(Vol.61 No.6 1997)
CO2 Sorption by Microbial Cells and Sterilization by High-pressure
CO2
Hitoshi Kumagai, Chiho Hata, and Kozo Nakamura 931
Analysis of Water Sorption Isotherms of Superabsorbent
Polymers by Solution Thermodynamics
Hitoshi Kumagai, Akinori Mizuno, Hitomi Kumagai, and Toshimasa Yano
936
Purification and Characterization of Soybean Allergen Gly
m Bd 28K
Hideaki Tsuji, Noriko Bando, Miki Hiemori, Rintaro Yamanishi, Masumi
Kimoto, Kiyoshi Nishikawa, and Tadashi Ogawa 942
Heterologous Protein Production in Acremonium chrysogenum:
Expression of Bacterial Cephalosporin C Acylase and Human Thrombomodulin
Genes
Goichi Honda, Akio Matsuda, Michitaka Zushi, Shuji Yamamoto, and Ken-ichi
Komatsu 948
Enzymatic Production of Pyrimidine Nucleotides Using Corynebacterium
ammoniagenes Cells and Recombinant Escherichia coli Cells: Enzymatic Production
of CDP-Choline from Orotic Acid and Choline Chloride (Part I)
Tatsuro Fujio and Akihiko Maruyama 956
Construction of a Plasmid Carrying both CTP Synthetase
and a Fused Gene Formed from Cholinephosphate Cytidylyltransferase and
Choline Kinase Genes and Its Application to Industrial CDP-Choline Production:
Enzymatic Production of@CDP-Choline from Orotic Acid (Part II)
Tatsuro Fujio, Sadao Teshiba, and Akihiko Maruyama 960
Purification and Characterization of a Glucose-tolerant
ΐ-Glucosidase from Aspergillus niger CCRC 31494
Tsong-Rong Yan and Chun-Lieh Lin 965
Long-term Culture of Primary Rat Hepatocytes on Heparin-
or Lambda Carrageenan-containing Collagen Gels
Kong Hua Lin, Sumio Maeda, Hidetoshi Inagaki, and Takao Saito 971
Elicitor Actions of N-Acetylchitooligosaccharides and Laminarioligosaccharides
for Chitinase and L-Phenylalanine Ammonia-lyase Induction in Rice Suspension
Culture
Hiroshi Inui, Yasuhiro Yamaguchi, and Shigehiro Hirano 975
The Sugar Specificity of a Na+/Glucose Cotransporter from
Rat Jejunum
Hitoshi Aoshima, Terufumi Yokoyama, Junko Tanizaki, Hanae Izu, and
Mamoru Yamada 979
Primary Structure of 6.5k-Arginine/Glutamate-rich Polypeptide
from the Seeds of Sponge Gourd (Luffa cylindrica)
Makoto Kimura, Sung-Soo Park, Ritsu Sakai, Nobuyuki Yamasaki, and Gunki
Funatsu 984
Metabolism of DFA III by Arthrobacter sp. H65-7: Purification
and Properties of a DFA III Hydrolysis Enzyme (DFA IIIase)
Hiroaki Sakurai, Atsushi Yokota, Yoko Sumita, Yumiko Mori, Hirokazu
Matsui, and Fusao Tomita 989
Actions of Pokeweed Antiviral Protein on Virus-infected
Protoplasts
Keiichi Watanabe, Tetsuji Kawasaki, Nobumichi Sako, and Gunki Funatsu
994
Changes in Proteasome Levels in Spinach (Spinacia oleracea)
Seeds during Imbibition and Germination
Miyuki Miyawaki, Misako Aito, Naoko Ito, Yuki Yanagawa, Richard E.
Kendrick, Keiji Tanaka, Takahide Sato, and Hiroki Nakagawa 998
Changes in Messenger RNA of Pancreatic Enzymes and Intestinal
Cholecystokinin after a 7-Day Bile-pancreatic Juice Diversion from the
Proximal Small Intestine in Rats
Hiroshi Hara, Yasuo Ochi, and Takanori Kasai 1002
Novel Amino Acid Metabolite Produced by Streptomyces sp.:
I. Taxonomy, Isolation, and Structural Elucidation
Tatsuharu Tajika, Isao Bando, Takaki Furuta, Noriko Moriya, Hiroyuki
Koshino, and Masakazu Uramoto 1007
Enhanced Peroxidation of Proteins of the Erythrocyte Membrane
and of Muscle Tissue by Dietary Oxidized Oil
Israela Hayam, Uri Cogan, and Shoshana Mokady 1011
Combined Effects of a Buffer and Solvent on Tetramethylpyrazine
Formation in a 3-Hydroxy-2-butanone/Ammonium Hydroxide System
Tzou-Chi Huang 1013
Randomly Amplified Polymorphic DNA (RAPD) for Rapid Identification
of the Spoilage Bacterium Alicyclobacillus acidoterrestris
Koji Yamazaki, Tsutomu Okubo, Norio Inoue, and Haruo Shinano 1016
Effects of DNA Topology on Transformation Efficiency of
Bacillus subtilis ISW1214 by Electroporation
Morimasa Ohse, Koji Kawade, and Hideo Kusaoke 1019
Inactivation of Bacillus Spores by the Supercritical Carbon
Dioxide Micro-bubble Method
Hiroya Ishikawa, Mitsuya Shimoda, Kei Tamaya, Akiyoshi Yonekura, Tamotsu
Kawano, and Yutaka Osajima 1022
Separation of Caffeine from Supercritical Carbon Dioxide
with a Zeolite Membrane
Yoshihiro Tokunaga, Tomoyuki Fujii, and Kozo Nakamura 1024
Apoptosis to HL-60 by Humulone
Hiroyasu Tobe, Mitsuaki Kubota, Mitsune Yamaguchi, Tomoji Kocha, and
Takaaki Aoyagi 1027
Inhibitory Effect of Alginic Acids on Hyaluronidase and
on Histamine Release from Mast Cells
Masahiro Asada, Makiko Sugie, Mami Inoue, Kazuya Nakagomi, Seiji Hongo,
Katsumi Murata, Shinji Irie, Toshio Takeuchi, Noboru Tomizuka, and Syuichi
Oka 1030
Structural Analysis of Disaccharides Synthesized by ΐ-D-Glucosidase
of Bifidobacterium breve clb and Their Assimilation by Bifidobacteria
Naoki Nunoura, Tomoyuki Fujita, Kohji Ohdan, Mitsunori Kirihata, Kenji
Yamamoto, and Hidehiko Kumagai 1033
Kinetics for the Autoxidation of Triacylglycerols Containing
Eicosapentaenoic Acid
Yasushi Endo, Sanae Hoshizaki, and Kenshiro Fujimoto 1036
Enantio- and Stereoselective Syntheses of Monodeuterium-labeled
Glycerols
Kenji Yagi, Hideaki Oikawa, and Akitami Ichihara 1038
Cloning, Expression, and Mutagenesis of Trypsin Inhibitor
ETIb from Erythrina variegata Seeds
Yoshiaki Kouzuma, Nobuyuki Yamasaki, and Makoto Kimura 1041
Competitive ELISA of Bovine Lactoferrin with Bispecific
Monoclonal Antibodies
Hiroshi Shinmoto, Masuko Kobori, Tojiro Tsushida, and Kazuki Shinohara
1044
Transduction of a Plasmid with an Inserted R4 Phage DNA
Fragment in Streptomyces lividans
Tomio Morino and Hideo Takahashi 1047
A Bacteriocin of Strain Pediococcus sp. ISK-1 Isolated
