(Vol.61 No.10 1997)
Purification and Properties of Two Isozymes of -Glutamyltranspeptidase
from Bacillus subtilis TAM-4
Kazuhiro Abe, Yoshihito Ito, Tetsuo Ohmachi, and Yoshihiro Asada 1621
Methods for Estimating the Parameters of Nonlinear Adsorption
Isotherms of Langmuir and Freundlich Types from a Response Curve of Pulse
Input of an Adsorbate
Shuji Adachi, Chaiya Panintrarux, and Ryuichi Matsuno 1626
Antioxidant Activity of Quercetin against Metmyoglobin-induced
Oxidation of Fish Oil-bile Salt Emulsion
Chimaki Hoshino, Yoko Tagawa, Shun Wada, Jun-Hwan Oh, Dong-Ki Park, Akihiko
Nagao, and Junji Terao 1634
Interaction of Mercury with Human and Bovine Milk Proteins
Luis Mata, Lourdes Sanchez, and Miguel Calvo 1641
Antioxidant Effects of Dopamine and Related Compounds
Gow-Chin Yen and Chiu-Luan Hsieh 1646
Synthesis of Novel 25-Substituted Milbemycin A(>24<)2
Derivatives and Their Acaricidal Activity against Tetranychus urticae
Yoshihisa Tsukamoto, Harumi Nakagawa, Hisaki Kajino, Kazuo Sato, Keiji
Tanaka, and Toshiaki Yanai 1650
Generation of Reactive Oxygen Species by Raphidophycean
Phytoplankton
Tatsuya Oda, Atsushi Nakamura, Midori Shikayama, Ienobu Kawano, Atsushi
Ishimatsu, and Tsuyoshi Muramatsu 1658
Analysis of Aggregate Structure in Food Protein Gels with
the Concept of Fractal
Tomoaki Hagiwara, Hitoshi Kumagai, Tomohide Matsunaga, and Kozo Nakamura
1663
Production of Recombinant Der f I with the Native IgE-Binding
Activity Using a Baculovirus Expression System
Hiroshi Shoji, Ichiro Shibuya, Mitsuo Hirai, Hiroyuki Horiuchi, and Masamichi
Takagi 1668
Measurement of Phenolic Compounds and Their Effect on
Shikonin Production in Lithospermum Cultured Cells
Kazufumi Yazaki, Hiroshi Fukui, Yumiko Nishikawa, and Mamoru Tabata 1674
Effects of Dietary Pyrazinamide on the Metabolism of Tryptophan
to Niacin in Streptozotocin-diabetic Rats
Katsumi Shibata, Aya Ishikawa, and Takako Kondo 1679
Efficient Production of -Polyglutamic Acid by Bacillus
subtilis (natto) in Jar Fermenters
Yoshihiro Ogawa, Fumio Yamaguchi, Katsumi Yuasa, and Yasutaka Tahara 1684
Role of Divalent Metal Ions on Activity and Stability
of Thermostable Dipeptidase from Bacillus stearothermophilus
Hong-Yon Cho, Katsuyuki Tanizawa, and Kenji Soda 1688
Formation Mechanism of Monodehydro-L-ascorbic Acid and
Superoxide Anion in the Autoxidation of L-Ascorbic Acid
Noriko Miyake, Miok Kim, and Tadao Kurata 1693
Synthesis of (R,Z)-7,15-Hexadecadien-4-olide, the Sex
Pheromone of the Yellowish Elongate Chafer (Heptophylla picea)
Shigefumi Kuwahara, Shiharu Hamade, Yukari Yoshinaga, Walter Soares Leal,
and Osamu Kodama 1696
Isolation and Some Properties of Sorbitol Oxidase from
Streptomyces sp. H-7775
Kazumi Hiraga, Mitsunori Kitazawa, Norihisa Kaneko, and Kohei Oda 1699
Purification of Levan Fructotransferase from Arthrobacter
nicotinovorans GS-9 and Production of DFA IV from Levan by the Enzyme
Katsuichi Saito, Hiroko Goto, Atsushi Yokota, and Fusao Tomita 1705
Cloning of Genes of the Aminopeptidase T Family from Thermus
thermophilus HB8 and Bacillus stearothermophilus NCIB8924: Apparent Similarity
to the Leucyl Aminopeptidase Family
Hidemasa Motoshima, Etsuo Minagawa, Fuji Tsukasaki, and Shuichi Kaminogawa
1710
Increase in Swimming Endurance Capacity of Mice by Capsaicin-induced
Adrenal Catecholamine Secretion
Kyung-Mi Kim, Teruo Kawada, Kengo Ishihara, Kazuo Inoue, and Tohru Fushiki
1718
Efficient Syntheses of the OPC Homologous Series, OPC-1
: 0, -3 : 0, -4 : 0, -5 : 0, -6 : 0, -7 : 0, and -8 : 0
Hiroaki Toshima, Shinji Nara, Hisateru Aramaki, Akitami Ichihara, Yasunori
Koda, and Yoshio Kikuta 1724
A Protein Factor Is Essential for in Situ Reactivation
of Glycerol-inactivated Adenosylcobalamin-dependent Diol Dehydratase
Koichi Mori, Takamasa Tobimatsu, and Tetsuo Toraya 1729
Note
Identification of Methyl -Glucopyranoside and Xylose as Soluble Sugar
Constituents in Roses (Rosa hybrida L.)
Kazuo Ichimura, Katsunori Kohata, Mamoru Koketsu, Yuichi Yamaguchi, Hiroyasu
Yamaguchi, and Kenichi Suto 1734
Note
Oxidative Stability of Liposomes Prepared from Soybean PC, Chicken Egg
PC, and Salmon Egg PC
Eiichi Nara, Kazuo Miyashita, and Toru Ota 1736
Note
Purification and Characterization of Konjac Glucomannan Degrading Enzyme
from Anaerobic Human Intestinal Bacterium, Clostridium butyricum-Clostridium
beijerinckii Group
Nobuyoshi Nakajima and Yasushi Matsuura 1739
Note
Anthraquinone Production by Cell Suspension Cultures of Rubia
akane NAKAI
Hiroshi Mizutani, Osamu Hashimoto, Ruka Nakashima, and Jun Nagai 1743
Note
Molecular Cloning of a New Type of cDNA for Pheromone Biosynthesis Activating
Neuropeptide in the Silkworm, Bombyx mori
Tsuyoshi Kawano, Hiroshi Kataoka, Hiromichi Nagasawa, Akira Isogai, and
Akinori Suzuki 1745
Note
Semi Quantification of Gibberellins in the Anthers of Thermosensitive Genetic
Male Sterile Rice (Oryza sativa L. cv. PL12)
Ichiro Honda, Shinya Iwasaki, Kazuhisa Sudo, Hiroshi Kato, Kiyoaki Maruyama,
Morifumi Hasegawa, Isomaro Yamaguchi, Noboru Murofushi, Tadashi Yanagisawa,
and Nobutaka Takahashi 1748
Note
A Sensitive and Rapid Method for Mapping Protein Bound to DNA by Atomic
Force Microscopy
Masato Tanigawa, Masayuki Machida, and Takao Okada 1751
Note
Prolyl Endopeptidase Inhibitors Derived from Actinomycetes
Ken-ichi Kimura, Fumiko Kanou, Yasushi Yamashita, Tadashi Yoshimoto, and
Makoto Yoshihama 1754
Note
Purification and Some Properties of an -Amylase from an Anaerobic Bacterium
Isolated from Coastal Sediment
Haruo Sugita, Akiko Kuruma, and Yoshiaki Deguchi 1757
Note
Sequence Analysis of Functional Regions of Homoserine Dehydrogenase Genes
from L-Lysine and L-Threonine-producing Mutants of Brevibacterium lactofermentum
Masakazu Sugimoto, Akiko Tanaka, Tomoko Suzuki, Hiroshi Matsui, Shigeru
Nakamori and Hiroshi Takagi 1760
Note
Gibberellin Metabolism in Intact Plants of Raphanus sativus L.
