Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.12 2001)

Sequential Comparison of Peptides Containing Half-cystine Residues
from Ovalbumins of Six Avian Species
Yuanxia SUN and Shigeru HAYAKAWA†　p.2589

Pressure Distribution Measurement in Biting Surimi Gels
with Molars Using a Multiple-point Sheet Sensor
Kaoru KOHYAMA,1,† Tomoko SAKAI,1 Teruaki AZUMA,2
Toru MIZUGUCHI,3 and Ikuo KIMURA3 p.2597

Induction of 4-Hydroxycinnamate Decarboxylase in Klebsiella oxytoca Cells
Exposed to Substrates and Non-substrate 4-Hydroxycinnamate Analogs
Yasuyuki HASHIDOKO,† Tomoko TANAKA, and Satoshi TAHARA p.2604

Stable Isotope-labeling Studies on the Oxidative Coupling
of Caffeic Acid via o-Quinone
Hiroyuki TAZAKI,† Daisuke TAGUCHI, Takaomi HAYASHIDA, and Kensuke NABETA p.2613

Isotope Ratio by HRGC-MS of Citrus junos Tanaka (Yuzu) Essential Oils:
m/z 137/136 of Terpene Hydrocarbons
Masayoshi SAWAMURA,1,† Toshiki ITO,1 Akitoshi UNE,1
Hiroyuki UKEDA,1 and Yuzo YAMASAKI2 p.2622

Synthesis of Southern (C1′--C11′) and Eastern (C8--C18) Fragments
of Pamamycin-607, an Aerial Mycelium-inducing Substance
of Streptomyces alboniger
Hiromasa KIYOTA,† Yukito FURUYA, Shigefumi KUWAHARA, and Takayuki ORITANI p.2630

Steric Effects on Interaction of Tea Catechins with Lipid Bilayers
Katsuko KAJIYA, Shigenori KUMAZAWA, and Tsutomu NAKAYAMA† p.2638

Purification and Characterization of Maltose Phosphorylase
from Bacillus sp. RK-1
Yasushi INOUE, Keiko ISHII, Tetsuji TOMITA, and Fumio FUKUI p.2644

Isolation, Growth, Ultrastructure, and Metal Tolerance of the Green Alga,
Chlamydomonas acidophila (Chlorophyta)
Kahoko NISHIKAWA and Noriko TOMINAGA p.2650

Purification and Identification of the Essential Ionizable Groups
of Honeybee, Apis mellifera L., Trehalase
Jin-Ha LEE, Masahisa TSUJI, Mitsuru NAKAMURA, Mamoru NISHIMOTO, Masayuki OKUYAMA,
Haruhide MORI, Atsuo KIMURA, Hirokazu MATSUI, and Seiya CHIBA† p.2657

High Yield Synthesis of Nitriles by a New Enzyme, Phenylacetaldoxime
Dehydratase, from Bacillus sp. Strain OxB-1
Sheng-Xue XIE, Yasuo KATO, and Yasuhisa ASANO† p.2666

Quantitative and Specific Detection of a Trichloroethylene-degrading
Methanotroph, Methylocystis sp. Strain M, by a Most Probable
Number-Polymerase Chain Reaction Method
Takeshi KIKUCHI,1 Kazuhiro IWASAKI,2 Hirofumi NISHIHARA,1
Yoshichika TAKAMURA,1 and Osami YAGI3 p.2673

Mutational and Comparative Analysis of Streptolysin O, an Oxygen-labile
Streptococcal Hemolysin
Ikkyu YAMAMOTO,1,† Hisashi KIMOTO,1 Yoriko TAKETO,2 and Akira TAKETO1,3 p.2682

Antihypertensive Effects of Acetic Acid and Vinegar
on Spontaneously Hypertensive Rats
Shino KONDO,1,† Kenji TAYAMA,1 Yoshinori TSUKAMOTO,1
Katsumi IKEDA,2 and Yukio YAMORI2 p.2690

Novel Substrate Specificity of Designer 3-Isopropylmalate Dehydrogenase
Derived from Thermus thermophilus HB8
Masaaki FUJITA, Hideyuki TAMEGAI, Tadashi EGUCHI,＊ and Katsumi KAKINUMA† p.2695

Cloning, Sequencing, and Expression of the Gene Encoding
4-Hydroxy-4-methyl-2-oxoglutarate Aldolase
from Pseudomonas ochraceae NGJ1
Kiyofumi MARUYAMA,1,† Michiko MIWA,1 Nobuyuki TSUJII,1 Tomoyuki NAGAI,1
Naotaka TOMITA,1 Toshiyuki HARADA,1 Hirobumi SOBAJIMA,1 and Hiroyuki SUGISAKI2
p.2701

Localization, Accumulation, and Antifungal Activity of Chitinases
in Rye (Secale cereale) Seed
Toki TAIRA,†,＊,＊＊ Takeshi YAMAGAMI,＊＊ Yoichi ASO,＊＊ Masatsune ISHIGURO,＊＊
and Masanobu ISHIHARA＊ p.2710

Substrate Specificity of β-Primeverosidase, A Key Enzyme in Aroma Formation
during Oolong Tea and Black Tea Manufacturing
Seung-Jin MA, Masaharu MIZUTANI, Jun HIRATAKE, Kentaro HAYASHI,＊
Kensuke YAGI,＊ Naoharu WATANABE,＊ and Kanzo SAKATA† p.2719

Sandwich Enzyme-linked Immunosorbent Assay System
for Micro-detection of the Wheat Allergen, Tri a Bd 17 K
Hiromi YAMASHITA,† Masumi KIMOTO, Miki HIEMORI, Misako OKITA,
Kazuhiko SUZUKI, and Hideaki TSUJI p.2730

Administration of Capsiate, a Non-Pungent Capsaicin Analog, Promotes
Energy Metabolism and Suppresses Body Fat Accumulation in Mice
Koichiro OHNUKI, Satoshi HARAMIZU, Kasumi OKI, Tatsuo WATANABE,＊
Susumu YAZAWA,＊＊ and Tohru FUSHIKI p.2735

A Gene Encoding Phosphatidylethanolamine N-Methyltransferase
from Acetobacter aceti and Some Properties of its Disruptant
Takashi HANADA,† Yasuhiro KASHIMA, Akihiko KOSUGI, Yukimichi KOIZUMI,
Fujiharu YANAGIDA, and Shigezo UDAKA†† p.2741

(4E)-Dehydrocitrals [(2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienals]
from Acarid Mite Histiogaster sp. A096 (Acari: Acaridae)†
Hiroyuki HIRAOKA, Naoki MORI, Ritsuo NISHIDA, and Yasumasa KUWAHARA†† p.2749