from Nukadoko, Bed of Fermented Rice Bran
Hirokazu Kimura, Rie Nagano, Hiromi Matsusaki, Kenji Sonomoto, and
Ayaaki Ishizaki 1049
Determination of Endogenous Peptides with in Vitro ACE
Inhibitory Activity in Normotensive Human Plasma by the Fluorometric HPLC
Method
Toshiro Matsui, Hiroshi Matsufuji, Terukazu Kawasaki, and Yutaka Osajima
1052
|Rapid Paper-
Purification and Properties of a New Enzyme, D-Carnitine
Dehydrogenase, from Agrobacterium sp. 525a
Siswa Setyahadi, Tomoko Ueyama, Takeshi Arimoto, Nobuhiro Mori, and
Yutaka Kitamoto 1055
-1-
CO2 Sorption by Microbial Cells and Sterilization by High-pressure
CO2
Hitoshi Kumagai,υ Chiho Hata, 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 September 20, 1996
@The amount of CO2 sorbed by microbial cells in a Saccharomyces cerevisiae-water
system was measured by a gravimetric method with a quartz spring, and the
correlation between CO2 sorption and the sterilization effect of high-pressure
CO2 was investigated. The sterilization rate of Saccharomyces cerevisiae
by high-pressure CO2 was measured by varying the water content and CO2
pressure, and analyzed by reaction kinetics. The sterilization rate could
be described by a first-order reaction, and the dependence of the sterilization
rate constant, k, on the water content and CO2 pressure was evaluated.
The amount of CO2 sorbed by the microbial cells reached equilibrium at
a constant CO2 pressure within a few minutes and was correlated well with
the value of k. In addition, the amount of unfreezable water was measured
by DSC as an index of the state of water in the cell-water system, this
being considered to be closely related to the amount of CO2 sorbed by the
microbial cells. The value of k increased with increasing water content;
however, the increase was only slight for a water content by which free
water existed.
Key words: sterilization; carbon dioxide; Saccharomyces cerevisiae; sorption;
water content
-2-
Analysis of Water Sorption Isotherms of Superabsorbent
Polymers by Solution Thermodynamics
Hitoshi Kumagai,υ Akinori Mizuno, Hitomi Kumagai,* and Toshimasa Yanoυυ
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
* Department of Agricultural and Biological Chemistry, Nihon University,
3-34-1 Shimouma, Setagaya-ku, Tokyo 154, Japan
Received November 13, 1996
@Water sorption isotherms of superabsorbent polymers were measured, and
their affinity for water was evaluated by solution thermodynamics. The
results provide basic data for the functional packaging of food to control
the water content of food during its transportation or storage. Water activity
above 0.9 was measured by adding a specific amount of water to the samples,
while that below 0.9 was measured with apparatus for evaluating water sorption
isotherms. Thus, water sorption isotherms for superabsorbent polymers were
obtained up to a water activity of approximately 0.98. The amount of water
sorbed by the superabsorbent polymers was influenced by the type of functional
groups in the polymers, and not by the degree of cross-linking in the polymers.
The integral Gibbs free energy, which is the most suitable parameter for
evaluating the affinity of a material for water, was evaluated from the
water sorption isotherms by using solution thermodynamics.
Key words: water activity; water sorption isotherm; superabsorbent polymer;
solution thermodynamics; Gibbs free energy
-3-
Purification and Characterization of Soybean Allergen Gly
m Bd 28K
Hideaki Tsuji,υ Noriko Bando, Miki Hiemori, Rintaro Yamanishi, Masumi Kimoto, Kiyoshi Nishikawa,* and Tadashi Ogawa
Department of Nutrition, School of Medicine, The University of Tokushima,
Kuramoto-cho, Tokushima 770, Japan
* Department of Pediatrics, National Kagawa Children's Hospital, Zentuji
765, Japan
Received October 11, 1996
@At least 15 allergenic proteins have been found in soybean using the
sera of soybean-sensitive patients with atopic dermatitis [T. Ogawa et
al., J. Nutr. Sci. Vitaminol., 35, 555-565 (1991)]. In the present study,
a monoclonal antibody (mAb) against Gly m Bd 28K, one of the major allergens
of soybean, was prepared, and Gly m Bd 28K was purified from defatted soybean
flakes by five purification steps, including immunoaffinity chromatography
with the mAb as a ligand. The purified allergen was found to be a glycoprotein
with a molecular mass of 26 kDa. During the purification process the allergen
was converted to more acidic proteins with the same molecular mass, suggesting
that the allergen is unstable. The sugar composition and amino acid sequence
of Gly m Bd 28K suggest that the allergen is a new glycoprotein with an
Asn-linked sugar moiety. The distribution of the allergen in soybean products
was examined by an immunoblotting technique with the mAb.
Key words: soybean allergen; Gly m Bd 28K; Gly m Bd 30K; Gly m Bd 68K
-4-
Heterologous Protein Production in Acremonium chrysogenum:
Expression of Bacterial Cephalosporin C Acylase and Human Thrombomodulin
Genes
Goichi Honda, Akio Matsuda, Michitaka Zushi, Shuji Yamamoto, and Ken-ichi Komatsu
Laboratory for Chemistry, Institute for Life Science Research, Asahi Chemical Industry Co., Ltd., Fuji, Shizuoka 416, Japan
Received October 18, 1996
@We have developed an efficient expression system for foreign genes in
Acremonium chrysogenum. After inserting the foreign gene between the phosphoglycerate
kinase (PGK) promoter and a terminator derived from A. chrysogenum, multiple
copies of this expression unit are tandemly ligated into cosmids and the
resultant cosmids are introduced into A. chrysogenum.