Takaaki Nishijima, Masaji Koshioka, Hiroko Yamazaki, and Lewis N. Mander
1763
Note
Construction of T-Tailed Vectors Derived from a pUC Plasmid: a Rapid System
for Direct Cloning of Unmodified PCR Products
Eiji Ido and Masanori Hayami 1766
Note
Carquinostatin B, a New Neuronal Cell-protecting Substance Produced by
Streptomyces exfoliatus
Kazuo Shin-ya, Toshihiro Kunigami, Jun-Sik Kim, Kazuo Furihata, Yoichi
Hayakawa, and Haruo Seto 1768
Note
Cytochrome b/f Complex is not Involved in Respiration in the Cyanobacterium
Synechocystis PCC 6803 Grown Photoautotrophycally
Tsuyoshi Endo 1770
Note
Antioxidative Activity of Water Extracts of Lagerstroemia speciosa Leaves
Tomonori Unno, Iwao Sakane, Toshiki Masumizu, Masahiro Kohno, and Takami
Kakuda 1772
Rapid Paper
Fluorometric Measurement of Yessotoxins in Shellfish by High-pressure Liquid
Chromatography
Takeshi Yasumoto and Azusa Takizawa 1775
Rapid Paper
A Novel Type of D-Mannitol Dehydrogenase from Acetobacter xylinum: Occurrence,
Purification, and Basic Properties
Tadao Oikawa, Junji Nakai, Yasuyuki Tsukagawa, and Kenji Soda 1778
Short Cmmunication
Identification of the Absolute Configuration of Pectenotoxin-6, a Polyether
Macrolide Compound, by NMR Spectroscopic Method Using a Chiral Anisotropic
Reagent, Phenylglycine Methyl Ester
Katsunori Sasaki, Masayuki Satake, and Takeshi Yasumoto 1783
Short Communication
Identification of the Minimum Segment Essential for the HII-Specific
Function of Staphylococcal -Hemolysin
Hirofumi Nariya and Yoshiyuki Kamio 1786
Short Communication
A Beta-Glucosidase Gene Downstream of the Cellulose Synthase Operon in
Cellulose-producing Acetobacter
Naoto Tonouchi, Naoki Tahara, Yukiko Kojima, Tomonori Nakai, Fukumi Sakai,
Takahisa Hayashi, Takayasu Tsuchida, and Fumihiro Yoshinaga 1789 Vol. 61.
No. 10(1997)
-1-
Purification and Properties of Two Isozymes of -Glutamyltranspeptidase
from Bacillus subtilis TAM-4
Kazuhiro Abe, Yoshihito Ito, Tetsuo Ohmachi, and Yoshihiro Asada
Department of Science of Bioresources, Faculty of Agriculture, Hirosaki University, Bunkyo-cho 3, Hirosaki 036, Japan
Received September 27, 1997
@Two isozymes of -glutamyltranspeptidase, GGT-A and GGT-B, were purified
to electrophoretic homogeneity from a culture broth of Bacillus subtilis
TAM-4, which produces poly(-glutamic acid) (PGA) de novo. GGT-A was composed
of three subunits with molecular weights of 23,000 (I), 39,000 (II), and
40,000 (III). GGT-B was composed of two subunits with molecular weights
of 22,000 (I) and 39,000 (II). The N-terminal amino acid sequences of GGT-A
subunit I and GGT-B subunit I were very similar. GGT-A subunit II and GGT-B
subunit II had an identical N-terminal amino acid sequence. That of GGT-A
subunit III showed no similarity to the other subunits. Both GGTs had similar
enzymatic properties (optimum pH and temperature: pH 8.8 and 55C) but
showed a significantly different thermal stability at 55C. Both GGT-A
and -B used D--glutamyl-p-nitroanilide as well as the L-isomer as the
-glutamyl donor and used various amino acids and peptides as the acceptor.
It was also found that the PGA produced by the strain was hydrolyzed to
glutamic acid by its own GGTs.
Key words: Bacillus subtilis; -glutamyltranspeptidase; poly(-glutamic
acid)
-2-
Methods for Estimating the Parameters of Nonlinear Adsorption
Isotherms of Langmuir and Freundlich Types from a Response Curve of Pulse
Input of an Adsorbate
Shuji Adachi, Chaiya Panintrarux, and Ryuichi Matsuno
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
Received January 14, 1997
@Methods for estimating the parameters of nonlinear adsorption isotherms
of Langmuir and Freundlich types from a pulse response curve are proposed
here based on the migration rate of an adsorbate at a constant concentration
and the mean residence time of the adsorbate in a bed. The methods were
used to estimate the parameters in isotherms for various combinations of
adsorbent and adsorbate. The isotherms estimated by the proposed methods
were compared with those estimated by conventional methods. It was demonstrated
that the proposed methods could evaluate the parameters with fairly good
precision when the type of isotherm was known. The criteria for discriminating
the type of isotherm from the pulse response curve are also described.
Key words: adsorption isotherm; Langmuir isotherm; Freundlich isotherm;
pulse response technique
-3-
Antioxidant Activity of Quercetin against Metmyoglobin-induced
Oxidation of Fish Oil-bile Salt Emulsion
Chimaki Hoshino, Yoko Tagawa,* Shun Wada,* Jun-Hwan Oh,** Dong-Ki Park,** Akihiko Nagao, and Junji Terao
National Food Research Institute, Ministry of Agriculture, Forestry
and Fisheries, Tsukuba, Ibaraki 305, Japan
* Department of Food Science and Technology, Tokyo University of Fisheries,
Minato-ku, Tokyo 108, Japan
** Department of Biochemistry, Kon-Kuk University, Choong Joo 380, Korea
Received January 28, 1997
@The antioxidative effect of quercetin was examined in metmyoglobin-induced
oxidation of a fish oil-bile salt emulsion (average diameter of particles;
2.0 m) to evaluate its effectiveness during the digestion of highly oxidizabile
oils. The activity of quercetin increased with the lowering of the initial
peroxide value (PV) of the oil and its effectiveness was superior to that
of -tocopherol. A synergistic antioxidant effect was observed upon the
addition of quercetin and -tocopherol irrespective of the initial PV
of the oils, and quercetin was consumed faster than -tocopherol. The
loss of quercetin was larger than that of -tocopherol when cumene hydroperoxide
and metmyoglobin were mixed in a trimyristin-bile salt emulsion. In an
ultrafiltration experiment on emulsified oil with a membrane filter of
100 nm pore size, the recovery of quercetin in the filtrate was higher
than that of -tocopherol. These data suggest that quercetin was an antioxidant
in the digestion of fish oil. The effectiveness seems to come from its
distribution in the emulsified oil, different from that of -tocopherol,
and its ability to scavenge radicals generated from the reaction of lipid
hydroperoxides with metmyoglobin.