Membrane-bound Quinoprotein D-Arabitol Dehydrogenase
of Gluconobacter suboxydans IFO 3257: A Versatile Enzyme
for the Oxidative Fermentation of Various Ketoses†
Osao ADACHI,＊ Yoshikazu FUJII, Mohamed F. GHALY,†† Hirohide TOYAMA,
Emiko SHINAGAWA,＊＊ and Kazunobu MATSUSHITA p.2755

Purification and Characterization of Membrane-bound Quinoprotein Cyclic
Alcohol Dehydrogenase from Gluconobacter frateurii CHM 9
Duangtip MOONMANGMEE,＃ Yoshikazu FUJII, Hirohide TOYAMA, Gunjana THEERAGOOL,＊
Napha LOTONG,＊ Kazunobu MATSUSHITA, and Osao ADACHI† p.2763

Note
Improvement of the Electro-transformation Efficiency of Facultatively Alkaliphilic Bacillus pseudofirmus OF4 by High Osmolarity
and Glycine Treatment
Masahiro ITO, and Makoto NAGANE p.2773

Note
Factors in Bovine Colostrum that enhance the Migration
of Human Fibroblasts in Type I Collagen Gels
Yoshiharu TAKAYAMA,† Katsuhiro KITSUNAI and Koko MIZUMACHI p.2776

Note
Analysis of the Molecular Species of Hydrogenase in the Cells
of an Obligately Chemolithoautotrophic, Marine Hydrogen-Oxidizing
Bacterium, Hydrogenovibrio marinus
Hirofumi NISHIHARA,1,† Yoshinori MIYATA,1 Youji MIYASHITA,1 Michael BERNHARD,2
Anne POHLMANN,2 B<0228>arbel FRIEDRICH,2 and Yoshichika TAKAMURA1 p.2780

Note
Isolation and Primary Structural Analysis of Two Conjugated Polyketone
Reductases from Candida parapsilosis
Aklani-Rose G. D. HIDALGO,1 Muhammad Ali AKOND,1,＊ Keiko KITA,2
Michihiko KATAOKA,1,† and Sakayu SHIMIZU1 p.2785

Note
Molecular Cloning and Characterization of Cotton cDNAs Expressed
in Developing Fiber Cells
Guang-Rong ZHAO, Jin-Yuan LIU,† and Xiong-Ming DU＊ p.2789

Note
Analysis of Upstream Region of Nicotianamine Synthase Gene
from Arabidopsis thaliana: Presence of Putative ERE-like Sequence
Kazuya SUZUKI, Hiromi NAKANISHI, Naoko K. NISHIZAWA, and Satoshi MORI p.2794

Note
Human Tyrosine tRNA Is Also Internally Cleavable by E. coli Ribonuclease
P RNA Ribozyme in Vitro
Tomoaki ANDO, Terumichi TANAKA,† Yoshiaki HORI, Etsuko SAKAI, and Yo KIKUCHI
p.2798

Note
Purification of an Aminopeptidase Preferentially Releasing N-terminal Alanine
from Cucumber Leaves and Its Identification as a Plant Aminopeptidase N
Yasuo YAMAUCHI,† Yukinori EJIRI, and Kiyoshi TANAKA p.2802

Note
Functional Similarities of Recombinant OLP and Cytokinin-Binding Protein 2
Daisuke IGARASHI,1 Hisashi KOIWA,2 Fumihiko SATO,2 Noboru ITO,1
Kazuo HARADA,1 and Koh KOBAYASHI1 p.2806

-1-
Sequential Comparison of Peptides Containing Half-cystine Residues
from Ovalbumins of Six Avian Species

Yuanxia SUN and Shigeru HAYAKAWA†

Department of Biochemistry and Food Science, Kagawa University, Ikenobe, Miki, Kagawa 761-0795, Japan

Received March 15, 2001; Accepted August 17, 2001
Hen ovalbumin contains one cystine disulfide (Cys73-
Cys120) and four cysteine sulfhydryl groups (Cys11, Cys30, Cys367, and Cys382) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys11, Cys367, and Cys382); and two for Pekin duck, mallard duck, and Emden goose (Cys11 and Cys331). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys73-Cys120). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.
Key words: ovalbumin; disulfide bond; sulfhydryl group; cysteine peptide; sequence comparison

-2-
Pressure Distribution Measurement in Biting Surimi Gels
with Molars Using a Multiple-point Sheet Sensor

Kaoru KOHYAMA,1,† Tomoko SAKAI,1 Teruaki AZUMA,2
Toru MIZUGUCHI,3 and Ikuo KIMURA3

1National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2RETS Division, Nitta Corporation, 8-2-1 Ginza, Chuo-ku, Tokyo 104-0061, Japan
3Nissui Central Research Laboratory, 559-6 Kitano-cho, Hachi-oji, Tokyo 192-0906, Japan

Received April 2, 2001; Accepted September 4, 2001
The bite force of three surimi gels with molars was measured in the mouth using a multiple-point sheet sensor. A peak force appeared at the breaking point of each sample, and then the force increased again, accompanied by a decrease in the opening between the upper and lower teeth. Low values in the peak force, pressure, and time at the first peak, the time at which the maximum contact area was engaged, impulse, and slope of bite curve were observed in samples with low breaking force and low breaking deformation found by the mechanical measurement of gel strength, and with less toughness in the sensory assessment. The duration of the bite force, the second peak time, and active bite pressure at the second peak did not change with a change in the surimi texture. The active pressure at the breaking point of each gel was affected by gel strength, while that at the second peak was independent of the gel strength.
Key words: surimi gels; texture; mastication; intra-oral force; gel strength

-3-
Induction of 4-Hydroxycinnamate Decarboxylase in Klebsiella oxytoca Cells
Exposed to Substrates and Non-substrate 4-Hydroxycinnamate Analogs