@We expressed Pseudomonas cephalosporin C acylase and a human thrombomodulin
mutant protein containing the fourth, fifth, and sixth epidermal growth
factor (EGF)-like structures (E456). The acylase activity in the transformants
obtained using our system was several times higher than that in the transformants
without the use of the system. The acylase proteins expressed had enzymatic
and immunochemical properties identical to those of authentic acylase.
The transformants with the expression plasmid for E456 secreted biologically
active E456 protein into the culture medium. The amino terminal sequence
of the purified E456 was identical to that of recombinant E456 obtained
using mammalian cells.
Key words: Acremonium chrysogenum; Pseudomonas; cephalosporin C acylase;
thrombomodulin; multiple-insert cosmid
-5-
Enzymatic Production of Pyrimidine Nucleotides Using Corynebacterium
ammoniagenes Cells and Recombinant Escherichia coli Cells: Enzymatic Production
of CDP-Choline from Orotic Acid and Choline Chloride (Part I)
Tatsuro Fujio* and Akihiko Maruyama**,υ
* Planning & Development Department/Food, Liquor & Food Division, and ** Fine Chemicals/Biochemicals Dept., Medicals Research and Development Center, Kyowa Hakko Kogyo Co., Ltd., 6-1 Ohtemachi, 1-chome, Chiyoda-ku, Tokyo 100,
Japan Received October 28, 1996
@Enzymatic production of cytidine diphosphate choline (CDP-choline) using
orotic acid and choline chloride as substrates was investigated using a
200-ml beaker as a reaction vessel. When Corynebacterium ammoniagenes KY13505
cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter
(77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP
and UTP were also accumulated, but CTP, a direct precursor of CDP-choline,
was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which
overproduce CTP synthetase by selfcloning of the pyrG gene, were used together
with cells of KY13505 for the enzymatic reaction using orotic acid as a
substrate. CTP was produced at 8.95 g/liter (15.1 mM) after 23 h of this
reaction. To produce CDP-choline, two additional enzyme activities were
needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline
kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKI gene)
and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase;
encoded by the CCT gene) respectively, were added to this CTP-producing
reaction system. After 23 h of the reaction using orotic acid and choline
chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated
without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and
PRPP required in the CDP-choline forming reaction system are biosynthesized
by those cells using glucose as a substrate.
Key words: CDP-choline; orotic acid; enzymatic production; ATP regeneration;
PRPP supply
-6-
Construction of a Plasmid Carrying both CTP Synthetase
and a Fused Gene Formed from Cholinephosphate Cytidylyltransferase and
Choline Kinase Genes and Its Application to Industrial CDP-Choline Production:
Enzymatic Production of CDP-Choline from Orotic Acid (Part II)
Tatsuro Fujio,* Sadao Teshiba,** and Akihiko Maruyama**,υ
* Planning & Development Department/Food, Liquor & Food Division, and ** Fine Chemicals/Biochemicals Dept., Medicals Research and Development Center, Kyowa Hakko Kogyo Co., Ltd., 6-1 Ohtemachi, 1-chome, Chiyoda-ku, Tokyo 100, Japan
Received October 28, 1996
@A new method for enzymatic production of cytidine diphosphate choline
(CDP-choline) from orotic acid and choline chloride was developed. To establish
an industrial manufacturing process, we constructed a plasmid, pCKG55,
which simultaneously expressed in Escherichia coli the three following
enzymes; CTP synthetase (encoded by the pyrG gene from E. coli), cholinephosphate
cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae),
and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and
CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused
protein. This CCT/CKI fused protein retained both activities and the thermal
stability of its cholinephosphate cytidylyltransferase activity was nearly
the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505
and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently.
Equal volumes of each broth were mixed in a 2-liter jar fermentor, and
then the enzymatic reaction was done using 47 mM orotic acid and 60 mM
choline chloride as substrates. After 23 h of the reaction at 32C, 21.5
mM (11 g/liter) of CDP-choline was accumulated.
Key words: CDP-choline; orotic acid; enzymatic production; ATP regeneration;
PRPP supply
-7-
Purification and Characterization of a Glucose-tolerant
ΐ-Glucosidase from Aspergillus niger CCRC 31494
Tsong-Rong Yanυ and Chun-Lieh Lin
Laboratory of Biochemistry, Department of Bioengineering, Tatung Institute of Technology, 40 Chung-Shang North Road 3rd Sec., Taipei, Taiwan 10451, Republic of China
Received November 11, 1996
@An extracellular glucose-tolerant ΐ-glucosidase was purified to homogeneity
by alcohol fractionation and preparative isoelectric focusing from Aspergillus
niger CCRC 31494. The enzyme was a dimeric protein with a subunit of 49,000,
and had its optimum activity at pH 5.0 and 55C. The enzyme was completely
inhibited by 5 mM Ag+. Thiol groups and serine residues were not essential
for its activity. Low concentrations of alcohols (10%) except for methanol
could activate the enzyme. It was very specific for para-nitrophenyl-ΐ-D-glucoside
(pNPG) and cellobiose. However, the enzyme also had some ΐ-xylosidase
activity, but showed no activity towards Ώ-linked glycosidic substrates.
The V(/)max of 124.4 U/mg and 21.6 U/mg were found for pNPG (Km=21.7 mM)
and para-nitrophenyl-ΐ-D-xyloside ( pNPX) (Km=14.2 mM), respectively.
The enzyme was tolerant to glucose inhibition with a Ki of 543 mM, while
fructose, galactose, mannose, and xylose were not inhibitory.
Key words: ΐ-glucosidase; Aspergillus niger; glucose-tolerance; purification;
characterization
-8-
Long-term Culture of Primary Rat Hepatocytes on Heparin-
or Lambda Carrageenan-containing Collagen Gels
Kong Hua Lin,υ Sumio Maeda,* Hidetoshi Inagaki,* and Takao Saito*
Biotechnology Department, Institute of Research and Innovation, 1201
Takada, Kashiwa, Chiba 277, Japan
* Department of Chemistry, National Industrial Research Institute of Nagoya,
Hirate-cho 1, Kita-ku, Nagoya 462, Japan
Received November 13, 1996
@The interactions of glycosaminoglycans (GAGs) with collagen are thought
to be important in cell adhesion and cell differentiation. To investigate
whether the interactions of GAG or sulfated polysaccharide with collagen
can maintain the functions of cultured primary rat hepatocytes, GAG- or
sulfated polysaccharide-containing collagen gels were reconstituted in
vitro and used for culture of hepatocytes. Among the GAGs and sulfated
polysaccharides examined, heparin- and lambda carrageenan-containing collagen
gels were found to be able to stimulate and sustain albumin synthesis,
while the other GAG- or sulfated polysaccharide-containing collagen gels
had almost no effect on maintenance of albumin synthesis. In the cultures
using collagen gels that contained 400 Κg/ml heparin or 100 Κg/ml lambda
carrageenan, albumin synthesis by rat hepatocytes was prolonged to about
4 and 5 weeks, respectively, but albumin synthesis was kept up for only
one week in the cultures using conventional collagen gels. These results
suggest that the interactions of heparin or lambda carrageenan with collagen
might be of importance for long-term maintenance of hepatocyte functions.