Key words: quercetin; -tocopherol; antioxidant; emulsion; lipid peroxidation
-4-
Interaction of Mercury with Human and Bovine Milk Proteins
Luis Mata, Lourdes Sanchez, and Miguel Calvo
Food Technology and Biochemistry Department, Faculty of Veterinary Science, University of Zaragoza, Miguel Servet 177, 50013-Zaragoza, Spain
Received February 10, 1997
@The interaction of inorganic mercury with human and bovine milk proteins
was studied. Gel filtration chromatography of skimmed milk and whey incubated
with mercury showed that, in human milk, mercury was mainly bound to caseins,
while a low proportion was bound to albumin. In bovine milk, mercury was
associated with two protein fractions, caseins and -lactoglobulin. Furthermore,
it was shown by electrophoresis that mercury induced the formation of dimers
of -lactoglobulin. Thus, in both human and bovine milk, mercury prossessed
greater ability to interact with milk proteins than to the low-molecular-weight
substances. However, the pattern of mercury distribution was different
between the milk of these two species.
Key words: mercury; human milk; bovine milk; milk proteins
-5-
Antioxidant Effects of Dopamine and Related Compounds
Gow-Chin Yen and Chiu-Luan Hsieh
Department of Food Science, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan, Republic of China
Received February 10, 1997
@The antioxidant and free radical scavenging effects of dopamine, noradrenaline,
tyramine, and tyrosine were investigated and compared with -tocopherol.
The antioxidant effect of dopamine and its related compounds on peroxidation
of linoleic acid were in the order of dopamine>-tocopherol=tyramine>tyrosine>noradrenaline
as measured by the thiocyanate method. These amine compounds had reducing
power, and a scavenging effect on reactive oxygen species, i.e., superoxide
anion and hydroxyl radical. The results for reducing power and scavenging
effect of these amine compounds had a similar trend as their inhibition
of linoleic acid peroxidation. The antioxidant activity of these amine
compounds in soybean oil was also evaluated by the Rancimat method. The
induction time to reach 100 meq/kg peroxide value (POV) of soybean oil
for dopamine, -tocopherol, tyramine, tyrosine, noradrenaline, and control
were 9.0, 8.2, 8.0, 6.4, 4.6, and 4.3 h, respectively. The antioxidant
efficacy of amine compounds seems to be correlated with the numbers of
hydroxy groups and their position on the phenolic ring.
Key words: antioxidant; dopamine; tyramine; noradrenaline; reactive oxygen
species
-6-
Synthesis of Novel 25-Substituted Milbemycin A4 Derivatives
and Their Acaricidal Activity against Tetranychus urticae
Yoshihisa Tsukamoto, Harumi Nakagawa, 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 March 5, 1997
@Novel 25-substituted milbemycin A4 derivatives were synthesized from
25a-hydroxymilbemycin A4 and 25b-hydroxymilbemycin A4, which had been obtained
by the microbial oxidation of milbemycin A4. The acaricidal activity of
each synthesized derivative was tested against Tetranychus urticae, and
all of the synthesized derivatives showed higher activity than parent milbemycin
A4. Some of the derivatives had higher acaricidal activity than milbemycin
D, which had higher acaricidal activity than milbemycin A4. Among them,
25b-methylmilbemycin A4 was the most active derivative, with 100% mortality
of the mite at a concentration of 1 ppm, and 63% mortality at 0.1 ppm.
Key words: milbemycin; 25a-substituted milbemycin A4 derivatives; 25b-substituted
milbemycin A4 derivatives; Tetranychus urticae; two-spotted spider mite
-7-
Generation of Reactive Oxygen Species by Raphidophycean
Phytoplankton
Tatsuya Oda, Atsushi Nakamura, Midori Shikayama, Ienobu Kawano, Atsushi Ishimatsu,* and Tsuyoshi Muramatsu
Division of Biochemistry, Faculty of Fisheries, Nagasaki University,
Nagasaki 852, Japan
* Nomo Fisheries Station, Nagasaki University, Nomozaki, Nagasaki 851-05,
Japan
Received March 10, 1997
@Chattonella marina, a raphidophycean flagellate, is one of the most toxic
red tide phytoplankton and causes severe damage to fish farming. Recent
studies demonstrated that Chattonella sp. generates superoxide (O-(/)2),
hydrogen peroxide (H2O2), and hydroxyl radicals (EOH), which may be responsible
for the toxicity of C. marina. In this study, we found that other raphidophycean
flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa
japonica also produce O-(/)2 and H2O2 under normal growth condition. Among
the flagellate species tested, Chattonella has the highest rates of production
of O-(/)2 and H2O2 as compared on the basis of cell number. This seems
to be partly due to differences in their cell sizes, since Chattonella
is larger than other flagellate species. The generation of O-(/)2 by these
flagellate species was also confirmed by a chemiluminescence assay by using
2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA).
All these raphidophycean flagellates inhibited the proliferation of a marine
bacterium, Vibrio alginolyticus, in a flagellates/bacteria co-culture system,
and their toxic effects were suppressed by the addition of superoxide dismutase
(SOD) or catalase. Our results suggest that the generation of reactive
oxygen species is a common feature of raphidophycean flagellates.
Key words: red tide plankton; Raphidophyceae; Chattonella marina; reactive
oxygen species; toxicity
-8-
Analysis of Aggregate Structure in Food Protein Gels with
the Concept of Fractal
Tomoaki Hagiwara, Hitoshi Kumagai, Tomohide Matsunaga, and Kozo Nakamura
Department of Applied Biological Chemistry, Division of Agriculture and Agricultural Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received March 14, 1997
@The fractal structure of the aggregates in food protein gels was analyzed.
Three kinds of food protein gels were prepared: (1) -lactoglobulin (-LG)
gel; (2) 11S soybean globulin gel; and (3) caseinate gel. From the concentration
dependence of the gel elasticity, the fractal dimensions Df of the aggregates
in the gels were evaluated, according to the theory of Shih et al. These
gels showed the weak-link behavior described in the theory of Shih et al.
The values obtained for Df were 2.6-2.7, which were larger than those predicted
by the cluster-cluster aggregation model for a dilute system. In addition,
for the -LG gels, the fractal dimension was also evaluated from the analysis
of the gel image obtained with a confocal scanning laser microscopy, the
value being close to that evaluated from the concentration dependence of
the gel elasticity. These results indicate that the elastic behavior of
the aggregate gels is a reflection of fractal structure of the aggregates
in the gels.
Key words: fractal; confocal scanning microscopy; elasticity; gel; aggregate
-9-
Production of Recombinant Der f I with the Native IgE-Binding
Activity Using a Baculovirus Expression System
Hiroshi Shoji, Ichiro Shibuya, Mitsuo Hirai, Hiroyuki Horiuchi,* and Masamichi Takagi*
Institute for Production Research and Development, The Nikka Whisky
Distilling Co., Ltd., 967, Matsuyama, Masuo, Kashiwa, Chiba 277, Japan
* Laboratory of Cellular Genetics, Department of Biotechnology, Division
of Agriculture and Agricultural Life Science, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received March 24, 1997
@Der f I is a cysteine protease contained in feces of mites and is one
of major mite allergens. Recombinant Der f I (reDer f I) that is produced
using a baculovirus expression system contains pro-sequences of different
lengths. Most of these can be removed by acid treatment. However, IgE-binding
activity of acid-treated reDer f I is lower than that of native Der f I
at high protein concentrations, and N-terminal amino acids of acid-treated
reDer f I are not uniform. Now, a method for processing of the pro-sequence
has been developed by producing reDer f I E(-1)K with baculovirus expression
system in which the carboxy terminal amino acid of the pro-sequence (glutamate)
was replaced by lysine using site directed mutagenesis. No difference in
the amount of production was observed upon introducing the mutation into
the pro-sequence. Addition of lysylendopeptidase into the culture medium
led to processing of the pro-sequence of reDer f I E(-1)K and proceeded
the degradation of the other proteins in the medium. Lysylendopeptidase-treated
reDer f I E(-1)K was easily purified with an anion exchange column, resulting
in 20% increase of the yield. Lysylendopeptidase-treated reDer f I E(-1)K
obtained through these processes was compared with the native Der f I.