Yasuyuki HASHIDOKO,† Tomoko TANAKA, and Satoshi TAHARA

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo 060-8589, Japan

Received April 12, 2001; Accepted July 17, 2001
The 4-hydroxycinnamate decarboxylase (4-HCD)-
inducing activity of several substrate analogs toward Klebsiella oxytoca was investigated. Four E-cinnamate-class compounds, E-4-hydroxycinnamic acid (1), caffeic acid (2), ferulic acid (3) and E-2,4-dihydroxycinnamic acid (4), all of which were accepted as substrates, all of which were accepted as substrates of 4-HCD, enable K. oxytoca cells to induce the decarboxylase at a 2.0 mM concentration, while five non-substrate compounds of the E-cinnamate class so far tested were completely inactive. However, 6-hydroxy-2-naphthoic acid (11) and 7-hydroxycoumarin 3-carboxylic acid (14), both of which are non-cinnamate-class analogs of the substrate, acted as strong 4-HCD inducers, even at a 0.5 mM concentration. The 4-HCD-inducing activities of compounds 11 and 14 at 0.5 mM were 10-12-fold higher than that of substrate 1. Compound 11 maintained its 4-HCD-inducing activity toward cultured cells through the late-log and stationary phases, unlike 1 that induced 4-HCD only in the early log phase. SDS-PAGE electrophoresis of protein mixtures from the cultured cells exposed to any 4-HCD inducer indicated that the 21.5 kDa protein was always present.
Key words: Klebsiella oxytoca; 4-hydroxycinnamate decarboxylase inducer; 6-hydroxy-2-naphthoic acid; 7-hydroxycoumarin 3-carboxylic acid; substrate analog

-4-
Stable Isotope-labeling Studies on the Oxidative Coupling
of Caffeic Acid via o-Quinone

Hiroyuki TAZAKI,† Daisuke TAGUCHI, Takaomi HAYASHIDA, and Kensuke NABETA

Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro 080-8555, Japan

Received may 9, 2001; Accepted July 30, 2001
The formation of ortho-quinone from ortho-diphenol is a key step in its dimerization. An NMR analysis of the oxidation of 3,4-dihydroxycinnamic acid (caffeic acid) by NaIO4 revealed the formation of 3-(3′,4′-dioxo-1′,5′-cyclohexadienyl) propenoic acid (o-quinone) prior to the formation of furofuran-type lignan 4,8-exo-bis
(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-
2,6-dione. Both electrolytic and enzymatic oxidation of caffeic acid also generated o-quinone. The yields of o-quinone from caffeic acid were quantified by NMR and HPLC analyses. A stable isotope-labeling study of the formation of lignans directly proved the random radical coupling of semiquinone radicals formed from a set of caffeic acid and o-quinone.
Key words: caffeic acid; oxidative coupling; stable isotope; phenoxy radical; o-quinone

-5-
Isotope Ratio by HRGC-MS of Citrus junos Tanaka (Yuzu) Essential Oils:
m/z 137/136 of Terpene Hydrocarbons

Masayoshi SAWAMURA,1,† Toshiki ITO,1 Akitoshi UNE,1
Hiroyuki UKEDA,1 and Yuzo YAMASAKI2

1Department of Bioresources Science, Faculty of Agriculture, Kochi University, B-200 Monobe,
Nankoku, Kochi 783-8502, Japan
2Kochi Prefectural Industrial Technology Center, 3992-3 Nunoshida, Kochi 781-5101, Japan

Received May 22, 2001; Accepted July 28, 2001
The isotope ratios of monoterpene hydrocarbons in Citrus junos Tanaka (yuzu) essential oils from different origins were determined by ordinary high-resolution gas chromatography-mass spectrometry (HRGC-MS). Both intensities of the molecular mass peaks (m/z 136) and of the isotope peaks (m/z 137) of monoterpene hydrocarbons were measured by single-ion monitoring with an MS analysis. The isotope ratios (m/z 137/136) of the ten monoterpene hydrocarbons commonly contained in citrus essential oils, α-pinene, β-pinene, sabinene, myrcene, α-phellandrene, α-terpinene, limonene, γ-terpinene, β-phellandrene and terpinolene, were determined in yuzu samples of the highest commercial quality from 42 different production districts. Statistical treatment of these data by the t-test and sign test revealed significant differences of the isotope effects in each yuzu sample. It is suggested that this technique will be applicable for evaluating the quality, genuineness and origin of citrus fruits and their products. The isotope fingerprints were also demonstrated in several citrus fruits other than the yuzu samples.
Key words: Citrus junos; yuzu; isotope effect; HRGC/MS; monoterpene hydrocarbon

-6-
Synthesis of Southern (C1′--C11′) and Eastern (C8--C18) Fragments
of Pamamycin-607, an Aerial Mycelium-inducing Substance
of Streptomyces alboniger

Hiromasa KIYOTA,† Yukito FURUYA, Shigefumi KUWAHARA, and Takayuki ORITANI

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Science, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan

Received May 24, 2001; Accepted July 30, 2001
Synthesis of the southern C1′--C11′ and eastern C8--C18 fragments of pamamycin-607, an aerial mycelium-inducing substance of Streptomyces alboniger, was achieved. The southern fragment was synthesized by using the Evans aldol reaction and cis-selective iodoetherification as the key steps in a 9.6％ overall yield (7 steps). The eastern fragment was constructed via the Julia coupling reaction and cis-selective iodoetherification in a 3.0％ overall yield (8 steps from the known epoxide).
Key words: pamamycin; antibiotic; aerial mycelium; synthesis; iodoetherification

-7-
Steric Effects on Interaction of Tea Catechins with Lipid Bilayers

Katsuko KAJIYA, Shigenori KUMAZAWA, and Tsutomu NAKAYAMA†

Department of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Received May 28, 2001; Accepted August 10, 2001
Interaction of tea catechins with lipid bilayers has been investigated with liposome systems. Tea catechins are classified into cis-type and trans-type from the configuration of the two hydrogens at the 2 and 3 positions on the C-ring. The amount of trans-type catechins incorporated into liposomes was less than that of the respective cis-type catechins. Furthermore, the order of the partition coefficients of catechins in an n-octanol/PBS system is the same as that of the amount incorporated into liposomes. These results indicate that in addition to the number of hydroxyl groups on the B-ring and the presence of the galloyl moiety, the stereochemical structure of the C-ring also governs the hydrophobicity and the affinity for lipid bilayers. Trans-type catechins with the galloyl moiety were located on the surface of the lipid bilayer, as well as cis-type catechins with the galloyl moiety, and perturbed the membrane structure. These different stereochemical structures should influence the affinity for lipid bilayers, the alteration of membrane structures, and the difference in the order of the biological activities.
Key words: tea catechins; liposome; lipid bilayer; stereochemical structure

-8-
Purification and Characterization of Maltose Phosphorylase
from Bacillus sp. RK-1

