Actomyosin
due to Storage under a High Salt Concentration and Low Temperature
Hiroyuki Tanji, Yoshihide Ikeuchi,*, Masatoshi Yoshizawa,* and Atsushi Suzuki *
Basic Research Department, Prima Meat Packers Co., Ltd., Tsuchiura,
Ibaraki 300, Japan
* Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata
University, Niigata 950-21, Japan
Received October 11, 1996
@Changes in the heat-induced gelation properties of reconstituted rabbit
skeletal actomyosin stored under a high salt concentration at pH 6.0 and
0C were investigated at different weight ratios of actin to myosin by
using dynamic rheological and biochemical measurements. The addition of
actin resulted in a pronounced peak maximum at about 50C and an accompanying
temporary reduction in the range at about 50C to 60C. The more the
initial actin concentration was increased, the greater was the area of
the peak/shoulder. However, this area was markedly diminished with increasing
storage time. As a result, the dynamic rheological pattern was transformed
from an actomyosin type into a myosin type. The relationship between the
G ' value at 80C and the actin/myosin weight ratio was curvilinear, with
a peak at the ratio of 0.05, immediately after storage was started. This
profile changed during storage, depending on the extent to denaturation
of actin and myosin in the reconstituted actomyosin (RAM). The G ' value
of actomyosin in 0.5 M KCl with a small actin/myosin ratio of 0.05 decreased
to one-half of its initial value after 7 days of storage, whereas the G
' value with a large actin/myosin ratio of 0.225 increased by about 1.6
times. In 1.5 M KCl, all the G ' values declined to the level with myosin
alone after 7 days of storage. The time-course plots of the remaining actin
concentration in RAM at different weight ratios of actin to myosin after
being treated with 0.5 M or 1.5 M KCl showed a decrease in the actin content
with increasing storage time, and an increase in the KCl concentration
to 1.5 M KCl promoted the denaturation of actin in RAM faster than with
0.5 M KCl. The surface hydrophobicity of each RAM sample progressively
increased with increasing storage time, while little significant increase
in the sulfhydryl (SH) content during storage was observed. It is concluded
that changes in the heat-induced gelation properties of actomyosin during
storage are largely attributable to the denaturation of actin rather than
to the denaturation of myosin or to quantitative changes in the SH content
and hydrophobicity.
Key words: thermal gelation; denaturation; myosin; actin; actomyosin
-8-
Characterization of 30-kDa Fragments Derived from -Conglycinin
Degradation Process during Germination and Seedling Growth of Soybean
Misako Kawai,*, Shun'ichi Suzuki,** Minao Asano,* Tetsuya Miwa,* and Hiroshiro Shibai**
* Food Research & Development Laboratories, and ** Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa 210, Japan
Received October 11, 1996
@The degradation process of -conglycinin, a vicilin-type glycosylated
storage protein of soybean seeds, during germination and seedling growth
was examined by concanavalin A blotting combined with polyacrylamide gel
electrophoresis. We detected and analyzed the structures of key intermediary
fragments of 30 kDa derived from -conglycinin degradation, they were
proved to be single-domain type subunits of -conglycinin. We show here
a degradation process of -conglycinin: -conglycinin is subjected to
limited proteolysis at exposed regions on the molecular surface, like domain
junctions, generating 30-kDa single-domain fragments before non-specific
proteolysis.
Key words: -conglycinin; soybean; degradation; proteolysis
-9-
Efficient Expression of Mono- and Diacylglycerol Lipase
Gene from Penicillium
camembertii U-150 in Aspergillus oryzae under the Control of Its Own Promoter
Shotaro Yamaguchi,*, Kazuyuki Takeuchi,** Tamio Mase,** and Akira Matsuura*,**
* Tsukuba Research Laboratories, Amano Pharmaceutical Co., Ltd., Miyukigaoka
22, Tsukuba, Ibaraki 305, Japan
** Research and Development Division, Amano Pharmaceutical Co., Ltd., Nishiharu,
Nishikasugai, Aichi 481, Japan
Received October 15, 1996
@The gene, mdlA, coding for mono- and diacylglycerol lipase from Penicillium
camembertii U-150 was expressed efficiently in Aspergillus oryzae under
the control of its own promoter. The gene product was secreted into the
culture medium with a highest productivity of 1 g/liter and correctly processed
at both N- and C-termini. KEX2-like processing was suggested to occur at
the C-terminus in both A. oryzae and P. camembertii. Specific activity
and substrate specificity of the purified recombinant protein were also
almost the same to that of native protein but the extent of N-glycosylation
in the recombinant protein was about half of that of the native protein.
The presence of introns did not seem to affect the gene expression. The
mdlA expression was induced by lipids and regulated transcriptionally in
A. oryzae as well as P. camembertii. Promoter deletion analysis showed
that the region between the positions at -382 and -554 bp from the translation
initiation point was important to the higher expression of mdlA. The promoter
sequence of mdlA was compared to that of the Geotrichum candidum lipase
gene, which is also reported to be inducible by lipids, with three commonly
observed oligonucleotide sequences.