Although some differences were found in protease activity and reactivity
with lectins, their N-terminal amino acid and the IgE-binding activity
were the same as those of the native one, indicating its usefulness for
diagnostic purpose.
Key words: allergen; baculovirus expression system; lysylendopeptidase;
lectin
-10-
Measurement of Phenolic Compounds and Their Effect on
Shikonin Production in Lithospermum Cultured Cells
Kazufumi Yazaki, Hiroshi Fukui, Yumiko Nishikawa, and Mamoru Tabata
Faculty of Pharmaceutical Sciences, Kyoto University, Yoshida, Kyoto 606-01, Japan
Received March 25, 1997
@Shikonin production by Lithospermum cell cultures is induced by transferring
the cells into production medium. Six phenolic compounds, p-hydroxybenzoic
acid, caffeic acid, sinapic acid, ferulic acid, syringaldehyde, and salicyclic
acid, were detected in both shikonin-producing and non-producing cells.
Their contents in the former were much lower than those in the latter except
for salicylic acid, the content of which strongly increased when cells
were producing shikonin. The cell wall fraction, after alkaline hydrolysis,
gave two phenolic compounds, p-hydroxybenzoic acid and caffeic acid. Their
contents were much higher in shikonin-producing cells than in shikonin-free
cells. Of these compounds, exogenous addition of p-hydroxybenzoic acid
increased shikonin production in the production medium. Although it is
a precursor of shikonin, the increment of shikonin produced was much larger
than the administered p-hydroxybenzoic acid, suggesting this compound has
a stimulatory effect on shikonin biosynthesis at a low concentration.
Key words: Lithospermum erythrorhizon; p-hydroxybenzoic acid; salicylic
acid; shikonin; biosynthesis
-11-
Effects of Dietary Pyrazinamide on the Metabolism of Tryptophan
to Niacin in Streptozotocin-diabetic Rats
Katsumi Shibata, Aya Ishikawa, and Takako Kondo
Department of Human Health Science, Faculty of Human Sciences, Osaka International University for Women, Moriguchi, Osaka 570, Japan
Received March 26, 1997
@We investigated the effects of feeding with a diet containing pyrazinamide
(PYR) on the metabolism of L-tryptophan (Trp) to nicotinamide in streptozotocin
(STZ)-diabetic rats and whether the diabetic action of STZ is prevented
by feeding with the PYR diet, which is known as an inhibitor of aminocarboxymuconate-semialdehyde
decarboxylase and poly(ADP)ribose synthetase and therefore, significantly
increases the formation of nicotinamide from Trp in normal rats. As was
expected, feeding with the PYR diet to the STZ-injected rats caused a significantly
increased excretion of nicotinamide and its metabolites like that in normal
rats. The body weight increased in the STZ-injected rats fed with the PYR
diet, while it was lost in the STZ-injected rats fed with the non-PYR diet.
However, the blood glucose level and the urinary excretion of glucose were
not improved even when the rats were fed with the PYR diet. Therefore,
it was suggested that chronically increasing the formation of nicotinamide
from Trp could not completely prevent the STZ-diabetic action.
Key words: pyrazinamide; streptozotocin; niacin; tryptophan; aminocarboxymuconate-semialdehyde
decarboxylase
-12-
Efficient Production of -Polyglutamic Acid by Bacillus
subtilis (natto) in Jar Fermenters
Yoshihiro Ogawa, Fumio Yamaguchi, Katsumi Yuasa, and Yasutaka Tahara*
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-shi,
Chiba 278, Japan
* Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka
University, 836 Ohya, Shizuoka-shi, Shizuoka 422, Japan
Received March 26, 1997
@The large scale fermentation of -polyglutamic acid (-PGA) by Bacillus
subtilis (natto) was done using a 30-liter jar fermenter. A stable cultivation
without foaming could be done with addition of 3% NaCl to the medium. The
-PGA productivity became higher with increasing speed of agitation and
amounts of glutamic acid added to the broth. Finally, we were able to obtain
about 35 mg/ml of -PGA under the optimum conditions. The glutamic acid
added to the medium was efficiently converted into -PGA in the stationary
phase. To discover the role of L-glutamic acid added to the medium for
-PGA biosynthesis by Bacillus subtilis (natto), the radioactivity incorporated
into -PGA from 14C-L-glutamic acid was measured. As a result, radioactive
-PGA was detected in the medium. Then, the glutamic acid in the medium
was transported into the cells and actually polymerized as the glutamic
acid unit of -PGA.
Key words: Bacillus subtilis (natto); -polyglutamic acid; salvage pathway
-13-
Role of Divalent Metal Ions on Activity and Stability
of Thermostable Dipeptidase from Bacillus stearothermophilus
Hong-Yon Cho, Katsuyuki Tanizawa,* and Kenji Soda**
Department of Food Science and Biotechnology, Graduate School of Biotechnology,
Korea University, Seoul 136-701, Korea
* Division of Biological Science, Institute of Scientific and Industrial
Research, Osaka University, Ibaraki, Osaka 567, Japan
** Department of Biotechnology, Faculty of Engineering, Kansai University,
Suita, Osaka 564, Japan
Received March 28, 1997
@Thermostable dipeptidase from Bacillus stearothermophilus, a typical
metalloenzyme containing 1.0 g atom of Zn per mole of subunit of the dimeric
enzyme was markedly activated by exogenous divalent metal ions such as
Mn2+, Co2+, and Cd2+. In contrast, several others including Ba2+, Hg2+,
and Cu2+ considerably inhibited the enzyme, even the inherent metal, Zn2+,
being slightly inhibitory. To study the metal-binding properties of this
dipeptidase, the enzyme was completely resolved to the inactive, Zn-free
apoenzyme by treatment with EDTA in the presence of guanidine hydrochloride
in a weakly acidic buffer. The apoenzyme was readily reconstituted by incubation
with either Zn2+, Mn2+, or Co2+, restoring the catalytic activity. The
Mn-reconstituted enzyme had nearly twice the activity of the original Zn-enzyme.
Combined with kinetic analyses of reconstitution of the apoenzyme with
metal ions, these results show that the enzyme has two non-identical metal-binding
sites, each with a different property. Furthermore, substitution of Mn2+
or Co2+ for Zn2+ considerably lowered the thermostability of the enzyme
without affecting the overall conformation of the enzyme protein, suggesting
that the prosthetic Zn is playing dual roles in conformational stability
and catalysis of the thermostable dipeptidase.
Key words: dipeptidase; metalloenzyme; thermostability; apoenzye; reconstitution
-14-
Formation Mechanism of Monodehydro-L-ascorbic Acid and
Superoxide Anion in the Autoxidation of L-Ascorbic Acid
Noriko Miyake, Miok Kim, and Tadao Kurata
Institute of Environmental Science for Human Life, Ochanomizu University, Bunkyo-ku, Tokyo 112, Japan
Received April 7, 1997
@The oxidation of L-ascorbic acid (ASA) by molecular oxygen was studied
in the absence of heavy metal ion catalysts. The formation of superoxide
anion was confirmed during the autoxidation of ASA not only in aqueous
solution but also in MeOH. The formation mechanism of superoxide anion
was discussed based on both experimental and molecular orbital (MO) calculation
results. It was proposed that ASA autoxidation proceeded via the C(2) oxygen
adduct of ASA, and superoxide anion would be directly released from the
C(2) oxygen adduct of ASA, forming monodehydro-ASA (MDASA).