Key words: hepatocytes; glycosaminoglycans; sulfated polysaccharides; collagen
gels; albumin synthesis
-9-
Elicitor Actions of N-Acetylchitooligosaccharides and Laminarioligosaccharides
for Chitinase and L-Phenylalanine Ammonia-lyase Induction in Rice Suspension
Culture
Hiroshi Inui,υ Yasuhiro Yamaguchi, and Shigehiro Hirano
Department of Agricultural Biochemistry and Biotechnology, Tottori University, Tottori 680, Japan
Received November 13, 1996
@When a series of chitin oligosaccharides was added into a rice suspension
culture, N-acetylchitohexaose, N-acetylchitopentaose, and N-acetylchitotetraose
caused an increase in extracellular chitinase activity, mainly due to induction
of a class III chitinase. In the case of N-acetylchitohexaose, a substantial
increase in the chitinase activity was observed at a concentration higher
than 0.01 Κg/ml, and a maximum effect was reached at 1 Κg/ml. In contrast,
N-acetylchitotriose, N-acetylchitobiose, N-acetyl-D-glucosamine, and chitohexaose
(a chitosan oligosaccharide) were not very effective. Chitinase induction
was also observed with laminarihexaose (a ΐ-1,3-glucan oligosaccharide),
but about a 10-fold higher concentration, compared with N-acetylchitohexaose,
was needed to get the maximum effect. ΐ-1,3-Glucanase activity was found
in cells (but not in medium), and the activity was increased by neither
N-acetylchitohexaose nor laminarihexaose. When cells were incubated with
N-acetylchitohexaose, L-phenylalanine ammonia-lyase (PAL) activity increased
promptly. A biphasic profile was obtained when a dose-dependent effect
of the elicitor on the PAL induction was examined; the first phase was
observed in a range from 0.01 to 1 Κg/ml and the second phase from 3 to
300 Κg/ml. Laminarihexaose also acted as an elicitor for PAL induction.
Key words: chitinase; l-phenylalanine ammonia-lyase; N-acetylchitooligosaccharide;
laminarioligosaccharide; rice (Oryza sativa L. var. Japonica)
-10-
The Sugar Specificity of a Na+/Glucose Cotransporter from
Rat Jejunum
Hitoshi Aoshima,* Terufumi Yokoyama,** Junko Tanizaki,** Hanae Izu,** and Mamoru Yamada**
* Department of Biology, Physics and Informatics, Faculty of Science, and ** Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753, Japan
Received November 18, 1996
@A cDNA for a Na+/glucose cotransporter was cloned from a rat jejunum
cDNA library. This transporter was expressed in Xenopus oocytes by injection
of cRNA synthesized from the cDNA, and the transporter ability was electrophysiologically
examined. The cotransporter had a very narrow sugar specificity. Only D-glucose,
D-galactose, and some of their derivatives elicited significant electrical
responses. These results of sugar specificity were compared with those
of the H+/hexose cotransporter of Chlorella. Dose-response relationships
of several sugars followed a simple Michaelis-Menten type of kinetics.
Both Vm and Km were dependent on the sugars. Not only the affinity of sugars
to the cotransporter but also the rate of conformational change of the
cotransporter loaded with the sugar and Na+, which translocates them from
outside to inside, possibly depends on the sugar structure. The rate-limiting
step of the transportation may be the conformational change, i.e., isomerization,
of the cotransporter that translocates both the sugar and Na+ from outside
to inside.
Key words: cloning; Na+/glucose cotransporter; rat jejunum; sugar specificity;
Xenopus oocyte
-11-
Primary Structure of 6.5k-Arginine/Glutamate-rich Polypeptide
from the Seeds of Sponge Gourd (Luffa cylindrica)
Makoto Kimura,* Sung-Soo Park,* Ritsu Sakai,* Nobuyuki Yamasaki,* and Gunki Funatsu**
* Laboratory of Biochemistry, Faculty of Agriculture, and ** Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture, Kyushu University, Fukuoka 812-81, Japan
Received November 20, 1996
@The amino acid sequence of 6.5k-arginine/glutamate rich polypeptide (6.5k-AGRP)
from the seeds of sponge gourd (Luffa cylindrica) has been determined.
The 6.5k-AGRP consists of a 47-residue polypeptide chain containing two
disulfide bonds, and a molecular mass calculated to be 5695 Da, which fully
coincides with a value of [M+H]+=m/z 5693.39 obtained by matrix-assisted
laser desorption ionization time of flight mass spectrometry (MALDI-TOF
MS). The mass spectrometric evidence indicated that 6.5k-AGRP is also present
partially truncated at the C-terminus. In our preparations, approximately
half of the polypeptide molecules have the C-terminal sequence Arg-Arg-Glu-Val-Asp;
the other half lack Val-Asp and end with the glutamic acid, making a total
of 45 residues in the polypeptide chain. The two disulfide bonds connect
Cys12 to Cys33 and Cys16 to Cys29. Comparison of the amino acid sequence
of 6.5k-AGRP with those of the other known proteins included in the PIR
protein sequence database showed that it is related to the amino acid sequence
of the N-terminal region encoded by the first exon of the cocoa (Theobroma
cacao) and cotton seeds vicilin genes, sharing a characteristic two Cys-Xaa-Xaa-Xaa-Cys
motif.
Key words: amino acid sequence; sponge gourd; Luffa cylindrica; storage
protein; arginine/glutamate-rich polypeptide
-12-
Metabolism of DFA III by Arthrobacter sp. H65-7: Purification
and Properties of a DFA III Hydrolysis Enzyme (DFA IIIase)
Hiroaki Sakurai, Atsushi Yokota, Yoko Sumita, Yumiko Mori, Hirokazu Matsui,* and Fusao Tomita
Laboratory of Applied Microbiology, Department of Bioscience and Chemistry,
Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
* Laboratory of Biochemistry, Department of Bioscience and Chemistry, Faculty
of Agriculture, Hokkaido University, Sapporo 060, Japan
Received November 27, 1996
@Assimilation of DFA III by Arthrobacter sp. H65-7 was found to consist
of two sequential enzyme steps, hydrolysis of DFA III to inulobiose (1-O-ΐ-D-fructofuranosyl-D-fructopyranose)
and inulobiose to fructose, that is, Ώ-(2¨3') and ΐ-(2'¨1) fructosidic
linkages were split separately. The enzyme catalyzing the first step, named
DFA III hydrolysis enzyme (DFA IIIase), has been purified from the cell-free
extracts of Arthrobacter sp. H65-7 to an electrophoretically pure state
by heat-treatment, ammonium sulfate fractionation, and chromatographies
on DEAE-Toyopearl 650M, Butyl-Sepharose 4B and TSKgel G3000SW(/)XL, and
Biophoresis III. The molecular weight of the purified enzyme was estimated
to be 125,000 by gel filtration, and 61,000 by SDS-polyacrylamide gel electrophoresis.