Key words: lipase: gene expression; Penicillium camembertii ; Aspergillus
oryzae; lipid induction
-10-
Synthesis of 24a-Substituted Milbemycin A4 Derivatives
and Their Acaricidal Activity
against Tetranychus urticae
Yoshihisa Tsukamoto, Hisaki Kajino, Kazuo Sato, Keiji Tanaka, and Toshiaki Yanai
Agroscience Research Laboratories, Sankyo Co., Ltd., 1041 Yasu, Yasu-cho, Yasu-gun, Shiga 520-23, Japan
Received October 17, 1996
@A series of 24a-substituted milbemycin A4 derivatives were synthesized
from 24a-hydroxymilbemycin A4, which had been obtained by the microbial
oxidation of milbemycin A4. The acaricidal activity of each synthesized
derivative against Tetranychus urticae was tested and some of the derivatives
showed higher activity than parent milbemycin A4. Among them, 24a-methylmilbemycin
A4 (22) was the most active derivative, with 100% mortality of the mite
at a concentration of 1 ppm and 50% mortality at a concentration of 0.1
ppm.
Key words: milbemycin; 24a-substituted milbemycin A4 derivatives; Tetranychus
urticae; two-spotted spider mite
-11-
Purification of Amylases and Other Enzymes by a Forced-affinity
Chromatography Method
Mikihiko Kobayashi, Yutaka Sasaki, and Shoichi Kobayashi
National Food Research Institute, Kannondai, Tsukuba 305, Japan
Received October 22, 1996
@An affinity matrix of soluble starch gel was prepared by cross-linking
catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA)
indicated that the amount of enzyme bound to the starch gel column increased
with increases in the ammonium sulfate (AmS) concentration in the equilibrating
buffer. TAA had an affinity for the gels with a starch structure, and desorbed
from the column with the buffer containing no AmS. Bound TAA was also eluted
with starch and cyclodextrin solution. The AmS stimulative effect was partially
replaced by polyethylene glycol and surfactants. Besides TAA, various other
amylases bound satisfactorily to the starch gel. Moreover, affinity purifications
of dextranase, cellulase, and pectinase were done by gels with dextran,
cellulose, and pectin structures, respectively. By the aid of forced effects
of AmS, various carbohydrases could be purified by the affinity gels of
polysaccharide linked by epichlorohydrin.
Key words: affinity chromatography; amylases; forced-affinity chromatography;
glucanases
-12-
Lipid Peroxidation in Linoleic Acid Micelles Caused by
H2O2 in the Presence of Myoglobin
Tsutomu Nakayama, Youhei Chiba, and Kei Hashimoto
School of Food and Nutritional Sciences, University of Shizuoka, Yada 52-1, Shizuoka 422, Japan
Received October 24, 1996
@We investigated the lipid peroxidation in linoleic acid micells caused
by H2O2 in the presence of metmyoglobin by monitoring the oxygen consumption.
O2 consumption usually consisted of two phases. In the first phase, it
occurred slowly and linearly until the concentration of linoleic acid hydroperoxide
reached a certain value, rapid consumption, presumably by a chain reaction,
then followed in the second phase. No effects of diethylenetriaminepentaacetic
acid (DTPA) on the induction period (the period during the first phase)
and the maximum oxygen consumption rate (MOCR) in the second phase indicate
that free ferric ions liberated from myoglobin had no role in any phases
during the lipid peroxidation. The differing dose effects of ascorbic acid,
-tocopherol, and sodium nitrite on the induction period and MOCR reflect
their respective antioxidative mechanisms during lipid peroxidation.
Key words: myoglobin; metmyoglobin; hydrogen peroxide; lipid peroxidation;
sodium nitrite
-13-
Incorporation of Farnesyl Pyrophosphate Derivertives into
Abscisic Acid and Its Biosynthetic Intermediates in Cercospora cruenta
Hirotaka Yamamoto and Takayuki Oritani
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981, Japan
Received October 24, 1996
@To investigate the transformation from (2E,6E )-farnesyl pyrophosphate
to (2Z,4E )--ionylideneethanol in the abscisic acid-producing fungi,
Cercospora cruenta, plausible [2-14C]-C15 intermediates were prepared and
fed. Substrates such as (2E,6E )-farnesyl pyrophosphate, (2Z,4E )--ionylideneethanol
and its pyrophosphate were incorporated into ABA and its known biosynthetic
precursors. It is suggested that (2E,6E )-farnesyl pyrophosphate is converted
to (2Z,4E )--ionylideneethanol in four consecutive steps: dehydrogenation,
isomerization, cyclization and hydrolysis.
Key words: Abscisic acid; Cercospora cruenta; -ionylideneethanol; -monocyclofarnesol;
farnesyl pyrophosphate
-14-
Enzymatic Synthesis of Mannosyl-cyclodextrin by -Mannosidase
from Jack Bean
Kenichi Hamayasu, Koji Hara, Koki Fujita, Yukio Kondo, Hitoshi Hashimoto, Toshiko Tanimoto,* Kyoko Koizumi,* Hirofumi Nakano,** and Sumio Kitahata**
Carbohydrate Research Laboratory, Ensuiko Sugar Refining Co., Ltd.,
13-46 Daikoku-cho, Tsurumi-ku, Yokohama 230, Japan
* School of Pharmaceutical Sciences, Mukogawa Women's University, 11-68
Koshien Kyuban-cho, Nishinomiya 663, Japan
** Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Jyoto-ku,
Osaka 536, Japan
Received October 25, 1996
@Mannosylated derivatives of cyclodextrins (CDs), mannosyl-, , and
CD were synthesized from a mixture of mannose and , , and CD by
the reverse action of -mannosidase from jack bean, respectively. Their
structures were analyzed by FAB-MS and 13C-NMR spectroscopies, and they
were identified as 6-O--D-mannosyl-, , and CD. The optimum conditions
for the production of 6-O--D-mannosyl-CD by -mannosidase were examined.