Key words: l-ascorbic acid; monodehydro-l-ascorbic acid; superoxide anion;
autoxidation; oxygen adduct of l-ascorbic acid
-15-
Synthesis of (R,Z)-7,15-Hexadecadien-4-olide, the Sex
Pheromone of the Yellowish Elongate Chafer (Heptophylla picea)
Shigefumi Kuwahara, Shiharu Hamade, Yukari Yoshinaga, Walter Soares Leal,* and Osamu Kodama
Laboratory of Agricultural Chemicals, Faculty of Agriculture, Ibaraki
University, Ami-machi, Inashiki-gun, Ibaraki 300-03, Japan
* Laboratory of Chemical Prospecting, National Institute of Sericultural
and Entomological Science, 1-2 Ohwashi, Tsukuba-shi 305, Japan
Received April 8, 1997
@(R,Z)-7,15-Hexadecadien-4-olide, the sex pheromone of the yellowish elongate
chafer (Heptophylla picea), was synthesized from L-malic acid in 15 steps.
The synthetic pheromone was identical with the natural product in its MS,
IR, GLC retention time, and biological activity.
Key words: sex pheromone; yellowish elongate chafer; Heptophylla picea;
7,15-hexadecadien-4-olide
-16-
Isolation and Some Properties of Sorbitol Oxidase from
Streptomyces sp. H-7775
Kazumi Hiraga, Mitsunori Kitazawa, Norihisa Kaneko, and Kohei Oda
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606, Japan
Received April 15, 1997
@A sorbitol oxidase (SOX) was found in the cell-free extract of a strain
isolated from soil. The strain was classified and designated as Streptomyces
sp. H-7775. SOX is constitutively expressed in the cell. The molecular
weight of SOX that purified from the cell-free extract was 45,000. The
optimum pH and the Km for sorbitol were 6.5-7.5 and 0.26 mM, respectively.
The prosthetic group was a covalently bound FAD. SOX catalyzed oxidation
of D-sorbitol to glucose and hydrogen peroxide without any requirements
of exogenous cofactors. SOX did not react with glucose, a reaction product
of D-sorbitol. This feature is useful in its application for diagnosis.
Key words: sorbitol oxidase; sorbitol; oxidase; Streptomyces sp.
-17-
Purification of Levan Fructotransferase from Arthrobacter
nicotinovorans GS-9 and Production of DFA IV from Levan by the Enzyme
Katsuichi Saito, Hiroko Goto, Atsushi Yokota, and Fusao Tomita
Laboratory of Applied Microbiology, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Received April 21, 1997
@A bacterial strain, GS-9, isolated from soil as a levan-degrading microorganism
produced an extracellular enzyme that converted levan into DFA IV. This
strain was identified as Arthrobacter nicotinovorans. The DFA IV-producing
enzyme was specifically induced by levan. The enzyme was purified 60-fold
from culture supernatant to give a single band on SDS-PAGE. The molecular
weight of this enzyme was 52,000 by SDS-PAGE and a monomer by gel filtration.
The enzyme gave DFA IV as a main product (>75%), and fructose, levanbiose,
and two unidentified oligosaccharides as minor products, and was identified
as a novel levan fructotransferase.
Key words: levan; levan fructotransferase; DFA IV; Arthrobacter nicotinovorans
GS-9
-18-
Cloning of Genes of the Aminopeptidase T Family from Thermus
thermophilus HB8 and Bacillus stearothermophilus NCIB8924: Apparent Similarity
to the Leucyl Aminopeptidase Family
Hidemasa Motoshima, Etsuo Minagawa, Fuji Tsukasaki, and Shuichi Kaminogawa*
Research Center, Yotsuba Milk Products Co., Ltd., 465-1, Wattsu, Kitahiroshima,
Hokkaido 061-12, Japan
* Department of Applied Biochemistry, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113, Japan
Received April 21, 1997
@To obtain genes with sequence similarity to aminopeptidase T (AP-T) of
Thermus aquaticus YT-1, we cloned the genes encoding aminopeptidase Th
(AP-Th) from Thermus thermophilus HB8 and aminopeptidase II (APII) from
Bacillus stearothermophilus NCIB8924. The AP-Th gene encoded a polypeptide
of 408 amino acid residues and the deduced molecular weight of this subunit
was 45,015. The APII gene encoded a polypeptide of 413 amino acid residues
with a deduced molecular weight of 46,207. The extent of amino acid sequence
similarity between AP-Th and AP-T was 86%, and that between APII and AP-T
was 43%. The substrate specificities of these expressed enzymes were similar,
and each efficiently hydrolyzed leucyl- or phenyl-peptide substrates. Since
the deduced amino acid sequence of these enzymes show no similarity to
other known aminopeptidases, they appear to comprise an independent family
of peptidases, designated the AP-T family. However, a conserved region
within the enzymes of the AP-T family shows similarity to the active site
signature of the leucyl aminopeptidase family, suggesting that these enzymes
may belong to the leucyl aminopeptidase superfamily.
Key words: aminopeptidase; Bacillus stearothermophilus; Thermus; aminopeptidase
T family
-19-
Increase in Swimming Endurance Capacity of Mice by Capsaicin-induced
Adrenal Catecholamine Secretion
Kyung-Mi Kim, Teruo Kawada, Kengo Ishihara, Kazuo Inoue, and Tohru Fushiki
Laboratory of Nutrition Chemistry, Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan
Received April 21, 1997
@Increase in endurance swimming capacity caused by capsaicin (CAP), a
pungent component of red pepper, -induced increase of fat metabolism in
mice was investigated using an adjustable-current water pool. The mice
administered CAP via a stomach tube, showed longer swimming time until
exhaustion than the control group of mice, in a dose-dependent manner.
The maximal effect was observed at a dose of 10 mg/kg while more than 15
mg/kg had no effect. The increase of endurance was observed only when CAP
was administered two hours before swimming. After the administration of
CAP, the serum glucose concentration rapidly increased and then decreased
within 60 min, while the concentration of serum-free fatty acids gradually
increased through 3 hours. The residual glycogen concentration of the gastrocnemius
muscle after 30 min of swimming was significantly higher in the CAP-administered
mice than in control mice, suggesting that use of the serum free fatty
acids spared muscle glycogen consumption. The serum adrenaline concentration
significantly increased with twin peaks at 30 min and two hours after administration
of CAP. An experiment using adrenalectomized mice was done to confirm that
the effect of CAP is due to increased energy metabolism through the secretion
of adrenaline from the adrenal gland. The swimming endurance capacity of
the adrenalectomized mice was not increased by CAP administration, although
adrenaline injection induced a 58% increase in the endurance time. These
results suggest that the increase of swimming endurance induced by CAP
in mice is caused by an increase in fatty acid utilization due to CAP-induced
adrenal catecholamine secretion.
Key words: capsaicin; swimming endurance capacity; adrenaline
-20-
Efficient Syntheses of the OPC Homologous Series, OPC-1
: 0, -3 : 0, -4 : 0, -5 : 0, -6 : 0, -7 : 0, and -8 : 0
Hiroaki Toshima, Shinji Nara, Hisateru Aramaki, Akitami Ichihara, Yasunori Koda,* and Yoshio Kikuta*
Department of Bioscience and Chemistry, and * Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Received April 25, 1997
@The OPC homologous series was synthesized from 2-[(Z )-2-pentenyl]cyclopenten-1-one
in short steps and with high yields. The carbon-carbon bond formation was
achieved by the 1,4-conjugate addition approach. This method makes it possible
to supply a sufficient amount of OPC homologues which would enable significant
information to be collected for plant physiological studies.