The enzyme showed the highest activity at pH 6.0 and 45C, and was stable
from pH 4.5 to 10.0 and up to 60C. The Km of this enzyme for DFA III
was 12.5 mM. The enzyme also catalyzed the reverse reaction, inulobiose
to DFA III.
Key words: inulin; Arthrobacter sp. H65-7; DFA III; inulobiose; DFA IIIase
-13-
Actions of Pokeweed Antiviral Protein on Virus-infected
Protoplasts
Keiichi Watanabe,υ Tetsuji Kawasaki, Nobumichi Sako, and Gunki Funatsu*
Department of Applied Biological Sciences, Saga University, Saga 840,
Japan
* Laboratory of Protein Chemistry and Engineering, Faculty of Agriculture,
Kyushu University, Fukuoka 812, Japan
Received December 9, 1996
@Pokeweed antiviral protein (PAP) belongs to a group of ribosome-inactivating
proteins (RIPs) that inactivate ribosomes by depurinating rRNA at a specific
site. To study the mechanism for the antiviral activity of PAP, the actions
of PAP on TMV-infected and uninfected tobacco protoplasts were investigated.
The addition of 0.33 ΚM PAP to TMV-inoculated protoplasts caused a complete
inhibition of TMV production. The same concentration of PAP was found to
inhibit protein synthesis in the virus-infected protoplasts and to kill
the cells, but it had no effect on the uninfected protoplasts. The concentration
dependence of protein synthesis-inhibition by PAP was related to that of
inhibition of viral multiplication. Furthermore, two other RIPs (ricin
A-chain and luffin-a), which showed 240 and 430-fold less activity on tobacco
ribosomes than PAP in a cell-free system, did not inhibit viral multiplication
even at a concentration of 3.3 ΚM. The analysis of RNAs from the virus-infected
and PAP-treated protoplasts demonstrated that 25S rRNA was depurinated
by PAP in the infected cells. These results suggest that PAP, which is
normally unable to penetrate the plasma membrane of uninfected protoplasts,
gains entrance to the cytosol of infected cells and prevents viral multiplication
by inactivating ribosomes.
Key words: pokeweed antiviral protein; ribosome-inactivating protein; RNA
N-glycosidase; tobacco mosaic virus; protoplasts
-14-
Changes in Proteasome Levels in Spinach (Spinacia oleracea)
Seeds during Imbibition and Germination
Miyuki Miyawaki, Misako Aito,* Naoko Ito,*,** Yuki Yanagawa,* Richard E. Kendrick,** Keiji Tanaka,*** Takahide Sato,* and Hiroki Nakagawa*,υ
* Department of Bioproduction Science, Faculty of Horticulture, Chiba
University, Matsudo, Chiba 271, Japan
** Laboratory for Signal Transduction and Photoperception, Frontier Research
Program, The Institute of Physical and Chemical Research (Riken), Wako,
Saitama 351-01, Japan
*** Division of Chemotherapy, The Tokyo Metropolitan Institute of Medical
Science, Tokyo 113, Japan
Received December 13, 1996
@Proteasomes are the major cytosolic protease complexes responsible for
energy-dependent and extra-lysosomal proteolysis. Ubiquitinated proteins
are degraded by the 26S proteasome which is composed of a 20S proteasome
and two regulatory complexes. Changes in the 20S and 26S proteasome activity
levels and protein abundance in spinach seeds during imbibition and germination
were examined by using glycerol density gradient centrifugation. The 26S
proteasome activity level decreased transiently during imbibition, reached
a minimum one day after starting imbibition, and then increased again.
During the period of minimal accumulation, the protein being detected with
an anti-26S proteasome antibody shifted to a lower sedimentation coefficient
fraction. In contrast, the 20S proteasome activity level increased during
imbibition, and remained high for two days. The change in the 20S proteasome
level corresponded to the change in the 20S proteasome activity level.
Key words: 26S proteasome; 20S proteasome; seed; germination; Spinacia
oleracea
-15-
Changes in Messenger RNA of Pancreatic Enzymes and Intestinal
Cholecystokinin after a 7-Day Bile-pancreatic Juice Diversion from the
Proximal Small Intestine in Rats
Hiroshi Hara,υ Yasuo Ochi, and Takanori Kasai
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Received December 24, 1996
@We have previously demonstrated the bile-pancreatic juice (BPJ)-independent
stimulation of pancreatic enzyme secretion in chronic BPJ-diverted rats.
Pancreatic and intestinal adaptation to 7-day BPJ diversion was next examined.
Pancreatic enzyme mRNA and cholecystokinin mRNA in the jejunal mucosa were
measured in rats with BPJ diverted into the ileum (PBD rats) in comparison
with the figures for rats with BPJ returned to the duodenum (normal rats)
or laparotomized (Intact) rats under well-nourished conditions. Amylase
mRNA in the pancreas was lower and trypsinogen plus chymotrypsinogen mRNA
was higher in the PBD rats than in the intact rats. The change in pancreatic
mRNA was similar to that in the specific activities of the enzymes after
a chronic BPJ diversion. This finding suggests that these pancreatic enzymes
were regulated by the mRNA level. The portal concentration of cholecystokinin
in the postabsorptive period (exogenously non-stimulated status) was 4-fold
higher in the PBD group than in the normal and intact groups. Cholecystokinin
mRNA in the jejunal mucosa of PBD rats was somewhat higher than that of
intact rats. These results suggest that intestinal cholecystokinin was
predominantly increased at the translational or later stage by chronic
BPJ diversion.
Key words: pancreatic enzyme; cholecystokinin; messenger RNA; bile-pancreatic
juice diversion; rat
-16-
Novel Amino Acid Metabolite Produced by Streptomyces sp.:
I. Taxonomy, Isolation, and Structural Elucidation
Tatsuharu Tajika, Isao Bando, Takaki Furuta, Noriko Moriya,* Hiroyuki Koshino,* and Masakazu Uramoto υ
Faculty of Agriculture, Tamagawa University, Machida, Tokyo 194, Japan
* The Institute of Physical and Chemical Research (RIKEN ) Wako, Saitama
351-01, Japan
Received January 8, 1997
@A strain of streptomycete isolated from a soil sample was found to produce
a novel amino acid metabolite. The compound was purified from the culture
fluid by column chromatography, using cation exchange resin, a synthetic
adsorbent, and finally by preparative HPLC with a reverse-phase column.