Yasushi INOUE, Keiko ISHII, Tetsuji TOMITA, and Fumio FUKUI

Showa Sangyo Co., Ltd., 16, Sakura 1-chome, Tsukubashi, Ibaraki 305-0003, Japan

Received June 4, 2001; Accepted August 20, 2001
Bacillus sp. RK-1 was isolated as a bacterium that produced maltose phosphorylase (MPase) in the culture supernatant. Screening was done from among about 400 isolates that could grow at 55°C in a medium containing maltose as the sole carbon source. The enzyme was purified to an electrophoretically homogeneous state and some properties were investigated. The Mr of the enzyme was estimated to be 170 kDa by gel filtration and 88.5 kDa by SDS-PAGE, suggesting that it consisted of two identical subunits. The enzyme showed optimum activity around pH 6.0--7.0 and the optimum temperature was about 65°C. The enzyme was stable in the range of pH 5.5--8.0 after keeping it at 4°C for 24 h and retained the activity up to about 55°C after keeping it for 15 min. This is the first report about an MPase that could be produced in the culture supernatant. Furthermore, these investigations showed that this MPase is one of the most thermostable ones reported so far.
Key words: maltose phosphorylase; Bacillus; thermostable; extracellular

-9-
Isolation, Growth, Ultrastructure, and Metal Tolerance of the Green Alga,
Chlamydomonas acidophila (Chlorophyta)

Kahoko NISHIKAWA and Noriko TOMINAGA

Institute of Environmental Science for Human Life, Ochanomizu University, 2-1-1 Otuka, Bunkyou-ku,
Tokyo 112-8610, Japan

Received June 4, 2001; Accepted August 13, 2001
An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance.
Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5--4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50％, EC50 were measured, after 72 h of static exposure. EC50s were 14.4 μM Cd2＋, 81.3 μM Co2＋, 141 μM Cu2＋, and 1.16 mM Zn2＋ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas.
Key words: acidophilic; Chlamydomonas; green alga; growth; metal tolerance

-10-
Purification and Identification of the Essential Ionizable Groups
of Honeybee, Apis mellifera L., Trehalase

Jin-Ha LEE, Masahisa TSUJI, Mitsuru NAKAMURA, Mamoru NISHIMOTO, Masayuki OKUYAMA,
Haruhide MORI, Atsuo KIMURA, Hirokazu MATSUI, and Seiya CHIBA†

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan

Received June 11, 2001; Accepted August 28, 2001
Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl β-glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1％ carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (Km) was 0.66 mM, and the molecular activity (k0) was 86.2 s−1. The enzyme was an <0192><0192>inverting′′ type which produced β-glucose from α, α-trehalose. Dependence of the V and Km values on pH gave values for the ionization constants, pKe1 and pKe2, of essential ionizable groups 1 and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe1, and pKe2 were shifted to higher values of ＋0.2 and ＋0.5 pH unit, respectively. The ionization heat (ΔH) of ionizable group 1 was estimated to be ＋1.8 kcal/mol, and the ΔH value of group 2 was ＋1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (--COO−, ionizable group 1) and the other a protonated type (--COOH, ionizable group 2), although the pKe2 value is high.
Key words: honeybee trehalase; α,α-trehalose; α,β-trehalose; essential ionizable group; Apis mellifera

-11-
High Yield Synthesis of Nitriles by a New Enzyme, Phenylacetaldoxime
Dehydratase, from Bacillus sp. Strain OxB-1

Sheng-Xue XIE, Yasuo KATO, and Yasuhisa ASANO†

Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi,
Toyama 939-0398, Japan

Received June 12, 2001; Accepted August 6, 2001
3-Phenylpropionitrile was synthesized from Z-3-phenylpropionaldoxime (0.75 M) in a quantitative yield (98 g/l) by the use of cells of Escherichia coli JM 109/pOxD-9OF, a transformant harboring a gene for a new enzyme, phenylacetaldoxime dehydratase, from Bacillus sp. strain OxB-1. Other arylalkyl- and alkyl-nitriles were also synthesized in high yields from the corresponding aldoximes. Moreover, 3-phenylpropionitrile was successfully synthesized by the recombinant cells in 70 and 100％ yields from 0.1 M unpurified E/Z-3-phenylpropionaldoxime, which is spontaneously formed from 3-phenylpropionaldehyde and hydroxylamine in a butyl acetate/water biphasic system and aqueous phase, respectively.
Key words: phenylacetaldoxime dehydratase; 3-phenylpropionitrile; aldehyde; 3-phenylpropionaldoxime; Bacillus sp. strain OxB-1

-12-
Quantitative and Specific Detection of a Trichloroethylene-degrading
Methanotroph, Methylocystis sp. Strain M, by a Most Probable
Number-Polymerase Chain Reaction Method

Takeshi KIKUCHI,1 Kazuhiro IWASAKI,2 Hirofumi NISHIHARA,1
Yoshichika TAKAMURA,1 and Osami YAGI3

1Department of Bioresource Sciences, School of Agriculture, Ibaraki University, Chuo 3-21-1, Ami-machi,
Inashiki-gun, Ibaraki-ken 300-0393, Japan
2Biotechnology Risk Assessment Team, Biodiversity Conservation Research Project, National Institute for
Environmental Studies, 16-2 Onogawa, Tsukuba-shi, Ibaraki-ken 305-8506, Japan
3Research Center for Water Environment Technology, Graduate School of Engineering, The University
of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

Received June 15, 2001; Accepted August 24, 2001
We developed a rapid and specific enumeration method for a trichloroethylene-degrading methanotroph, Methylocystis sp. strain M, based on a most probable number-polymerase chain reaction method for monitoring the bacterium at bioremediation sites. The primers designed for the mmoC gene of the soluble methane monooxygenase gene cluster were specific to strain M. Recovery of the cells with a membrane filter enabled us to detect strain M in trichloroethylene-
contaminated groundwater. We used the enumeration method to monitor the number of strain M cells in effluent from soil columns supplied with trichloroethylene-contaminated groundwater. The number of strain M cells in the effluent depended on the amount of the strain M inoculated and the number of cells measured by the most probable number-polymerase chain reaction method was correlated with that measured by a culture method. The detection limit for strain M in effluent detected by MPN-PCR method was 4 to 8×102 cells/ml.
Key words: MPN-PCR; methanotroph; trichloroethylene

-13-
Mutational and Comparative Analysis of Streptolysin O, an Oxygen-labile
Streptococcal Hemolysin

Ikkyu YAMAMOTO,1,† Hisashi KIMOTO,1 Yoriko TAKETO,2 and Akira TAKETO1,3

1Department of Biochemistry I, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan
2Department of Pharmacology, Kanazawa University School of Medicine, Kanazawa 920-0935, Japan
3Department of Applied Physics and Chemistry, Fukui University of Technology, Gakuen,
Fukui 910-8505, Japan