Key words: jasmonoid; octadecanoid; 12-oxo-PDA; OPC; 1,4-conjugate addition
-21-
A Protein Factor Is Essential for in Situ Reactivation
of Glycerol-inactivated Adenosylcobalamin-dependent Diol Dehydratase
Koichi Mori, Takamasa Tobimatsu, and Tetsuo Toraya
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700, Japan
Received May 2, 1997
@The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca
undergoes suicidal inactivation by glycerol during catalysis involving
irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated
holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid
containing the diol dehydratase genes and their flanking regions was rapidly
reactivated in the presence of free AdoCbl, ATP, and Mg2+. ,-Methylene
ATP was not able to replace ATP. Inactive complexes of the enzyme with
aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl,
ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest
that the inactivated holoenzyme is reactivated in situ in the presence
of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine
moiety for free intact AdoCbl. The in situ reactivation was also observed
when an analog lacking the -ribose moiety of the nucleotide loop was
used as coenzyme. The results with a recombinant E. coli strains carrying
a deletion mutant plasmid demonstrate that certain protein(s) encoded by
the 3'-flanking region of the diol dehydratase genes are essential for
the in situ reactivation of inactivated diol dehydratase.
Key words: reactivating factor; adenosylcobalamin; diol dehydratase; vitamin
B12; suicide inactivation
-22-
Note
Identification of Methyl -Glucopyranoside and Xylose as Soluble Sugar
Constituents in Roses (Rosa hybrida L.)
Kazuo Ichimura, Katsunori Kohata, Mamoru Koketsu,* Yuichi Yamaguchi, Hiroyasu Yamaguchi, and Kenichi Suto
National Research Institute of Vegetables, Ornamental Plants and Tea,
Ano, Mie 514-23, Japan
* Faculty of Engineering, Gifu University, Gifu 501-11, Japan
Received December 13, 1996
@Two unidentified sugars were isolated from rose petals using HPLC. The
isolated compounds were identified as methyl -glucopyranoside and xylose
using 1H-NMR, 13C-NMR, and GC-MS. Methyl -glucopyranoside and xylose
were distributed in three cultivars tested relatively in large amounts.
These results indicate that methyl -glucopyranoside and xylose occur
universally as soluble sugar constituents in roses.
Key words: methyl -glucopyranoside; xylose; soluble sugar; rose; cut
flower
-23-
Note
Oxidative Stability of Liposomes Prepared from Soybean PC, Chicken Egg
PC, and Salmon Egg PC
Eiichi Nara, Kazuo Miyashita, and Toru Ota
Department of Chemistry, Faculty of Fisheries, Hokkaido University, 3-1-1 Minatocho, Hakodate 041, Japan
Received February 14, 1997
@The oxidative stability of phosphatidylcholines (PCs) from soybean, chicken
egg, and salmon egg in liposomes was compared with that in aqueous micelles.
When each PC was oxidized in aqueous micelles, salmon egg PC was the most
oxidatively stable, followed by chicken egg PC and soybean PC, however,
no significant difference in the oxidative stability was apparent between
chicken egg PC and salmon egg PC in liposomes. The main molecular species
of soybean PC was 1,2-dilinoleoyl-PC, while most of the PUFAs in chicken
egg PC and salmon egg PC were not esterified at the sn-1 position but at
the sn-2 position. Therefore, it is suggested that the oxidative stability
of PC liposomes would be strongly influenced by the positional distribution
of PUFAs in the PC molecule. Further studies on the oxidation of PC liposomes
showed that chicken egg albumin and soybean protein protected PC bilayers
against attack by free radicals generated in the aqueous phase.
Key words: PC liposome; oxidative stability; molecular species; antioxidant
effect of proteins
-24-
Note
Purification and Characterization of Konjac Glucomannan Degrading Enzyme
from Anaerobic Human Intestinal Bacterium, Clostridium butyricum-Clostridium
beijerinckii Group
Nobuyoshi Nakajima and Yasushi Matsuura
Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-11, Japan
Received March 6, 1997
@Konjac glucomannan degrading enzyme was purified to homogeneity from
the culture broth of an anaerobic human intestinal bacterium, Clostridium
butyricum-Clostridium beijerinckii group. The enzyme was composed of a
single polypeptide chain with a molecular weight of 50,000-53,000. The
enzyme was an endo--mannanase that acted specifically on the polysaccharides
such as konjac glucomannan and coffee mannan, producing exclusively their
smaller oligosaccharides and the monosaccharides. The optimal pH of the
enzyme for the hydrolysis of konjac glucomannan was around 7-8 and the
enzyme was stable in rather alkaline pH range of 8-10. The enzyme reaction
was activated by the addition of CaCl2 and dithiothreitol. It was suggested
that the enzyme might contribute to the decomposition of konjac glucomannan
in human digestive tract.
Key words: konjac glucomannan; coffee mannan; extracellular endo--mannanase;
Clostridium butyricum-Clostridium beijerinckii group; anaerobic intestinal
bacteria
-25-
Note
Anthraquinone Production by Cell Suspension Cultures of Rubia akane NAKAI
Hiroshi Mizutani,*,** Osamu Hashimoto,* Ruka Nakashima,* and Jun Nagai**
* Sumi Research and Development Center, Konobu-Nakashima, Bisai 494,
Japan
** Faculty of Engineering, Tottori University, Koyama, Tottori 680, Japan
Received March 7, 1997
@The effects of various plant growth regulators and nutrients on cell
growth and anthraquinone production in cell suspension cultures of Rubia
akane NAKAI were investigated. Use of an optimized medium resulted in a
two-fold increase in the anthraquinone content. From the cell cultures
1,2-dihydroxyanthraquinone was isolated as the main constituent.
Key words: Rubia akane Nakai; rubiaceae; anthraquinone; cell suspension
cultures
-26-
Note
Molecular Cloning of a New Type of cDNA for Pheromone Biosynthesis Activating
Neuropeptide in the Silkworm, Bombyx mori
Tsuyoshi Kawano, Hiroshi Kataoka,*, Hiromichi Nagasawa, Akira Isogai, and Akinori Suzuki
Department of Applied Biological Chemistry, and * Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Received March 11, 1997
@A neuropeptide named pheromone biosynthesis activating neuropeptide (PBAN)
stimulates the pheromone production of lepidopteran insects. We have identified
a different type of cDNA for PBAN, which shows two amino acid replacements
in the region of the PBAN sequence previously reported. Single strand conformation
polymorphism (SSCP) analysis revealed that the two types of cDNA originated
from two allelic variants of the gene for PBAN.