The structure of the compound was established as N Β-(5-methyl-4-oxo-2-imidazolin-2-yl)-L-ornithine
on the basis of an analysis of the spectral data and chemical degradation.
This was confirmed by comparing the NMR spectrum of the metabolite with
that of the compound synthesized by treating methylglyoxal and N Ώ-acetyl-L-arginine.
The substance did not show any antimicrobial activity against bacteria,
fungi and yeasts by the agar plate method, but exhibited a weak preventive
effect on cucumber powdery mildew disease in a pot test.
Key words: amino acid; Streptomyces; arginine analogue; imidazoline derivative
-17-
Note
Enhanced Peroxidation of Proteins of the Erythrocyte Membrane
and of Muscle Tissue by Dietary Oxidized Oil
Israela Hayam, Uri Cogan, and Shoshana Mokadyυ
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel
Received May 27, 1996
@Male weanling rats were fed on diets containing 10% of either oxidized
or fresh (control) soybean oil for periods of 4 and 7 weeks. The ingestion
of oxidized oil was found to reduce the content of free thiols in the erythrocyte
membrane and to increase the amount of membrane-extractable spectrin. The
level of reactive carbonyl groups was higher in the proteins of the ghosts
and of the muscle tissue derived from the experimental animals. These alterations,
which are characteristic of peroxidized proteins, suggest that oxidative
stress caused by oxidized dietary lipids may damage tissue proteins.
Key words: dietary oxidized oil; protein peroxidation; erythrocyte membrane;
muscle tissue
-18-
Note
Combined Effects of a Buffer and Solvent on Tetramethylpyrazine
Formation in a 3-Hydroxy-2-butanone/Ammonium Hydroxide System
Tzou-Chi Huang
Department of Food Science and Technology, National Pingtung Polytechnic Institute, Pingtung 912, Taiwan, Republic of China
Received August 13, 1996
@A phosphate buffer was found to significantly promote tetramethylpyrazine
(TMP) formation in an acetoin (3-hydroxy-2-butanone)/ammonium hydroxide
system. The effect of the phosphate ion on TMP formation was additive in
the range of 0.05-0.2 M. The change in pH value of the system reveals that
a proton-coupled redox type of reaction occurred during TMP formation.
Phosphate serves both as proton donor and acceptor to facilitate proton
transfer during the Schiff base formation between ammonia and 3-hydroxy-2-butanone.
Protic solvents, methanol, and ethanol, were found to attract the water
released from the system. The combination of a phosphate buffer and protic
solvent led to the completion of TMP formation. The TMP formation mechanism
in a phosphate buffer (pH 7.2) is proposed.
Key words: tetramethylpyrazine; phosphate buffer; protic solvent; buffer
capacity
-19-
Note
Randomly Amplified Polymorphic DNA (RAPD) for Rapid Identification
of the Spoilage Bacterium Alicyclobacillus acidoterrestris
Koji Yamazaki, Tsutomu Okubo,* Norio Inoue, and Haruo Shinano
Department of Marine Bioresources Chemistry, Faculty of Fisheries, Hokkaido
University, Hakodate 041, Japan
* Central Research Laboratory, Taiyo Kagaku Co., Ltd., Yokkaichi, 510,
Japan
Received August 27, 1996
@RAPD (randomly amplified polymorphic DNA) analysis was developed for
rapid identification of the spoilage bacterium, Alicyclobacillus acidoterrestris.
Three primers, Ba-10, F-61, and F-64, allowed adequate discrimination between
strains of A. acidoterrestris and related bacteria in RAPD analyses. As
compared with the conventional method and a RAPD assay against the isolates
from various environmental and food samples, the results obtained from
the RAPD pattern were identical to the identification by the conventional
method. We suggest that the RAPD analysis is a rapid and reliable technique
to distinguish A. acidoterrestris from other related bacteria.
Key words: thermoacidophiles; Alicyclobacillus acidoterrestris; RAPD; acidic
beverages; spoilage bacterium
-20-
Note
Effects of DNA Topology on Transformation Efficiency of
Bacillus subtilis ISW1214 by Electroporation
Morimasa Ohse, Koji Kawade, and Hideo Kusaoke
Department of Applied Physics and Chemistry, Faculty of Engineering, Fukui University of Technology, Gakuen-cho 3-6-1, Fukui 910, Japan
Received September 2, 1996
@We report an investigation of electrotransformation by three different
topological isomers, circular supercoiled (sc DNA), circular relaxed (cr
DNA), and linearized (ln DNA) forms of the plasmids pUB110 (4.5 kbp) and
pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214.
Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity
and the DNA assumed the relaxed circular form. Treatment of sc DNA with
restriction endonuclease linearized the DNA. The transformation with the
sc DNA of pUB110 resulted in the maximum efficiency of (2.6}0.6)~105
transformants per Κg DNA higher than that ((2.0}0.3)~104 transformants
per Κg DNA) for the cr DNA, using the DNA concentration of 20 Κg/ml at
an electric field strength of 7 kV/cm and a capacitance of 10 ΚF with
a single decayed pulse. The transformation efficiency (TE) for the ln DNA
was zero. The variations of TE for different topological forms of DNA reflected
their relative stability in the host cells. The molecular efficiency (ME,
transformants per molecule) for sc DNA was nearly one order of magnitude
greater for the lower molecular size of pUB110 DNA than that for the higher
molecular size of pBDR331T DNA.
Key words: topology; transformation efficiency; molecular efficiency; electroporation;
electrotransformation; Bacillus subtilis
-21-
Note
Inactivation of Bacillus Spores by the Supercritical Carbon
Dioxide Micro-bubble Method
Hiroya Ishikawa, Mitsuya Shimoda, Kei Tamaya, Akiyoshi Yonekura, Tamotsu Kawano,* and Yutaka Osajima
Department of Food Science and Technology, Faculty of Agriculture, Kyushu
University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan
* Nihon Tansan Co., Ltd., 1-8-8 Shinkawa, Chuo-ku, Tokyo 104, Japan
Received September 5, 1996
@Bacillus spores were effectively inactivated by the supercritical (SC)
CO2 micro-bubble method. The micro-bubble SC CO2 treatment of B. cereus,
B. subtilis, B. megaterium, B. polymyxa, and B. coagulans at 40C and
30 MPa for 30 min produced greater reduction (about 3 log cycles of reduction)
than a similar treatment without a filter. The SC CO2 treatment of B. polymyxa,
B. cereus, and B. subtilis spores at 45C, 50C, and 55C, respectively,
and 30 MPa for 60 min resulted in a 6-log cycle reduction of survival.