Optimum pH and temperature were pH 4.5 and 60C, respectively. Yield of
mannosyl-CD increased with increasing mannose concentration and reached
more than 35% (mol/mol) at the concentration of 2 M mannose and 0.4 M CD.
Key words: CD; mannosyl-CD; -mannosidase; Jack bean; reverse action
-15-
High-level Expression of the Chemically Synthesized Gene
for Microbial Transglutaminase from Streptoverticillium in Escherichia
coli
Misako Kawai, Shino Takehana, and Hiroshi Takagi ,*
Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan
Received October 28, 1996
@We developed a novel approach for the high-level production of a microbial
transglutaminase (TGase) from Streptoverticillium in E. coli. The direct
expression of the TGase gene in E. coli cells did not cause overproduction,
probably due to the harmful influence of TGase activity, which introduces
covalent crosslinks between proteins. Therefore, we fused the chemically
synthesized TGase gene coding for the entire 331 amino acid residues at
the amino terminus to a bacteriophage T7 gene 10 leader peptide (260 amino
acids) using an inducible expression vector. The TGase gene was expressed
as inclusion bodies in the E. coli cytoplasm. Restoring 15 amino acid residues
upstream of the amino terminus of the mature TGase by a two-step deletion
of the fusion sequence facilitated solubilization and subsequent proteolytic
cleavage, thus releasing mature TGase. Although the mature form had less
TGase activity than native TGase, because of the poor refolding rate, these
results suggest that this system is suitable for the efficient production
of TGase.
Key words: transglutaminase; gene synthesis; high-level expression; fused
protein; refolding
-16-
Effects of Dietary Sesaminol and Sesamin on Eicosanoid
Production and Immunoglobulin Level in Rats Given Ethanol
Michiko Nonaka, Kanae Yamashita,* Yoshie Iizuka,* Mitsuo Namiki,* and Michihiro Sugano
Laboratory of Food Science, Department of Food Science and Technology,
School of Agriculture, Kyushu University, Higashi-ku, Fukuoka 812-81, Japan
* Department of Food and Nutrition, School of Life Studies, Sugiyama Jogakuen
University, Nagoya 464, Japan
Received November 5, 1996
@The effects of sesaminol and sesamin on the ethanol-induced modulation
of immune indices related to food allergy were examined in rats given a
low (10%)-casein diet. Chronic ethanol drinking, at the dietary level of
23% (w/w), significantly increased the plasma IgA and IgM concentrations,
irrespective of the presence of 0.1% and 0.2% sesaminol, but the effects
disappeared with 0.2% sesamin. A significant IgG-elevating effect of these
lignans was also found. In contrast, the concentration of plasma IgE was
not influenced by the dietary manipulation. Although ethanol drinking did
not influence splenic leukotriene B4 production, sesaminol tended to decrease
it dose dependently, while sesamin increased the plasma prostaglandin E2
concentration. These results suggest that sesaminol and sesamin seems to
have a diverse effect on the plasma levels of immunoglobulins and eicosanoids.
Key words: ethanol; sesaminol; sesamin; immunoglobulin; eicosanoids; rats
-17-
High Level Expression of XMP Aminase in Escherichia coli
and Its Application for the Industrial Production of 5'-Guanylic Acid
Tatsuro Fujio, Tatsunari Nishi,** Seiga Ito,*** and Akihiko Maruyama*,
Planning & Development Department/Food, Liquor & Food Division,
and * Fine Chemicals/Biochemicals Dept., Medicals Research and Development
Center, Kyowa Hakko Kogyo Co., Ltd., 6-1-1 Ohtemachi, Chiyoda-ku, Tokyo
100, Japan
** Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi,
Machida-shi, Tokyo 194, Japan
*** Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd.,
1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka-ken, 411, Japan
Received November 8, 1996
@To improve the efficiency of the enzymatic conversion of 5'-xanthylic
acid (XMP) to 5'-guanylic acid (GMP), we attempted to increase the activity
of the conversion enzyme, XMP aminase (GMP synthetase) encoded by the guaA
gene in Escherichia coli. By connecting the PL promoter of phage, the
SD sequence of trpL of E. coli, and ATG, at a suitable position upstream
of the guaA gene, we obtained plasmid pPLA66. Sequencing of the nucleotides
of the upstream region of the guaA gene on pPLA66 showed that the C-terminal
region of the guaB gene, which encodes IMP dehydrogenase, was conserved
and a short peptide consisted of 14 amino acids was coded. E. coli MP347/pPLA66
showed an increase in the activity of approximately 370 times when compared
with that of the strain MM294, and the amount of the enzyme protein represented
approx. 34% of the total cellular protein. Strain MP347/pPLA66 was cultivated
in a 5-liter jar fermentor using a medium which contained mainly corn steep
liquor. The culture broth had high XMP aminase activity. In the conversion
reaction using mixed broths consisted of 600 ml of XMP-fermentation broth
of Corynebacterium ammoniagenes KY13203 and 30 ml of cultured broth of
E. coli MP347/pPLA66, a surfactant, Nymeen S-215 and xylene were added
to the reaction mixture to make the cell membrane permeable to nucleotides.