Received June 19, 2001; Accepted August 30, 2001
The structural gene of streptolysin O was cloned from Streptococcus pyogenes strain Sa and S. equisimilis H46A, and the nucleotide sequences were compared with those of strain Richards. To obtain the minimal active fragment of the toxin and to elucidate structure-function relationships in hemolytic function, streptolysin O mutants deleted in N- and C-terminal regions were constructed. Internal amino acid residues were also replaced by introduction of point mutations. Analyses of these mutants showed that considerable activity was retained even after deletion of the N-terminal 107 residues, but genetic removal of the ultimate C-terminal residue resulted in a marked decrease in hemolytic function. By removal in succession, hemolytic activity declined exponentially, and only 0.002％ of the activity remained after deletion of the C-terminal four residues. Nucleotide replacement experiments indicated pivotal roles of I202, V217, D324--L325, V339, and H469 residues in hemolysis.
Key words: streptolysin O; hemolysin; mutational analysis; Streptococcus

-14-
Antihypertensive Effects of Acetic Acid and Vinegar
on Spontaneously Hypertensive Rats

Shino KONDO,1,† Kenji TAYAMA,1 Yoshinori TSUKAMOTO,1
Katsumi IKEDA,2 and Yukio YAMORI2

1Central Research Institute, Mitsukan Group Corporation, Aichi 475-8585, Japan
2Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan

Received June 22, 2001; Accepted September 3, 2001
To clarify the possibility of a preventive effect of dietary vinegar on blood pressure, long-term administration of vinegar or the acetic acid to SHR was examined. As a result, it was observed that acetic acid itself, the main component of vinegar, significantly reduced both blood pressure (p＜0.05) and renin activity (p＜0.01) compared to controls given no acetic acid or vinegar, as well as vinegar. There were no significant differences in angiotensin I-converting enzyme activity in various organs. As for the mechanism of this function, it was suggested that this reduction in blood pressure may be caused by the significant reduction in renin activity and the subsequent decrease in angiotensin II. From this study, it was also suggested that the antihypertensive effect of vinegar is mainly due to the acetic acid in it.
Key words: SHR; vinegar; acetic acid; antihypertensive effect; renin activity

-15-
Novel Substrate Specificity of Designer 3-Isopropylmalate Dehydrogenase
Derived from Thermus thermophilus HB8

Masaaki FUJITA, Hideyuki TAMEGAI, Tadashi EGUCHI,＊ and Katsumi KAKINUMA†

Department of Chemistry and ＊Department of Chemistry and Material Science, Tokyo Institute of Technology,
O-okayama, Meguro-ku, Tokyo 152-8551, Japan

Received June 22, 2001; Accepted August 23, 2001
Redesigning of an enzyme for a new catalytic reaction and modified substrate specificity was exploited with 3-isopropylmalate dehydrogenase (IPMDH). Point-mutation on Gly-89, which is not in the catalytic site but near it, was done by changing it to Ala, Ser, Val, and Pro, and all the mutations changed the substrate specificity. The mutant enzymes showed higher catalytic efficiency (kcat/Km) than the native IPMDH when malate was used as a substrate instead of 3-isopropylmalate. More interestingly, an additional insertion of Gly between Gly-89 and Leu-90 significantly altered the substrate-specificity, although the overall catalytic activity was decreased. Particularly, this mutant turned out to efficiently accept D-lactic acid, which was not accepted as a substrate by wild-type IPMDH at all. These results demonstrate the opportunity for creating novel enzymes by modification of amino acid residues that do not directly participate in catalysis, or by insertion of additional residues.
Key words: 3-isopropylmalate dehydrogenase; designed catalytic reaction; point mutation

-16-
Cloning, Sequencing, and Expression of the Gene Encoding
4-Hydroxy-4-methyl-2-oxoglutarate Aldolase
from Pseudomonas ochraceae NGJ1

Kiyofumi MARUYAMA,1,† Michiko MIWA,1 Nobuyuki TSUJII,1 Tomoyuki NAGAI,1
Naotaka TOMITA,1 Toshiyuki HARADA,1 Hirobumi SOBAJIMA,1 and Hiroyuki SUGISAKI2

1Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1,
Gifu 501-1193, Japan
2Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Received June 25, 2001; Accepted August 11, 2001
A DNA fragment that carried the gene (proA) encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase was cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1, and the coding region was assigned to the nucleotide sequence based on the N-terminal amino acid sequence of the enzyme purified from the organism. The proA gene was 684 bp long, corresponding to a protein of 227 amino acid residues with a calculated molecular mass of 24,067 Da. The genes encoding a putative transporter and a 4-oxalomesaconate hydratase were upstream, and a 3′-truncated gene encoding 2-pyrone-4,6-dicarboxylate lactonase was downstream from the proA gene in the same orientation on the DNA fragment. The proA gene product was overproduced in Escherichia coli and briefly purified to homogeneity from the crude extract by a two-step purification. The molecular and catalytic properties of the gene product were similar to those of the P. ochraceae enzyme.
Key words: class II aldolase; gene cloning; over-expression; protocatechuate meta-degradation operon

-17-
Localization, Accumulation, and Antifungal Activity of Chitinases
in Rye (Secale cereale) Seed

Toki TAIRA,†,＊,＊＊ Takeshi YAMAGAMI,＊＊ Yoichi ASO,＊＊ Masatsune ISHIGURO,＊＊
and Masanobu ISHIHARA＊

＊Department of Bioscience and Biotechnology, Faculty of Agriculture, Ryukyu University,
Okinawa 903-0213, Japan
＊＊Laboratory of Protein Chemistry and Engineering, Graduate School of Genetic Resources Technology,
Kyushu University, Fukuoka 812-8581, Japan

Received June 28, 2001; Accepted August 29, 2001
In order to understand a physiological role of chitinases in rye, the localization and accumulation of rye seed chitinase-a and -c (RSC-a and -c) in the seeds were studied by immunochemical methods. An antiserum specific to the chitin-binding domain (CB-domain), which is an N-terminal part of RSC-a, and an antiserum specific to the catalytic region of RSC-a and RSC-c were used. An immunoblot analysis detected both RSC-a and RSC-c in the endosperm of the rye seed. Immunohistochemical staining indicated that RSC-a was localized in only the aleurone cells, whereas RSC-c existed at least in the starchy endosperm and was also likely to exist in the aleurone cells. It was found by ELISA and an immunoblot analysis that RSC-a and -c accumulated in the seed during the later stage of development. Both chitinases and the Cat-domain exhibited antifungal activity toward Trichoderma species, while the CB-domain did not. Observation of the inhibition of hyphal growth of the T. species suggests that the two chitinases acted in different ways.
Key words: chitinase; chitin-binding domain; rye seed; aleurone; antifungal activity