The SC CO2 treatment under the foregoing conditions should offer higher
efficiency than that of heat treatment at 100C for 60 min. In addition,
the SC CO2 treatment (30 MPa, 60C, 30 min) of B. polymyxa and B. cereus
spores also produced a 6-log cycle reduction.
Key words: supercritical CO2; micro-bubble; inactivation; Bacillus; spore
-22-
Note
Separation of Caffeine from Supercritical Carbon Dioxide
with a Zeolite Membrane
Yoshihiro Tokunaga, Tomoyuki Fujii,υ and Kozo Nakamura
Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received September 18, 1996
@Caffeine was separated from supercritical carbon dioxide with a zeolite
membrane that had been tested for pervaporation. The pore size of the NaY
zeolite membrane was evaluated to be of subnanometer scale from the result
of a Dubinin-Astakhov analysis. The rejection of caffeine was 0.98 by the
zeolite membrane which would make it applicable for SCCO2 membrane separation.
Key words: supercritical carbon dioxide; membrane separation; zeolite membrane;
Dubinin-Astakhov analysis; caffeine
-23-
Note
Apoptosis to HL-60 by Humulone
Hiroyasu Tobe,υ Mitsuaki Kubota, Mitsune Yamaguchi, Tomoji Kocha, and Takaaki Aoyagi
Showa College of Pharmaceutical Sciences, 3165 Higashitamagawagakuen 3-chome, Machida-shi, Tokyo 194, Japan
Received September 26, 1996
@Humulone, a bone resorption inhibitor isolated from hop extract, induced
apoptosis in the premyocytic leukemia cell line HL-60 between 1 and 100
Κg/ml. Our data suggested that there was a correlation between the apoptosis-inducing
activity of humulone and its antioxidative activity.
Key words: apoptosis; bone resorption inhibitor; humulone; antioxidant;
HL-60
-24-
Note
Inhibitory Effect of Alginic Acids on Hyaluronidase and
on Histamine Release from Mast Cells
Masahiro Asada, Makiko Sugie, Mami Inoue, Kazuya Nakagomi,* Seiji Hongo,* Katsumi Murata,** Shinji Irie,** Toshio Takeuchi,** Noboru Tomizuka, and Syuichi Okaυ
National Institute of Bioscience and Human-Technology, 1-1 Higashi,
Tsukuba, Ibaraki 305, Japan
* Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama,
Toyama 930-01, Japan
** Kibun Food Chemifa Co., Ltd., 1-11-8 Shinsayama, Sayama, Saitama 350-13,
Japan
Received October 2, 1996
@The effects of various types of alginic acid consisting of L-guluronic
acids (G) and D-mannuronic acids (M) on hyaluronidase and mast cell degranulation
were examined. Alginic acid with an M/G ratio of 1.0 exhibited the strongest
inhibition of both activities, the higher molecular weight alginic acids
of 150 to 370 kDa being preferable in both cases. Esterification of the
carboxyl residue enhanced the latter activity.
Key words: alginic acid; uronic acid; inhibitor; hyaluronidase; mast cell
degranulation
-25-
Note
Structural Analysis of Disaccharides Synthesized by ΐ-D-Glucosidase
of Bifidobacterium breve clb and Their Assimilation by Bifidobacteria
Naoki Nunoura,υ Tomoyuki Fujita,* Kohji Ohdan, Mitsunori Kirihata,* Kenji Yamamoto, and Hidehiko Kumagaiυυ
Department of Food Science and Technology, Faculty of Agriculture, Kyoto
University, Kitashirakawa, Sakyo-ku, Kyoto 606-01, Japan
* Department of Applied Biochemistry, College of Agriculture, University
of Osaka Prefecture, Sakai, Osaka 593, Japan
Received November 18, 1996
@Circulation of a solution of 1 M D-fucose and 1 M D-glucose through a
reaction system consisting of serial columns of immobilized recombinant
ΐ-D-glucosidase of Bifidobacterium breve clb and activated charcoal gave
two oligosaccharides. Structural analysis identified these oligosaccharides
as D-fucosylglucose (6-O-ΐ-D-Fucopyranosyl-D-glucose) and gentiobiose
(6-O-ΐ-D-Glucopyranosyl-D-glucose).
@The D-fucosylglucose obtained was well assimilated by many Bifidobacteria
but not by the other intestinal bacteria tested.
Key words: ΐ-d-glucosidase; immobilized enzyme; Bifidus factor; condensation
reaction; d-fucosylglucose
-26-
Note
Kinetics for the Autoxidation of Triacylglycerols Containing
Eicosapentaenoic Acid
Yasushi Endo,υ Sanae Hoshizaki, and Kenshiro Fujimoto
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba, Sendai 981, Japan
Received November 26, 1996
@The oxidative rate of three kinds of synthetic triacylglycerols (TAGs)
consisting of eicosapentaenoic acid (EPA) and palmitic acid was measured
by the induction period method to understand the mechanism for the autoxidation
of TAGs containing highly unsaturated fatty acids (HUFAs) such as marine
oils. The propagation rate, oxidizability, and kinetic chain length increased
with the number of moles of EPA in a single TAG molecule, although the
initiation rate was almost the same for all the TAG samples. These results
demonstrate that the oxidative rate of EPA in TAGs was affected by the
TAG structure and that EPA highly concentrated in a TAG molecule was very
susceptible to free-radical oxidation.
Key words: autoxidation; eicosapentaenoic acid; highly unsaturated fatty
acid; kinetics; triacylglycerol
-27-
Note
Enantio- and Stereoselective Syntheses of Monodeuterium-labeled
Glycerols
Kenji Yagi, Hideaki Oikawa, and Akitami Ichihara
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan
Received December 3, 1996
@An efficient method is described for synthesizing and four possible diastereomers
of stereochemically defined monodeuterated glycerols by utilizing Sharpless
asymmetric dihydroxylation.