After 23 h of the reaction, 70 mg/ml (131 mM) of GMPENa2E7H2O was accumulated
from 83 mg/ml (155 mM) of XMPENa3E7H2O, without addition of ATP. The
molar conversion yield was approx. 85%. The facts that the cell membrane
was treated to allow nucleotides to permeate and that the conversion reaction
proceeded well enough in spite of a small amount of E. coli cells indicate
ATP was regenerated from AMP by C. ammoniagenes cells and supplied to E.
coli cells. Therefore, it was considered that the coupling reaction between
these two kind of strains was established.
Key words: 5'-guanylic acid; XMP aminase (GMP synthetase); genetic engineering;
enzymatic conversion; Escherichia coli
-18-
Sequential Binding of Staphylococcal -Hemolysin to Human
Erythrocytes and Complex Formation of the Hemolysin on the Cell Surface
Jun Kaneko, Toshiko Ozawa, Toshio Tomita, and Yoshiyuki Kamio
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981, Japan
Received November 8, 1996
@Staphylococcal -hemolysin consists of two protein components, F (or
HI) and HII. To elucidate the mode of action of -hemolysin, we studied
the binding order of F and HII to human erythrocytes and the cell-bound
state of the two components. The binding of F to human erythrocytes preceded
the binding of HII to the cells, and thereafter hemolysis occurred. Western
immunoblot analysis of the cell-bound -hemolysin indicated that F and
HII components form high-molecular-mass (150-250 kDa) complexes on the
erythrocytes. The toxin complexes were recovered in a Triton X-100-insoluble
fraction of the erythrocytes, which contains cytoskeleton proteins. Neither
the formation of the toxin complex(es) nor hemolysis occurred when the
erythrocytes were treated with proteinase K. Abortion of the complex formation
on the proteinase K-treated erythrocytes may be due to the failure of the
binding of HII to the cells, because F bound to the proteinase K-treated
erythrocytes to the same extent as to the non-treated erythrocytes.
Key words: -hemolysin; Staphylococcus aureus; human erythrocytes; binding,
complex formation
-19-
Protoplast Formation from Schizophyllum commune by a Culture
Filtrate of Bacillus circulans KA-304 Grown on a Cell-wall Preparation
of S. commune as a Carbon Source
Katsushige Mizuno, Osamu Kimura, and Takashi Tachiki
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu 525-77, Japan
Received November 11, 1996
@From microorganisms growing on a cell-wall preparation (CWP) of Schizophyllum
commune as a carbon source, Bacillus circulans KA-304 was isolated on the
bases of activities in culture filtrate to decrease turbidity of the CWP-suspension
and to form protoplasts from S. commune. The culture filtrate was also
active in hydrolyzing p-nitrophenyl ( p-NP)--D-glucoside, p-NP--D-glucoside,
and p-NP--D-N-acetylglucosaminide. The protoplast-forming and the p-NP-glycoside-hydrolyzing
activities were increased by the addition of CWP of S. commune to the culture
medium, and this was not observed for other bacteria tested (15 genera,
80 species). B. circulans KA-304 was shown on gel filtration to produce
at least two enzyme species for hydrolyzing both p-NP--D-glucoside and
p-NP--D-glucoside. The protoplast-forming activity was retained for at
least 6 months at 5C as an ammonium sulfate (90% saturation) precipitate
or at -20C as a freeze-dried preparation (KA-preparation). The activity
was stable at pH 6.5-7.0, and remained after 10 min of treatment at 40C.
Protoplast formation proceeded optimally at pH 6.5 with 50 mM potassium
phosphate buffer and 0.5 M mannitol as an osmotic stabilizer. B. circulans
KA-304 seems to be a suitable strain producing enzyme(s) to prepare protoplasts
from S. commune.
Key words: Schizophyllum commune; protoplast formation; Bacillus circulans
KA-304
-20-
Purification and Characterization of a Dipeptidyl Carboxypeptidase
from Pseudomonas sp. WO24
Wataru Ogasawara, Nobuhide Abe, Takashi Hagio, Hirofumi Okada, and Yasushi Morikawa
Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan
Received November 11, 1996
@A dipeptidyl carboxypeptidase (DCP) activity was detected in cell-free
extracts of Pseudomonas sp. WO24. After purification and characterization
the enzyme was found to be homogeneous by SDS-PAGE, and had a molecular
mass of 74,000 Da by SDS-PAGE and 72,000 Da by gel filtration, indicating
that it is monomeric. The isoelectric point was 5.2 and optimum pH was
6.5-7.0. It showed a specific activity of 780 mol/min/mg, which is the
highest of the values shown by known enzymes. The enzyme hydrolyzed angiotensin
I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from
the C-terminus bradykinin. The DCP could not cleave imido-bonds, Gly-Gly
bonds, or tripeptides. The enzymatic activity was completely inhibited
by 0.001 mM EDTA and 0.1 mM o-phenanthroline, but it was not affected by
general serine and cysteine protease inhibitors. Addition of Zn2+ completely
restored the original activity of the inactivated DCP treated with EDTA.
These results suggest that this enzyme is a zinc metalloprotease. The characteristics
of the purified enzyme are slightly different from those of the DCPs from
Escherichia coli, Pseudomonas maltophilia, and Corynebacterium equi, and
considerably from those of the DCP from Bacillus pumilus.
Key words: dipeptidyl carboxypeptidase; DCP; Pseudomonas; angiotensin-I
converting enzyme; ACE
-21-
Effects of Ethylene and Gibberellins on the Elongation
of Rice Seedlings (Oryza sativa L.)