-18-
Substrate Specificity of β-Primeverosidase, A Key Enzyme in Aroma Formation
during Oolong Tea and Black Tea Manufacturing

Seung-Jin MA, Masaharu MIZUTANI, Jun HIRATAKE, Kentaro HAYASHI,＊
Kensuke YAGI,＊ Naoharu WATANABE,＊ and Kanzo SAKATA†

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
＊Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan

Received July 4, 2001; Accepted August 27, 2001
We synthesized nine kinds of diglycosides and a monoglycoside of 2-phenylethanol to investigate the substrate specificity of the purified β-primeverosidase from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita) in comparison with the apparent substrate specificity of the crude enzyme extract from tea leaves. The crude enzyme extract mainly showed β-primeverosidase activity, although monoglycosidases activity was present to some extent. The purified β-primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, and especially prominent specificity for the β-primeverosyl (6-O-β-D-xylopyranosyl-β-D-glucopyranosyl) moiety. The enzymes hydrolyzed naturally occurring diglycosides such as β-primeveroside, β-vicianoside, β-acuminoside, β-gentiobioside and 6-O-α-L-arabinofuranosyl-β-D-glucopyranoside, but were unable to hydrolyze synthetic unnatural diglycosides. The purified enzyme was inactive toward 2-phenylethyl β-D-glucopyranoside. The enzyme hydrolyzed each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate the β-primeverosidase, a diglycosidase, to be a key enzyme involved in aroma formation during the tea manufacturing process.
Key words: substrate specificity; β-primeverosidase; diglycosidase; β-primeveroside; Camellia sinensis

-19-
Sandwich Enzyme-linked Immunosorbent Assay System
for Micro-detection of the Wheat Allergen, Tri a Bd 17 K

Hiromi YAMASHITA,† Masumi KIMOTO, Miki HIEMORI, Misako OKITA,
Kazuhiko SUZUKI, and Hideaki TSUJI

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University,
Soja, Okayama 719-1197, Japan

Received July 4, 2001; Accepted August 23, 2001
Seven monoclonal antibodies (mAbs) against the wheat allergen, Tri a Bd 17 K, were prepared to obtain mAbs suitable for a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for determination of the allergen. Two of the mAbs strongly immunoblotted the allergen purified from wheat flour. However, only one (1G11) of them was found to be suitable for sandwich ELISA. Epitope mapping against mAb-1G11 on the allergen showed that the mAb recognized the peptide containing Lys-38 and Gln-39 of the allergen. We developed a sandwich ELISA method consisting of Aleuria aurantia lectin for fixing the allergen and 1G11 as the first antibody that enabled 4--4,000 ng/well of the allergen to be determined.
Key words: wheat allergen; Tri a Bd 17 K; monoclonal antibody; sandwich ELISA; lectin

-20-
Administration of Capsiate, a Non-Pungent Capsaicin Analog, Promotes
Energy Metabolism and Suppresses Body Fat Accumulation in Mice

Koichiro OHNUKI, Satoshi HARAMIZU, Kasumi OKI, Tatsuo WATANABE,＊
Susumu YAZAWA,＊＊ and Tohru FUSHIKI

Laboratory of Nutrition Chemistry, Department of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan
＊School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
＊＊Laboratory of Vegetable and Ornamental Horticulture, Division of Agriculture, Graduate School
of Agriculture, Kyoto University, Kyoto 606-8585, Japan

Received June 6, 2001; Accepted August 20, 2001
We investigated the effects of a single oral administration of capsiate, which is found in the fruits of a non-pungent cultivar of pepper, CH-19 Sweet, and has the same structure as capsaicin except for replacement of NH by O in the alkyl chain, on the thermogenesis and fat accumulation in mice. The oxygen consumption and serum adrenalin concentration were higher in both the capsaicin (10 mg/kg-body weight) and capsiate (10 mg/kg-body weight) groups than those in the control group. We also examined the effects of 2 weeks of administration of capsaicin and capsiate on body fat accumulation. Eevery day for 2 weeks administration of capsiate (10, 50 mg/kg-body weight/day) markedly suppressed body fat accumulation as well as capsaicin (10 mg/kg-body weight/day). These results suggest that capsiate promotes energy metabolism and suppresses body fat accumulation as does capsaicin.
Key words: CH-19 Sweet; capsiate; capsaicin; body fat; oxygen consumption

-21-
A Gene Encoding Phosphatidylethanolamine N-Methyltransferase
from Acetobacter aceti and Some Properties of its Disruptant

Takashi HANADA,† Yasuhiro KASHIMA, Akihiko KOSUGI, Yukimichi KOIZUMI,
Fujiharu YANAGIDA, and Shigezo UDAKA††

Department of Fermentation Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku,
Tokyo 156-8502, Japan

Received July 9, 2001; Accepted August 27, 2001
Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced.
One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3％ identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.
Key words: Acetobacter aceti; phosphatidylethanolamine N-methyltransferase; phosphatidylcholine synthesis; acetic acid tolerance; Pmt gene

-22-
(4E)-Dehydrocitrals [(2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienals]
from Acarid Mite Histiogaster sp. A096 (Acari: Acaridae)†

Hiroyuki HIRAOKA, Naoki MORI, Ritsuo NISHIDA, and Yasumasa KUWAHARA††

Laboratory of Chemical Ecology, Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received July 12, 2001; Accepted August 28, 2001
A mixture of two monoterpenes was obtained as the opisthonotal gland secretion from unidentified Histiogaster sp. A096 (Acari: Acaridae), and their structures were elucidated to be (4E)-dehydrocitrals [(2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienals] by GC/MS, GC/FT-IR, UV and 1H-NMR spectra. Both isomers of (4E)-dehydrocitral prepared by syntheses in 4 steps from 3-methyl-2-butenal with 34.2％ yields (based on the ylide) were separated by column chromatography into the (2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-
octatrienal. Mass spectra together with GC retention times of the purified natural (4E)-dehydrocitrals were identical with those of synthetic (2E,4E)-3,7-dimethyl-2,4,6-octatrienal and (2Z,4E)-3,7-dimethyl-2,4,6-
octatrienal. The geometry at the 2-C position of both synthetic (4E)-dehydrocitrals was confirmed by NOESY analyses. This is the first identification of (4E)-
dehydrocitrals from the animal kingdom.
Key words: astigmatid mite; Histiogaster sp. A096; monoterpene aldehyde; (4E)-dehydrocitral; (2E,4E)- and (2Z,4E)-3,7-dimethyl-2,4,6-octatrienal