Key words: chirally monodeuterated glycerols; Sharpless asymmetric dihydroxylation
-28-
Note
Cloning, Expression, and Mutagenesis of Trypsin Inhibitor
ETIb from Erythrina variegata Seeds
Yoshiaki Kouzuma,υ Nobuyuki Yamasaki, and Makoto Kimura
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-81, Japan
Received December 10, 1996
Erythrina variegata trypsin inhibitors designated ETIa and ETIb belong
to the Kunitz family trypsin inhibitor, but ETIa is unique in its ability
to inhibit tissue-type plasminogen activator, while ETIb is not. The cDNA
clone encoding ETIb was isolated from the seed cDNA library constructed
in the lambda phage Ιgt11. The ETIb cDNA insert consists of 765 bp, including
an open reading frame of 606 pb from ATG to TGA codons. The deduced amino
acid sequence consists of 202 amino acids, having the signal peptides of
22 amino acids in the N-terminus and 2 amino acids in C-terminus. The cDNA
fragment encoding the mature form of ETIb was introduced into an expression
vector, pET-22b, and expressed in Escherichia coli BL21 (ED3) in a functional
form. Furthermore, the ETIb mutant bP61R/F62L, in which Pro61 and Phe62
in ETIb were changed to the corresponding amino acid residues Arg and Leu,
respectively, as in ETIa, was constructed, and its inhibitory potency toward
tPA was assayed. This mutant showed significant tPA inhibitory activity,
albeit less than ETIa. The result demonstrates that the Arg61 and Leu62
residues in ETIa are important in inhibiting tPA, and also suggests that
beside these two residues, the other amino acid(s) or other structural
element may be involved in interaction of ETIa with tPA.
Key words: Erythrina variegata; trypsin inhibitor; tissue-type plasminogen
activator inhibitor; cDNA cloning; expression
-29-
Note
Competitive ELISA of Bovine Lactoferrin with Bispecific
Monoclonal Antibodies
Hiroshi Shinmoto,υ Masuko Kobori, Tojiro Tsushida, and Kazuki Shinohara*
National Food Research Institute, Ministry of Agriculture, Forestry,
and Fisheries, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan
* National Research Institute of Fisheries Science, Fisheries Agency, 2-12-4
Fukuura, Yokohama, Kanagawa 236, Japan
Received December 11, 1996
@A mouse hybrid-hybridoma, HH1-4-3, secreting IgG1 class bispecific antibodies
to bovine lactoferrin (bLF) and horseradish peroxidase (HRPO), was established
previously. The competitive enzyme-linked immunosorbent assay (ELISA) of
bLF using the HH1-4-3 culture supernatant was not sensitive enough to measure
bLF concentration in biological fluids. To improve the sensitivity of the
competitive ELISA, we fractionated the bispecific antibodies by antigen
affinity column chromatography. A column immobilized with bLF adsorbed
60% of the antibodies of the HH1-4-3 supernatant, and the amount of antibodies
adsorbed on a column immobilized with HRPO was less than 5%. The competitive
ELISA of bLF using the affinity purified bispecific antibodies through
an HRPO-immobilized column chromatography showed a good standard curve
at bLF concentrations of 10 ng/ml to 100 Κg/ml.
Key words: bispecific antibody; hybrid-hybridoma; ELISA
-30-
Note
Transduction of a Plasmid with an Inserted R4 Phage DNA
Fragment in Streptomyces lividans
Tomio Morinoυ and Hideo Takahashi *
Research and Development Division, Pharmaceuticals Group, Nippon Kayaku,
Co., Ltd., 3-31-12 Shimo, Kita-ku, Tokyo 115, Japan
* Institute of Molecular and Cellular Biosciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received December 18, 1996
@A Streptomyces plasmid, pR4C2, with an inserted DNA fragment of R4 phage,
was encapsidated into R4 phage particles in vivo and transduced to Streptomyces
lividans at 3~10-6CFU/PFU. Formation of transducing phage was dependent
on the inserted R4 DNA, and some of the transducing phages had larger DNA
than R4 phage. A possible transduction mechanism through plasmid-phage
cointegrate formation in vivo is discussed.
Key words: plasmid transduction; R4 phage; phage density; in vivo packaging;
Streptomyces
-31-
Note
A Bacteriocin of Strain Pediococcus sp. ISK-1 Isolated
from Nukadoko, Bed of Fermented Rice Bran
Hirokazu Kimura, Rie Nagano, Hiromi Matsusaki, Kenji Sonomoto, and Ayaaki Ishizakiυ
Laboratory of Microbial Technology, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-81, Japan
Received January 8, 1997
@Pediococcus sp. ISK-1 isolated in our laboratory from well-aged Nukadoko,
produces a bacteriocin which has a unique antimicrobial spectrum among
pediocins. The bacteriocin was stable at acidic pH, and more than 60% of
the antimicrobial activity still remained even after being autoclaved at
121C for 20 min in the pH range of 3 to 8. This is the first report dealing
with a bacteriocin produced by lactic acid bacteria isolated from Nukadoko.
Key words: Pediococcus; bacteriocin; Nukadoko; pediocin
-32-
Note
Determination of Endogenous Peptides with in Vitro ACE
Inhibitory Activity in Normotensive Human Plasma by the Fluorometric HPLC
Method
Toshiro Matsui,υ Hiroshi Matsufuji, Terukazu Kawasaki,* and Yutaka Osajima
Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812, Japan
* Institute of Health Science, Kyushu University, 6-1 Kasugakouen, Kasuga,
Fukuoka 816, Japan
Received January 20, 1997
@An in vitro degradation test of angiotensin (ANG) II or III in normotensive
supine human plasma from 9 healthy male subjects confirmed the production
of smaller ANG metabolites with angiotensin I-converting enzyme inhibitory
activity. These metabolites were identified as ANG (3-8), ANG (4-8), ANG
(5-8), and ANG (3-4), whose respective peptide concentrations were determined
by our proposed naphthalene-2,3-dialdehyde (NDA)-HPLC method to be 64}9,
39}5, 176}22, and 197}35 fmol/ml of plasma.
Key words: ACE; angiotensin metabolites; HPLC
-33-
Rapid Paper
Purification and Properties of a New Enzyme, D-Carnitine
Dehydrogenase, from Agrobacterium sp. 525a
Siswa Setyahadi, Tomoko Ueyama, Takeshi Arimoto, Nobuhiro Mori, and Yutaka Kitamoto
Department of Biochemistry and Biotechnology, Faculty of Agriculture, Tottori University, Tottori 680, Japan
Received November 8, 1996
@A new enzyme, D-carnitine dehydrogenase from Agrobacterium sp. 525a,
was purified by DEAE-Toyopearl, ammonium sulfate fractionation, Sephadex
G-75, affinity chromatography, and Mono Q and TSK-gel filtration column
chromatography. The enzyme had the molecular mass of 89 kDa and consisted
of three identical subunits. The optimum pH for the oxidation reaction
was 9.3. The Michaelis constants for D-carnitine and NAD+ were 3.1 and
0.07 mM, respectively. The N-terminal 20 amino acids were sequenced.
Key words: d-carnitine dehydrogenase; degradation of d-carnitine