Koji Furukawa,*,**, Young-Yell Yang,** Ichiro Honda,***, Tadashi Yanagisawa,*** Akira Sakurai,*,*** Nobutaka Takahashi,** and Yuji Kamiya**
* Graduate School of Science and Engineering, Saitama University, Urawa,
Saitama 338, Japan
** Frontier Research Program, The Institute of Physical and Chemical Research
(RIKEN ), Wako, Saitama 351-01, Japan
*** Department of Agricultural Chemistry, Faculty of Agriculture, Utsunomiya
University, Utsunomiya, Tochigi 321, Japan
**** The Institute of Physical and Chemical Research (RIKEN ), Wako, Saitama
351-01, Japan
Received November 21, 1996
@Three-day-old rice seedlings treated with ethylene showed elongation
of the 2nd and 3rd leaves. This ethylene-stimulated elongation was not
observed in the presence of uniconazole-P or prohexadione, both gibberellin
(GA) biosynthesis inhibitors, suggesting that GA was involved in the response.
An analysis of endogenous GAs by GC-MS revealed that the GA1 level was
reduced in the 3rd leaf in response to ethylene. Dose-response experiments
showed that the responsiveness to GA1 was enhanced by ethylene. Feeding
experiments of 14C-GA1 with ethylene-treated seedlings showed that ethylene
may increase the conversion of GA1 to GA8. These results suggest that,
in young seedlings of rice, ethylene stimulates leaf elongation by increasing
the responsiveness to GA1 and the turnover of GA1.
Key words: elongation; ethylene: GC-SIM; gibberellin; Oryza sativa L.
-22-
Acyl Amino Acid Derivatives as Novel Inhibitors of Influenza
Neuraminidase
Mitsuyo Kondoh, Toshiyuki Furutani,* Masayuki Azuma, Hiroshi Ooshima, and Jyoji Kato
Department of Bioapplied Chemistry, Osaka City University, Sugimoto,
Sumiyoshi-ku, Osaka 558, Japan
* Pharmaceutical Development Research Laboratory, Tanabe Seiyaku Co., Ltd,
Kashima, Yodogawa-ku, Osaka 532, Japan
Received November 28, 1996
@We searched our chemical collection to identify non-N-acetylneuraminate
(NeuAC) inhibitors of influenza neuraminidase (NA). Of the 62 acyl derivatives
tested, several acyl amino acids, but not acyl alkanolamine derivatives,
were effective and inhibited the NA activity in a dose-dependent manner.
N-3-Hydroxymyristoyl D-cysteine and N-myristoyl-O-caproyl-D-serine were
the more potent compounds and inhibited the enzyme in a noncompetitive
manner (Ki=102 and 125 M, respectively) without respect to the substrate.
An important consideration for the choice of inhibitor is the selectivity
of the inhibition. These compounds were selective inhibitors of viral NA
and effective for any variant enzyme, but the enzymes from V. cholerae
and human placenta were insensitive. Accordingly, the acyl amino acid derivatives
may be expected to be inhibitors without cellular toxicity and may serve
as lead compounds for anti-influenza agents.
Key words: influenza neuraminidase; noncompetitive inhibitor; acyl amino
acid derivatives
-23-
Cloning and Sequencing of a cDNA Encoding -Glucosidase
from Sugar Beet
Hirokazu Matsui, Shunsuke Iwanami, Hiroyuki Ito, Haruhide Mori, Mamoru Honma, and Seiya Chiba
Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Sapporo 060, Japan
Received December 4, 1996
@A cDNA encoding sugar beet -glucosidase was cloned from a library constructed
from mRNA of suspension-cultured cells. The cDNA, 3056 bp in length, had
an open reading frame encoding a polypeptide of 913 amino acid residues
with a molecular mass of 102,078 Da, included only one of four regions
which were conserved in the -amylase family of enzymes. The deduced amino
acid sequence from the analysis of the cDNA contained the sequences of
the proteolysis peptides and the active site region peptide of sugar beet
-glucosidase. The primary structure indicated relatively high homology
in the range of 28.2 to 54.3% to those for other -glucosidases. The highest
homology was found in barley -glucosidase.
Key words: -glucosidase; sugar beet; cDNA cloning; nucleotide sequence
-24-
Absolute Configurations of Some Hydroxy-fatty Acids Produced
by the Insect Genus Laccifer
Akio Ichikawa, Haruko Takahashi,* Takashi Ooi,* and Takenori Kusumi*
National Institute of Sericultural and Entomological Science, 1-2 Owashi,
Tsukuba, Ibaraki 305, Japan
* Faculty of Pharmaceutical Sciences, Tokushima University, 1-78 Sho-machi,
Tokushima-shi, Tokushima 770, Japan
Received August 21, 1996
@The absolute configurations of some hydroxy-fatty acids were examined
by the modified Mosher's method proposed by Ohtani et al. The absolute
configurations of the major components were determined from NMR data of
their MTPA esters and 2-NMA esters. The application of Mosher's method
for the anti-glycol is discussed.