Key words: PBAN (pheromone biosynthesis activating neuropeptide); Bombyx
mori ; SSCP analysis; insect hormone
-27-
Note
Semi Quantification of Gibberellins in the Anthers of Thermosensitive Genetic
Male Sterile Rice (Oryza sativa L. cv. PL12)
Ichiro Honda,, Shinya Iwasaki, Kazuhisa Sudo, Hiroshi Kato,* Kiyoaki Maruyama,* Morifumi Hasegawa,** Isomaro Yamaguchi,** Noboru Murofushi,** Tadashi Yanagisawa, and Nobutaka Takahashi***
Department of Applied Biochemistry, Utsunomiya University, 350 Mine-machi,
Utsunomiya 321, Japan
* National Agriculture Research Center, Ministry of Agriculture, Forestry,
and Fisheries, 3-1-1 Kannondai, Tsukuba 305, Japan
** Department of Applied Biological Chemistry, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
*** Frontier Research Program, The Institute of Physical and Chemical Research
(RIKEN), 2-1 Hirosawa, Wako 350-01, Japan
Received March 13, 1997
@The levels of endogenous GAs in the anthers of rice (Oryza sativa L.,
cv. Reimei (normal)) and in those of sterile and fertile plants of a thermosensitive
genetic male sterile line (Norin PL12; derived from Reimei) were measured
by enzyme-linked immunosorbent assay (ELISA). High levels of GA4#7 were
detected in the anthers of Reimei (normal fertility) and fertile (growing
under fertile conditions) PL12, while such levels were markedly reduced
with increased sterility in PL12. The anthers of the plants exposed to
the sterile conditions contained undeveloped pollens. These results suggest
that the occurrence of GA4#7 was closely related to the expression of the
sterility gene and the growth and/or development of the anther and/or pollen
PL12.
Key words: Oryza sativa; sterile; thermosensitive genetic male sterility;
gibberellin; ELISA
-28-
Note
A Sensitive and Rapid Method for Mapping Protein Bound to DNA by Atomic
Force Microscopy
Masato Tanigawa, Masayuki Machida,*, and Takao Okada
Joint Research Center for Atom Technology, Higashi 1-1-4, Tsukuba, Ibaraki
305, Japan
* Department of Molecular Biology, National Institute of Bioscience and
Human-Technology, Higashi 1-1, Tsukuba, Ibaraki 305, Japan
Received March 13, 1997
@We addressed the strategies of mapping protein binding sites on a DNA
fragment by atomic force microscopy (AFM). The protein binding site was
uniquely mapped by distinguishing two termini of a linear DNA fragment.
Our simple methods were found very useful to get information on transcription
regulatory regions by taking advantage of long range and quick scanning
by AFM.
Key words: DNA mapping; atomic force microscopy; Sp1; DNA-protein complex
-29-
Note
Prolyl Endopeptidase Inhibitors Derived from Actinomycetes
Ken-ichi Kimura, Fumiko Kanou, Yasushi Yamashita, Tadashi Yoshimoto,* and Makoto Yoshihama
Research Institute of Life Science, Snow Brand Milk Products Co., Ltd.,
Ishibashi-machi, Shimotsuga-gun, Tochigi 329-05, Japan
* School of Pharmaceutical Sciences, Nagasaki University, Nagasaki, Nagasaki
852, Japan
Received March 24, 1997
@Four prolyl endopeptidase inhibitors isolated from actinomycetes, named
propeptin, SNA-8073-B, staurosporine, and enduracidin were classified into
3 groups on the basis of their inhibition potency against prolyl endopeptidase
from a bacterium (Flavobacterium) and a mammal (human placenta). Staurosporine
inhibited the enzyme from Flavobacterium more strongly than that from human
placenta. Enduracidin inhibited the enzyme from human placenta more strongly
than that from Flavobacterium. Propeptin and SNA-8073-B, both new compounds,
inhibited the enzymes from both origins to the same extent.
Key words: prolyl endopeptidase; inhibitor; actinomycetes; natural product
-30-
Note
Purification and Some Properties of an -Amylase from an Anaerobic Bacterium
Isolated from Coastal Sediment
Haruo Sugita, Akiko Kuruma, and Yoshiaki Deguchi
Department of Marine Science and Resources, Nihon University, Kameino, Fujisawa, Kanagawa 252, Japan
Received March 25, 1997
@A marine, obligate anaerobic bacterium, SS71, isolated from a coastal
sediment, was a Gram-positive, asporogenous, rod-shaped organism with a
G+C mol% of 37.3}0.1. This bacterium produced an -amylase with a molecular
mass of 91 kDa and an isoelectric point of 4.3. The -amylase had an optimal
pH of 7.0 and an optimal temperature of 35C. The enzyme activity was
promoted by 0.5-2.0% NaCl.
Key words: -amylase; marine bacteria; obligate anaerobe; coastal sediment
-31-
Note
Sequence Analysis of Functional Regions of Homoserine Dehydrogenase Genes
from L-Lysine and L-Threonine-producing Mutants of Brevibacterium lactofermentum
Masakazu Sugimoto, Akiko Tanaka, Tomoko Suzuki, Hiroshi Matsui, Shigeru Nakamori, and Hiroshi Takagi ,
Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan
Received March 28, 1997
@The homoserine dehydrogenase (HD) genes from Brevibacterium lactofermentum
lysine- and threonine-producing mutants were cloned, using the polymerase
chain reaction, and sequenced. We found the amino acid substitutions, Val104Ile
in the lysine-producing mutants in which HD may cause leaky mutation and
Ser393Phe in the threonine-producing mutant with feedback-insensitive HD.
Key words: homoserine dehydrogenase gene; nucleotide sequence; Brevibacterium
lactofermentum
-32-
Note
Gibberellin Metabolism in Intact Plants of Raphanus sativus L.
Takaaki Nishijima, Masaji Koshioka, Hiroko Yamazaki, and Lewis N. Mander *
National Research Institute of Vegetables, Ornamental Plants and Tea,
360 Kusawa, Ano-cho, Age-gun, Mie 514-23, Japan
* Research School of Chemistry, The Australian National University, G.P.O.
Box 4, Canberra A.C.T. 2601, Australia
Received April 7, 1997
@The metabolism of gibberelline (GAs) in intact plants of Raphanus sativus
was investigated. With [2H]GA feeds, [2H]GA1 from [2H]GA4, and [2H]GA4
and [2H]GA20 from [2H]GA9 were metabolized. Since [2H]GA20 was not converted
into [2H]GA1, endogenous GA1 may have been biosynthesized from GA9 via
GA4 rather than from GA20. The radioactivity of [3H]GA9 and [3H]GA20 was
much more strongly transported among the plant organs than that of [3H]GA1
and [3H]GA4.
Key words: gibberellin; Raphanus sativus; metabolism; [2H]gibberellin;
[3H]gibberellin
-33-
Note
Construction of T-Tailed Vectors Derived from a pUC Plasmid: a Rapid System
for Direct Cloning of Unmodified PCR Products
Eiji Ido and Masanori Hayami
Laboratory of Pathogenic Viruses, Institute for Virus Research, Kyoto University, 53 Shogoin-kawaracho, Sakyo-ku, Kyoto 606, Japan
Received April 7, 1997
@We consructed two new T-vectors called pUCTA119 and pUCTA18, derived
from pUC18. The vectors were designed to produce single thymidine (T)-overhangs
when digested with a restriction enzyme Eam1105I. The use of the vectors
provides a very rapid system for direct TA cloning and subsequent sequencing
of unmodified PCR products since Taq DNA polymerase preferentially adds
an adenosine (A) residue to the 3' end of the products under standard PCR
conditions.
Key words: T-vector; PCR; TA cloning; pUC
-34-
Note
Carquinostatin B, a New Neuronal Cell-protecting Substance Produced by
Streptomyces exfoliatus
Kazuo Shin-ya, Toshihiro Kunigami, Jun-Sik Kim, Kazuo Furihata,* Yoichi Hayakawa, and Haruo Seto
Institute of Molecular and Cellular Biosciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113, Japan
* Division of Agriculture and Agricultural Life Sciences, The University
of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Received April 18, 1997
@Brain ischemia injury is elicited by the excitotoxicity of L-glutamate.
Carquinostatin B was isolated from Streptomyces exfoliatus 2419-SVT2 as
a potent neuroprotective substance which protests neuronal hybridoma N18-RE-105
cells from L-glutamate toxicity. The structure of carquinostatin B was
established principally by NMR studies to be a carbazole derivative with
an ortho quinone function.