-23-
Membrane-bound Quinoprotein D-Arabitol Dehydrogenase
of Gluconobacter suboxydans IFO 3257: A Versatile Enzyme
for the Oxidative Fermentation of Various Ketoses†

Osao ADACHI,＊ Yoshikazu FUJII, Mohamed F. GHALY,†† Hirohide TOYAMA,
Emiko SHINAGAWA,＊＊ and Kazunobu MATSUSHITA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
＊＊Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai,
Ube 755-8555, Japan

Received July 16, 2001; Accepted August 17, 2001
Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purifiy ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2＋, and of a buffer system involving acetate buffer supplemented with Ca2＋, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2＋. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2＋. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.
Key words: acetic acid bacteria; D-arabitol dehydrogenase of membrane-bound; Gluconobacter suboxydans; oxidative fermentation; quinoprotein

-24-
Purification and Characterization of Membrane-bound Quinoprotein Cyclic
Alcohol Dehydrogenase from Gluconobacter frateurii CHM 9

Duangtip MOONMANGMEE,＃ Yoshikazu FUJII, Hirohide TOYAMA, Gunjana THEERAGOOL,＊
Napha LOTONG,＊ Kazunobu MATSUSHITA, and Osao ADACHI†

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
＊Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand

Received July 18, 2001; Accepted August 8, 2001
A quinoprotein catalyzing oxidation of cyclic alcohols was found in the membrane fraction for the first time, after extensive screening among aerobic bacteria. Gluconobacter frateurii CHM 9 was finally selected in this study. The enzyme tentatively named membrane-bound cyclic alcohol dehydrogenase (MCAD) was found to occur specifically in the membrane fraction, and pyrroloquinoline quinone (PQQ) was functional as the primary coenzyme in the enzyme activity. MCAD catalyzed only oxidation reaction of cyclic alcohols irreversibly to corresponding ketones. Unlike already known cytosolic NAD(P)H-dependent alcohol-aldehyde or alcohol-ketone oxidoreductases, MCAD was unable to catalyze the reverse reaction of cyclic ketones or aldehydes to cyclic alcohols. MCAD was solubilized and purified from the membrane fraction of the organism to homogeneity. Differential solubilization to eliminate the predominant quinoprotein alcohol dehydrogenase (ADH), and the subsequent two steps of column chromatographies, brought MCAD to homogeneity. Purified MCAD had a molecular mass of 83 kDa by SDS-PAGE. Substrate specificity showed that MCAD was an enzyme oxidizing a wide variety of cyclic alcohols. Some minor enzyme activity was found with aliphatic secondary alcohols and sugar alcohols, but not primary alcohols, differentiating MCAD from quinoprotein ADH. NAD-dependent cytosolic cyclic alcohol dehydrogenase (CCAD) in the same organism was crystallized and its catalytic and physicochemical properties were characterized. Judging from the catalytic properties of CCAD, it was apparent that CCAD was distinct from MCAD in many respects and seemed to make no contributions to cyclic alcohol oxidation.
Key words: acetic acid bacteria; cyclic alcohol oxidation; membrane-bound dehydrogenase; NAD-dependent cyclic alcohol dehydrogenase; quinoprotein cyclic alcohol dehydrogenase

-25-
Note
Improvement of the Electro-transformation Efficiency of Facultatively Alkaliphilic Bacillus pseudofirmus OF4 by High Osmolarity
and Glycine Treatment

Masahiro ITO, and Makoto NAGANE

Faculty of Life Sciences, Toyo University, Izumino, Itakura-machi, Oura-gun, Gunma 374-0193, Japan

Received April 10, 2001; Accepted August 8, 2001
An electro-transformation method was established for facultatively alkaliphilic Bacillus pseudofirmus OF4. A high osmolarity electroporation medium with a high electric field strength was effective. Transformation efficiency improved 110-fold in glycine-treated cells compared to non-glycine-treated cells. Under optimum conditions, the transformation efficiency was 1.69×106 transformants per μg of pUB110.
Key words: alkaliphiles; electroporation; Bacillus; glycine-treatment

-26-
Note
Factors in Bovine Colostrum that enhance the Migration
of Human Fibroblasts in Type I Collagen Gels

Yoshiharu TAKAYAMA,† Katsuhiro KITSUNAI and Koko MIZUMACHI

National Institute of Livestock and Grassland Science, Ikenodai 2, Kukizaki, Inashiki, Ibaraki 305-0901 Japan

Received April 12, 2001; Accepted June 27, 2001
Bovine colostrum has an activity that increases the migration of WI38 fibroblasts. We evaluated the motility of fibroblasts by their ability to contract collagen gels. Part of the activity was absorbed by anion-exchange chromatography at pH 6.4, and eluted by 0.2--0.3 M sodium chloride. The activity was separated into many fractions corresponding to 20--150 kDa by gel filtration chromatography under acidic conditions. The major peak of the activity coincided with 50--70 kDa.
Key words: colostrum; collagen gel contraction; anion-exchange chromatography

-27-
Note
Analysis of the Molecular Species of Hydrogenase in the Cells
of an Obligately Chemolithoautotrophic, Marine Hydrogen-Oxidizing
Bacterium, Hydrogenovibrio marinus

Hirofumi NISHIHARA,1,† Yoshinori MIYATA,1 Youji MIYASHITA,1 Michael BERNHARD,2
Anne POHLMANN,2 B<0228>arbel FRIEDRICH,2 and Yoshichika TAKAMURA1

1Department of Bioresource Science, College of Agriculture, Ibaraki University, 3-21-1 Chu-ou, Ami-machi,
Ibaraki 300-0393, Japan
2Institut f<0228>ur Biologie, Humboldt Universit<0228>at zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany

Received May 8, 2001; Accepted August 28, 2001
Hydrogenovibrio marinus was suggested to have only membrane-bound hydrogenase (MBH). The change of cultivation pO2 did not affect the molecular species of hydrogenase expressed. We propose the MBH is grouped in class I [NiFe] MBH according to the subunit composition, size (Mw 38,000 and Mw 74,000 subunits) and N-terminal sequences of the subunits, and arrangement of the structural genes. Ni-requirement for the autotrophic growth on H2 also suggested the MBH is the Ni-containing type. Southern hybridization analysis using a part of the MBH gene showed a possibility of the presence of two highly homologous MBHs which were not separated by SDS-PAGE.
Key words: class I [NiFe] membrane-bound hydrogenase; Hydrogenovibrio marinus; aerobic hydrogen-oxidizing bacterium; marine environment; N-terminal amino acid sequence