Key words: Mosher's method; absolute configuration; MTPA; 2-NMA; Laccifer
-25-
Isolation and Partial Amino Acid Sequence of Bacteriocins
Produced by Lactobacillus acidophilus
Takatsugu Tahara and Kazuo Kanatani
Research Laboratory, Tamon Sake Brewing Co., Ltd., 1-13-11 Higashi-cho, Nishinomiya, Hyogo 662, Japan
Received September 4, 1996
@Bacteriocins produced by Lactobacillus acidophilus JCM 1023, JCM 1028,
JCM 1021, JCM 1229, and JCM 5342 were active against closely related lactobacilli.
These bacteriocins were purified and partial sequenced. Bacteriocin activities
of L. acidophilus JCM 1023 and JCM 1028 were associated with two components.
On the basis of N-terminal amino acid sequencing and the molecular masses,
it is interpreted that these two-component bacteriocins are identical to
acidocin J1132, a bacteriocin from L. acidophilus JCM 1132 [Tahara et al.,
Appl. Environ. Microbiol., 62, 892-897 (1996)]. Other bacteriocins were
single-peptide bacteriocins.
Key words: Lactobacillus acidophilus ; bacteriocin; amino acid sequence
-26-
Stimulating Effect of Ileal Pancreaticobiliary Secretion
on Ileal Apolipoprotein A-IV mRNA Expression in Fasted Rats
Kei Sonoyama, Reiko Fujiwara, and Yoritaka Aoyama
Laboratory of Food Biochemistry, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Received September 25, 1996
@The effect of pancreaticobiliary secretion on the intestinal expression
of the apo A-IV gene was examined in fasted rats. Pancreaticobiliary diversion,
but not biliary diversion alone, into the ileum increased the ileal apo
A-IV mRNA expression by 24 h post-operation. Jejunal apo A-IV mRNA was
reduced by biliary exclusion. The data suggest that the biliary constituent
plays an important role in the apo A-IV gene expression in the entire length
of the small intestine, and that up-regulation of the apo A-IV gene requires
exocrine pancreatic in addition to biliary secretion.
Key words: apolipoprotein A-IV; intestine; bile; rat
-27-
Biological Activity of Purpurogallin
Yoshihiko Inamori, Chikaaki Muro, Eiko Sajima, Motoharu Katagiri, Yukiko Okamoto, Hajime Tanaka, Yoshikazu Sakagami,* and Hiroshi Tsujibo
Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki-shi,
Osaka 569-11, Japan
* Osaka Prefectural Institute of Public Health, Nakamichi, Higashinari-ku,
Osaka 537, Japan
Received October 14, 1996
@Purpurogallin showed antibacterial activity toward gram-positive bacteria.
Strong activity against methicillin-resistant Staphylococcus aureus [minimal
inhibitory concentration (MIC) against methicillin of 1600 g/ml] was
found, with MIC of 11.0 g/ml. Purpurogallin inhibited the growth of all
tested plants and decreased the chlorophyll content in the cotyledons of
Brassica campestris subsp. rapa. It showed potent inhibitory activity against
prolyl endopeptidase (the 50% inhibitory concentration was 1.6~10-5 M),
unlike its analogues, hinokitiol and tropolone.
Key words: purpurogallin; antibacterial activity; methicillin-resistant
Staphylococcus aureus;plant growth-inhibitory activity;prolyl endopeptidase
-28-
Preparation of Glutaraldehyde Cross-linked Complex from
Support
Toshiro Matsui, Tomoyuki Oki, Kiyoshi Matsumoto, and Yutaka Osajima
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-81, Japan
Received October 16, 1996
@A glutaraldehyde (GA) spacer complex on enzyme immobilization was cleaved
from the support introduced N -9-fluorenylmethyloxycarbonylglycine. By
the 1H-NMR spectroscopic measurement of the desired complex, glycine-GA-phenethylamine
(GGP), GA was thought to be involved in enzyme immobilization as a hetero-polymer
with the molecular weight range of about 600 to 1300.
Key words: glutaraldehyde; cross-linking; NMR; immobilization
-29-
Occurrence of Cobalamin Coenzymes in the Photosynthetic
Green Alga, Chlorella vulgaris
Fumio Watanabe, Katsuo Abe, Shigeo Takenaka,* Yoshiyuki Tamura,* Isao Maruyama,** and Yoshihisa Nakano***
Department of Food and Nutrition, Kochi Women's University, Kochi 780,
Japan
* Department of Nutrition and Food Science, Hagoromo-gakuen College, Sakai,
Osaka 592, Japan
** Chlorella Industry Co., Ltd., Fukuoka 833, Japan
*** Department of Applied Biological Chemistry, Osaka Prefecture University,
Sakai, Osaka 593 Japan
Received October 18, 1996
@To analyze cobalamin metabolism in photosynthetic green algae, the effects
of cobalamin on growth of Chlorella vulgaris C-30 were studied and the
algal cobalamin contents were assayed. Cobalamin significantly stimulated
growth of the Chlorella cells, but biologically inactive cobalamin analogues
did not. Chlorella grown in a cobalamin-free medium (control) contained
cobalamin coenzymes, 5'-deoxyadenosylcobalamin (7.95}0.31 ng/g wet weight)
and methylcobalamin (2.72}0.45 ng/g wet weight), of which the levels were
increased significantly in cobalamin-supplemented cells. These results
indicate that the alga has ability to take up exogenous cobalamin and synthesize
the coenzyme forms.