Key words: ischemia injury; l-glutamate toxicity; neuroprotection; carquinostatin
B; antioxidative substance
-35-
Note
Cytochrome b/f Complex Is Not Involved in Respiration in the Cyanobacterium
Synechocystis PCC 6803 Grown Photoautotrophycally
Tsuyoshi Endo
The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan
Received May 14, 1997
@Respiratory oxygen uptake was not suppressed by the inhibitor of the
quinol oxidation site of cytochrome b/f complex 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone
(DBMIB), but by far-red illumination in Synechocystis PCC 6803 cells grown
photoautotrophically. It is proposed that cytochrome b/f complex is not
involved in the respiratory electron transport.
Key words: cytochrome b/f complex; cyanobacterium; photosynthesis; respiration;
Synechocystis PCC 6803
-36-
Note
Antioxidative Activity of Water Extracts of Lagerstroemia speciosa Leaves
Tomonori Unno, Iwao Sakane, Toshiki Masumizu,* Masahiro Kohno,* and Takami Kakuda
Central Research Institute, Itoen Ltd., 21 Mekami, Sagara-cho, Haibara-gun,
Shizuoka 421-05, Japan
* Analytical Instruments Division, JEOL Ltd., 1-2 Musashino 3-chome, Akishima,
Tokyo 196, Japan
Received May 28, 1997
@In order to develop naturally occurring antioxidants from edible plants,
the antioxidative effect of hot water extracts of Lagerstroemia speciosa
leaves, known by the Tagalog name of banaba in the Phillipines, was studied.
The content of tannin in banaba extract was 36.8% in dry weight. Banaba
extract showed strong antioxidative activity in a linoleic acid autoxidation
system. Banaba extract was found to have a potent radical scavenging action
on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and superoxide radicals
(O2-) generated by a hypoxanthine (HPX)/xanthine oxidase (XOD) system.
In vitro lipid peroxidation of rat liver homogenate induced by tert-butyl
hydroperoxide (BHP) was inhibited by the addition of banaba extract in
a dose-dependent manner. From these results, banaba extract was demonstrated
to be useful as an antioxidant or free radical scavenger to protect biological
systems against oxidative stress.
Key words: Lagerstroemia speciosa; banaba; antioxidative activity; lipid
peroxidation; radical scavenging activity
-37-
Rapid Paper
Fluorometric Measurement of Yessotoxins in Shellfish by High-pressure Liquid
Chromatography
Takeshi Yasumoto and Azusa Takizawa
Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981, Japan
Received May 20, 1997
@A rapid HPLC method with fluorescence detection of yessotoxin (YTX) and
its two analogs (45-OHYTX and norYTX) in mussels and scallops is presented.
A dienophile reagent, DMEQ-TAD, was used for fluorescence labeling. YTX
was measured in the range 1-100 ng. The method confirmed the occurrence
of YTX and 45-OHYTX for the first time in mussels from Chile and New Zealand.
Key words: fluorometric measurement; DMEQ-TAD; yessotoxin; HPLC; mussels
-38-
Rapid Paper
A Novel Type of D-Mannitol Dehydrogenase from Acetobacter xylinum: Occurrence,
Purification, and Basic Properties
Tadao Oikawa,*,**, Junji Nakai,* Yasuyuki Tsukagawa,* and Kenji Soda*,**
* Department of Biotechnology, Faculty of Engineering, Kansai University, and ** Kansai University High Technology Research Center, Suita-shi, Osaka 564, Japan
Received May 21, 1997
@We purified a novel type of D-mannitol dehydrogenase, which contains
a c-type cytochrome and an unknown chromophore in the soluble fraction
of an acetic acid bacterium, Acetobacter xylinum KU-1, to homogeneity.
The enzyme showed the maximum activity at pH 5 and 40C. It was stable
up to 60C at pH 6, and was inhibited by Hg2+ and p-quinone (Ki=0.18 mM).
The molecular weight of the enzyme was about 140,000, and those of the
subunits were 69,000, 51,000, and 20,000; the enzyme is hetero-trimeric
and contained 8 g-atoms of Fe per mole. The -helix content was estimated
to be about 52.9%. The enzyme catalyzed phenazine methosulfate dependent
oxidation of D-mannitol with an apparent Km of 98 M (for D-mannitol)
and Vmax of 213 mol/min/mg. The reduced form of the enzyme showed the
absorption maxima at 386, 416, 480, 518, 550, and 586 nm, which are attributable
to a c-type cytochrome in the enzyme.
Key words: Acetobacter xylinum; d-mannitol dehydrogenase; c-type cytochrome
-39-
Short Communication
Identification of the Absolute Configuration of Pectenotoxin-6, a Polyether
Macrolide Compound, by NMR Spectroscopic Method Using a Chiral Anisotropic
Reagent, Phenylglycine Methyl Ester
Katsunori Sasaki, Masayuki Satake, and Takeshi Yasumoto
Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiya, Aoba-ku, Sendai 981, Japan
Received May 20, 1997
@The absolute configuration of pectenotoxin-6 (4, PTX6), found in association
with diarrhetic shellfish poisoning, was identified by NMR spectroscopy
using a chiral anisotropic reagent, phenylglycine methyl ester (PGME),
which was condensed with a carboxyl group of 4.
Key words: diarrhetic shellfish poisoning (DSP); pectenotoxin (PTX); cytotoxicity;
macrolide; phenylglycine methyl ester (PGME)
-40-
Short Communication
Identification of the Minimum Segment Essential for the HII-Specific
Function of Staphylococcal -Hemolysin
Hirofumi Nariya and Yoshiyuki Kamio
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981, Japan
Received May 26, 1997
@Staphylococcal -hemolysin consists of HI (or LukF) of 34 kDa and
HII of 32 kDa, which cooperatively lyse human erythrocytes. Our previous
data showed that the N-terminal 57-residue segment of HII is the essential
region for the HII function [H. Nariya and Y. Kamio, Biosci. Biotech.
Biochem., 59, 1603-1604 (1995)]. To identify the minimum amino acid residues
in the 57-residue segment responsible for the specific hemolytic activity,
a series of mutant genes were constructed and expressed in Escherichia
coli. The mutant proteins were purified and assayed for their hemolytic
activity. The results indicate that the 5-residue segment (K23R24L25A26I27)
of HII is the minimum region essential for the HII function.
Key words: staphylococcal leukocidin; -hemolysin; bi-component cytolysin;
pore-forming toxin
-41-
Short Communication
A Beta-Glucosidase Gene Downstream of the Cellulose Synthase Operon in
Cellulose-producing Acetobacter
Naoto Tonouchi, Naoki Tahara, Yukiko Kojima, Tomonori Nakai,* Fukumi Sakai,* Takahisa Hayashi,* Takayasu Tsuchida, and Fumihiro Yoshinaga
Bio-Polymer Research Co., Ltd., 3-2-1 Sakato, Takatsu-ku, Kawasaki 213,
Japan
* Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611, Japan
Received July 4, 1997
@An open reading frame was found 214 bp downstream of the cellulose synthase
operon of Acetobacter. The encoded amino acid sequence was found to be
similar to some beta-glucosidases (G3ases). We detected G3ase activity
in the culture medium and analysis of the N-terminal amino acid sequence
showed that this gene encodes the enzyme. Therefore, it is possible that
this region is a gene cluster for cellulose synthesis.
Key words: Acetobacter; cellulose synthesis; beta-glucosidase; gene