-28-
Note
Isolation and Primary Structural Analysis of Two Conjugated Polyketone
Reductases from Candida parapsilosis

Aklani-Rose G. D. HIDALGO,1 Muhammad Ali AKOND,1,＊ Keiko KITA,2
Michihiko KATAOKA,1,† and Sakayu SHIMIZU1

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2Department of Biotechnology, Faculty of Engineering, Tottori University, 4-101 Koyama, Tottori 680-8552,
Japan

Received June 6, 2001; Accepted August 20, 2001
Two conjugated polyketone reductases (CPRs) were isolated from Candida parapsilosis IFO 0708. The primary structures of CPRs (C1 and C2) were analyzed by amino acid sequencing. The amino acid sequences of both enzymes had high similarity to those of several proteins of the aldo-keto-reductase (AKR) superfamily. However, several amino acid residues in the putative active sites of AKRs were not conserved in CPRs-C1 and -C2.
Key words: conjugated polyketone reductase; aldo-keto reductase; asymmetric reduction; Candida parapsilosis; optically active alcohols

-29-
Note
Molecular Cloning and Characterization of Cotton cDNAs Expressed
in Developing Fiber Cells

Guang-Rong ZHAO, Jin-Yuan LIU,† and Xiong-Ming DU＊

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
＊Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang 455112, Henan, China

Received June 19, 2001; Accepted August 22, 2001
Using the FDD-PCR technique, ten new fiber-specific cDNAs were isolated from developing cotton fiber cells and showed high amino acid identity to previously recorded cDNAs. Five cDNAs encoding bisphosphate nucleotidase, α-tubulin, β-galactosidase, annexin, and reversibly glycosylated polypeptide were identified while the functions of five other cDNAs were undetermined. Dot blot analysis showed that all transcripts of the 10 cDNAs accumulated preferentially in fiber cells and the majority were expressed in the early phase of cotton fiber development, except for F14 which accumulated at a high level during the late phase of developing fiber cells, indicating that all of the genes were involved in the process of fiber development.
Key words: cDNA sequence; developing fiber cells; differential gene expression; Gossypium hirsutum

-30-
Note
Analysis of Upstream Region of Nicotianamine Synthase Gene
from Arabidopsis thaliana: Presence of Putative ERE-like Sequence

Kazuya SUZUKI, Hiromi NAKANISHI, Naoko K. NISHIZAWA, and Satoshi MORI

Laboratory of Plant Molecular Physiology, Department of Applied Biological Chemistry, The University
of Tokyo, 1-1 Yayoi, Bunkyo-ku, 113-8657 Tokyo, Japan

Received July, 3, 2001; Accepted August 15, 2001
Nicotianamine (NA) is present in all plants so far examined, and is thought to chelate transition metal ions. Previously, we isolated three nicotianamine synthase (NAS) genes of Arabidopsis thaliana (AtNAS1, 2, and 3) and showed that each NAS gene is differentially expressed. Deletion analysis of the 5′ flanking region of AtNAS3 found a putative ethylene-responsive sequence, ATTTTCAAA.
Key words: Arabidopsis thaliana; ethylene-responsive element; nicotianamine; nicotianamine synthase

-31-
Note
Human Tyrosine tRNA Is Also Internally Cleavable by E. coli Ribonuclease
P RNA Ribozyme in Vitro

Tomoaki ANDO, Terumichi TANAKA,† Yoshiaki HORI, Etsuko SAKAI, and Yo KIKUCHI

Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University
of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan

Received July 3, 2001; Accepted August 8, 2001
Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNAAla, tRNAHis, and tRNAMeti) molecules fit to form another, non-cloverleaf, structure in which the 3′-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool to detect destablized tRNA molecules from any species.
Key words: Escherichia coli; tRNA; RNase P; hyperprocessing; tyrosine

-32-
Note
Purification of an Aminopeptidase Preferentially Releasing N-terminal Alanine
from Cucumber Leaves and Its Identification as a Plant Aminopeptidase N

Yasuo YAMAUCHI,† Yukinori EJIRI, and Kiyoshi TANAKA

Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan

Received July 5, 2001; Accepted September 6, 2001
In this study, a highly active foliar aminopeptidase preferentially releasing N-terminal alanine from artificial substrates was purified and characterized from cucumber (Cucumis sativus L. suyo). The enzyme had a molecular mass of 200 kDa consisting of two subunits of 95 kDa. It was a metalloprotease the pH optimum of which was 8 to 9. It cleaved Ala-, Gly-, Met-, Ser-, Leu-, Lys-, and Arg artificial substrates. An internal amino acid sequence was similar to those of aminopeptidase N (clan MA, family M1) of microorganisms, and was very similar to that of a putative aminopeptidase N of Arabidopsis thaliana. From these results, the highly active aminopeptidase in cucumber leaves was identified to be a plant aminopepitdase N.
Key words: Cucumis sativus; cucumber; purification; aminopeptidase N; naphthylamidase

-33-
Note
Functional Similarities of Recombinant OLP and Cytokinin-Binding Protein 2
Daisuke IGARASHI,1 Hisashi KOIWA,2 Fumihiko SATO,2 Noboru ITO,1

Kazuo HARADA,1 and Koh KOBAYASHI1

1Laboratory of Life Sciences, Tokyo Gakugei University, Tokyo 184-8501, Japan
2Department of Applied Life Sciences, Kyoto University, Kyoto 606-8224, Japan

Received Augast 3, 2001; Accepted August 22, 2001
CBP1 and CBP2 are cytokinin-binding proteins isolated from tobacco callus. In particularly, CBP2 is a 26-kDa protein with high affinity (Kd＝1.08×10-6 M) for cytokinin [Kobayashi et al. Plant Cell Physiol.41(2): 148--157 (2000)] and the N-terminal amino acid analysis of CBP2 showed high sequence homology (92.9％) to tobacco osmotin-like protein (OLP). To compare the properties of OLP and CBP2, recombinant OLP was purified, and binding to benzyladenine (BA) was examined. The inclusion bodies of recombinant OLP were solubilized in 8 M urea and purified on an SP-Sepharose column. SDS-PAGE analysis of the purified recombinant OLP revealed a single band of 26 kDa. The Kd of solublized recombinant OLP to BA obtained from a Scachard plot was 1.10×10−6 M, which was similar to the Kd of CBP2 to BA (1.08×10−6 M).
Key words: cytokinin-binding protein 2 (CBP2); Kd; immunoblotting; inclusion bodies; recombinant OLP