Key words: alga; Chlorella vulgaris; cobalamin; cobalamin coenzymes; stimulation
of growth
-30-
Action of a Thermostable Trehalose Synthase from Thermus
aquaticus on Sucrose
Tomoyuki Nishimoto, Tetsuya Nakada, Hiroto Chaen, Shigeharu Fukuda, Toshiyuki Sugimoto, Masashi Kurimoto, 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
@A thermostable trehalose synthase from Thermus aquaticus ATCC 33923,
which catalyzes the interconversion between maltose and trehalose by intramolecular
transglucosylation, converted sucrose into trehalulose (1-O--D-glucopyranosyl-D-fructose).
The trehalulose-forming activity of the enzyme was very low compared with
that of maltose and trehalose. Kinetic studies showed that sucrose competitively
inhibited the interconversion activity between maltose and trehalose. Consequently,
these three substrates, maltose, trehalose, and sucrose, are thought to
bind the same active site of trehalose synthase.
Key words: trehalose synthase; sucrose; trehalulose
-31-
Selective Incorporation of Polyunsaturated Fatty Acids
into Organelle Phospholipids of Animal Cells
Hironori Masui, Reiko Urade,* and Makoto Kito*
Department of Food and Life Science, Yamaguchi Prefectural University,
3-2-1 Sakurabatake, Yamaguchi 753, Japan
* Research Institute for Food Science, Kyoto University, Uji, Kyoto 611,
Japan
Received November 1, 1996
@The selective incorporation of linoleic (18:2(n-6)) and docosahexaenoic
(22:6(n-3)) acids into phospholipids of mitochondria, endoplasmic reticulum,
and plasma membrane was investigated by changing the ratio of 22:6(n-3)
against 18:2(n-6) in a medium, in which Chinese hamster V79-R cells were
grown.
@In those organelles, 18:2(n-6) and its elongation product (eicosadienoic
acid) (20:2 (n-6)) were predominantly incorporated into phosphatidylcholine.
However, 22:6(n-3) was incorporated more selectively into phosphatidylethanolamine
than 18:2(n-6) and 20:2(n-6).
Key words: unsaturated fatty acids; phospholipids; Chinese hamster V79-R
-32-
Inhibition of Glucan Synthesis by Flavipin-crosslinked
Casein Polymers
Osamu Hayashida, Yamaji Nakano, Keiji Hasumi, and Akira Endo
Department of Applied Biological Science, Faculty of Agriculture, Tokyo Noko University, Fuchu-shi, Tokyo 183, Japan
Received November 7, 1996
@Mutastein, a casein polymer that inhibits insoluble glucan synthesis
by Streptococcus sobrinus, was produced when Aspergillus terreus was grown
in a medium containing -casein. In these experiments, it was shown that
flavipin (3,4,5-trihydroxy-6-methyl-o-phthalaldehyde) isolated from cultures
of A. terreus caused polymerization of -casein. Like mutastein, the polymerization
products were active in inhibiting insoluble glucan synthesis by S. sobrinus.
Key words: glucan synthesis; flavipin; casein; mutastein
-33-
Zinc Finger-like Motif Conserved in a Family of RNA Binding
Proteins
Yuichi Matsushima,* Kiyoshi Matsumura,* and Yasuo Kitagawa,*,**,
* Graduate Program of Biochemical Regulation, School of Agricultural Sciences, Nagoya University, and ** Nagoya University BioScience Center, Nagoya 464-01, Japan
Received November 8, 1996
@NP220s compose a family of RNA binding proteins together with matrin
3, one of major proteins of the nuclear matrix. They have repeats of RNA
recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear
RNPs I/L in addition to MH1 and MH3 with unknown function. In search of
additional homologous sequences, we found the reported sequence of rat
matrin 3 is partially incorrect. Correction of this sequence showed that
the NP220 family has a fourth homologous motif with the characteristics
of a Cys2-His2 zinc finger-like motif. The sequence of this motif is perfectly
conserved in human and mouse NP220s despite their 75% overall sequence
homology.
Key words: nuclear protein; nuclear matrix; RNA binding motif; zinc finger
-34-
Expression of Gene Encoding Endo-1,4--glucanase in Suspension-cultured
Poplar (Poplus alba L.) Cells
Takumi Takeda, Fukumi Sakai, and Takahisa Hayashi
Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611, Japan
Received November 8, 1996
@The level of mRNA for endo-1,4--glucanase was increased before the
exponential phase of growth and decreased rapidly during the exponential
phase in suspension-cultured poplar cells. The level of mRNA was increased
for a short period after the addition of either 2,4-D or sucrose to the
culture medium at the stationary phase. The level was increased to the
maximal level when both 2,4-D and sucrose were provided together, but one
did not increase the effect of the other. These findings suggest that expression
of gene encoding poplar endo-1,4--glucanase is controlled during cell
growth by independent systems activated by auxin and sucrose.
Key words: Poplus alba L.; endo-1,4--glucanase; 2,4-dichlorophenoxyacetic
acid; sucrose
-35-
A Gene Homologous to the Streptomyces Chymotrypsin-like
Protease (SAM-P20) Gene Is Tandemly Located
Seiichi Taguchi, Takahiro Ogawa, Takeshi Endo, and Haruo Momose
Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba 278, Japan
Received November 12, 1996
@A gene encoding a homolog of the Streptomyces chymotrypsin-like serine
protease, SAM-P20, was identified downstream of the sam-p20 gene and designated
SAM-P20D. This gene has two tandem Shine-Dalgarno sequences and two initiation
codons. We have established vector systems with the function of tyrosinase
gene-bone melanin pigmentation as a reporter for sam-p20D gene expression
in Streptomyces coelicolor in order to identify the promoter and terminator
activities. Using this system, the sam-p20D gene was suggested to be transcribed
monocistronically.
Key words: Streptomyces; protease; protease inhibitor; SSI; tandem location
-36-
New Dihydroquinolinone Toxic to Artemia salina Produced
by Penicillium sp. NTC-47
Hideo Hayashi, Tadashi Nakatani, Yoshiki Inoue, Mitsuru Nakayama, and Hiroshi Nozaki*
Department of Applied Biological Chemistry, College of Agriculture,
Osaka Prefecture University, Sakai, Osaka 593, Japan
* Department of Biological Chemistry, Faculty of Science, Okayama University
of Science, Ridai-cho, Okayama 700, Japan
Received November 25, 1996
@Penicillium sp. NTC-47, which had been isolated from a soil sample, produced
novel dihydroquinolinones when cultured with okara (the insoluble residue
of whole soybean). These metabolites, 1 and 2, were crystalline products
with molecular formulas of C17H17NO5 and C17H17NO4, respectively. The structure
of 1 was established to be 3-methoxy-4,5-dihydroxy-4-(4'-methoxyphenyl)quinolinone
by spectroscopic evidence and by an X-ray crystallographic analysis. Spectral
data indicated the structure of 2 to be 5-deoxy-1. Compound 1 demonstrated
toxicity against brine shrimp with an LC(/)50 value of 20 g/ml, but 2
exhibited no activity at a dose of 100 g/ml.
Key words: dihydroquinolinone; toxicity; brine shrimp; Penicillium sp.
-37-
Peculiar Archaea Found in Japanese Paddy Soils
Yuko Kudo, Satoshi Shibata, Taro Miyaki, Toshihiro Aono, and Hiroshi Oyaizu
Graduate School of Agriculture and Agricultural Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 112, Japan
Received November 25, 1996
@Archaeal 16S rDNA clones retrieved from paddy soil DNA were sequenced.
Among 100 clones, 88 clones were assigned to methanogens and nine clones
were assigned to crenarchaeota. However, three of the nine clones were
phylogenetically far from the cultured crenarchaeota and closely related
to marine planktonic archaea. The other three clones showed extremely novel
16S rDNA sequences and were phylogenetically far from both Crenarchaeota
and Euryarchaeota. This paper reports the ubiquitous presence of crenarchaeotal
and extremely novel clones in paddy soils.
Key words: methanogen; archaeal clone; novel archaea; 16S rDNA
-38-
Grandinal, a New Phloroglucinol Dimer from Eucalyptus
grandis
Inder Pal Singh, Rie Hayakawa,* Hideo Etoh,*, Midori Takasaki,** and Takao Konoshima**
The United Graduate School of Agricultural Sciences, Gifu University
(Shizuoka University), Shizuoka 422, Japan
* Department of Applied Biological Chemistry, Shizuoka University, 836
Ohya, Shizuoka 422, Japan
** Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607,
Japan
Received November 29, 1996
@Grandinal, a new dimeric phloroglucinol compound, was isolated from Eucalyptus
grandis and characterized by spectral techniques. Tautomeric structures
1, 2, and 3 were assigned to grandinal. Biogenetically, 1 is proposed to
be formed from intermediates derived from grandinol and jensenone.
Key words: Eucalyptus grandis ; grandinal; phloroglucinol; sideroxylonal
A; grandinol
-39-
Occurrence of Free N-Glycans in Pea (Pisum sativum. L)
Seedlings
Yoshinobu Kimura,*, Shigeaki Takagi,* and Tomonori Shiraishi**
* Department of Bioresources Chemistry, and ** Department of Biological Function and Genetic Resources Science, Faculty of Agriculture, Okayama University, Tsushima-Naka, Okayama 700, Japan
Received January 16, 1997
@Free N-glycans have been found in pea seedlings. These free N-glycans
were coupled with 2-aminopyridine and purified by gel filtration, Con A-Sepharose
affinity chromatography, and size fractionation HPLC. These structures
of pyridylaminated free N-glycans were analyzed by exomannosidase digestions
and ion-spray tandem mass spectrometry. The structural analyses showed
that the several oligomannose-type sugar chains having one GlcNAc residue
at the reducing-end side occur in the seedlings, suggesting the endo--N-acetylglucosaminidase
PS [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 228-232 (1996)] should
be involved in the release of oligomannose-type N-glycans from the storage
glycoproteins [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 1841-1850
(1996)] during the germination of pea seeds.
1.5Key words: free N-glycans; pea seedling; endo--N-acetylglucosaminidase;
peptide : N-glycanase; ion-spray mass spectrometry
-40-
Structure and Activity of a New Form of the Glutamate
Transporter of the Nematode Caenorhabditis elegans
Tsuyoshi Kawano,* Kyoko Takuwa, and Terumi Nakajima
Suntory Institute for Bioorganic Research, Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka 618, Japan
Received January 16, 1997
@A Caenorhabditis elegans cDNA for a glutamate transporter was cloned
and examined in this study. The predicted protein is 11 residues shorter
at the N-terminus than Ceglut-1, which we previously reported. The protein,
when expressed in Xenopus laevis oocytes, showed much higher glutamate
transport activity than Ceglut-1, suggesting that the N-terminal sequence
is critical in glutamate transport.
Key words: nematode; cDNA cloning; glutamate; transporter; expression