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板垣, 英治 ; Itagaki, Eiji
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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正橋剛二(解説) 山本渓山(著)『入越日記』能登・越中・立山に薬草を求めて桂書房, 2017年12月20日初版発行, A5判, pp.197, 定価3,000円(税別)
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板垣, 英治 ; itagaki, Eiji
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板垣, 英治 ; itagaki, Eiji
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板垣, 英治 ; Itagaki, Eiji
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Itagaki, Eiji ; Taniguchi, Shigehiko ; 板垣, 英治
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Itagaki, Eiji ; Fujita, Takeshi ; Sato, Ryo ; 板垣, 英治
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A preparation containing formate dehydrogenase, cytochrome b1 and nitrate reductas_??_ was solubilized and partially purified from the particulate fraction of E. coli cells growr_??_ anaerobically under the conditions favorable
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for nitrate respiration. Some properties o_??_ formate dehydrogenase and cytochrome b1 in this purified preparation were investigated It was suggested that formate dehydrogenase is a metalloflavoprotein with essential sulf_??_ hydryl groups. Its activity was strongly bu_??_ reversibly inhibited by molecular oxyge_??_ The prosthetic group of cytochrome b1 was decisively identified as protoheme, and it was found that the cytochrome is autoxidizable. The reduction of cytochrome b1 by formate was shown to require, in addition to formate dehydrogenase, a lipid-soluble factor which could be replaced by vitamin K3 in the solubilized system.We are indefted to Drs. H. Maeno and S. Sakakibara for generous gifts of crude snake venonand crystalline protohemin, respectively.
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Fujita, Takeshi ; Itagaki, Eiji ; Sato, Ryo ; 板垣, 英治
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Cytochrome b1 was solubilized from the particulate fraction of Escherichia coli with the aid of snake venom and deoxycho
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late and was extensively purified by ammonium sul-fate fractionation and hydroxylapatite column chromatography. The purifed preparation was still contaminated by a colorless impurity which sedimented more slowly than the cytochrome in the ultracentrifugal field; the purity being estimated to be 50 to 70 per cent. A molecular weight of 600, 000 to 800, 000 could be expected for the cytochrome from its sedimentation constant. On the other hand, aminimum molecular weight of about 160, 000 was obtained from the heme content of the cytochrome. The cytochrome could be split by acid acetone into protoheme and an apoprotein moiety. The recombination of the two components could be observed spectro-photometrically, but the reconstructed cytochrome was no more native as evidenced by its capabiliiy to combine with CO. The normal oxidation-reduction potential of purified cytochrome bt was determined to be about -20 mV at pH 7.0 and 25°C. Spectral properties, reactivity and chemical composition of the purified preparation were also studied.We wish to thank Dr. A. Ohsaka of the Natio-nal Institute of Health, Tokyo, for a generous gift of snake venom, and Dr. K. Kakiuchi of the Institute for Protein Research, Osaka University, Osaka, for carrying out the ultracentrifugal analyses.
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Itagaki, Eiji ; Fujita, Takeshi ; Sato, Ryo ; 板垣, 英治
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The reactions of cytochrome b1 and nitrate reductase in an enzyme preparation solubiliz-ed and partially purified from the particulate fraction of Escherichia coli were investigated. The cytochrome b1 reduced by the addition of formate and
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vitamin K3 was found to be reoxidizable by oxygen, nitrate and chlorate. While the oxidation by oxygen was insensitive to cyanide and azide (the autoxidizability of the cytochrome), these reagents strongly inhi-bited the reaction by nitrate and chlorate suggesting the intervention of nitrate reduc-tase. With chlorate as the oxidant, the de-formation of the absorption spectrum of the cytochrome was observed. The nitrate reduc-tion by formate in the solubilized system was found to require the addition of vitamin K3, but flavins had no effects on this reaction. Since the vitamin is required also for thereduction of cytochrome b1 in this prepara-tion, the involvement of the cytochrome in the nitrate-reducing mechanism was further substantiated. The formate-nitrate reaction was strongly inhibited by 2-heptyl-4-hydroxy-quinoline-N-oxide (HOQNO), but this inhibi-tion was not competitive with respect to vitamin K3. It was spectrophotometrically revealed that HOQNO inhibits the reoxidation of cytochrome bt by nitrate, but not the re-duction by formate plus vitamin K3. In the particulate fraction, the formate-nitrate reduc-tase activity was inhibited much more remark-ably than the formate oxidase activity. Some of the implications of these results are dis-cussed.We wish to thank Dr. S. Taniguchi for a gener-ous gift of 2-heptyl-4-hydoxyquinoline-N-oxide.
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Itagaki, Eiji ; 板垣, 英治
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A pale-brown powder preparation of the particulate fraction of E. coli cells grown with nitrate respiration was obtained with cold acetone treatment. This preparation, was similar to the solubilized and partially purified enzyme
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preparation, had high activities of for-mate dehydrogenase and nitrate reductase and contained cytochrome b1, but no activity of formate-nitrate reductase system. By addition of lipids prepared from this organism with aid of a detergent, PADA, the activity of formate-nitrate reductase system was completely restored. The restoration efficiencies of enzyme activity by UQ8 and K2 which were quinones found in this organism, were 2.6 to 1 per mole.The experiments with the UQ8, K2 or both specifically deprived, and ultraviolet irradiated lipid preparations showed that UQ8, rather than K2, acted more effectively as an electron carrier.UQ8 was anaerobically reduced slowly by the enzyme preparation wit formate, and UQ8H2 was rapidly oxidized by nitrate with simultaneous production of stoichiometric amounts of nitrite. From these results, it is concluded taht UQ8 acts principally as hydrogen acceptor for formate dehydrogenase of E. coli.The author is very grateful to Professer R. Sato of this laboratory for his helpful advice, to Mr. T. Fujita of his continous discussion to this work, to Dr. S. Ikeda of the Faculty of Science, Osaka University for his helpful advice to the synthesis of PADA, to Dr. K. Tanaka of Takeda Pharmaceutical Industries Ltd., Osaka and Dr. K. Uehara of the Pharmac-eutical Faculty of Osaka University for materials, and to Dr. K. Kusai of Nagase Sangyo Co. Ltd. for large culture of the organism.
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Itagaki, Eiji ; Suzuki, Sakaru ; 板垣, 英治
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An enzymatic method is described for the estimation of formic acid formed during the periodate oxidation of carbohydrate
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s in the 10-2μmole range. The method is based on the coupled action of formic acid dehydro-genase and nitrate reductase obtained as a particulate fraction from Escherichia coli strains. The enzymes catalyze the quantitative reduc-tion of nitrate with formic acid to yield nitrite. The nitrite is estimated by a photometric me-thod. The results obtained using this method on D-glucose, L-rhamnose, N-acetyl-D-glucos-amine and UDP-glucose are given.The authors express their thanks to Mr. M. Iwatsuka for his technical assistance.
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Suzuki, Yasuyuki ; Itagaki, Eiji ; Mori, Hiromu ; Hosoya, Toichiro ; 板垣, 英治
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The testosterone-binding globulin (TeBG) from bovine serum was purified by affinity chromatography and hydroxylapatite c
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hromatography. The affinity column used was prepared by coupling 17α-carboxyethynyl-l7-hydroxy-4-androsten-3-one to aminoethyl-Sepharose. The compound was replaceable by 17α-carboxyethynyl-l7-hydroxy-5α-androstan-3-one, but not by testosterone 17-hemisuccinate, estradiol 17-hemisuccinate, or testosterone 3-(O-carboxy-methyl)oxime. The TeBG isolated was homogeneous on analytical polyacrylamide gel electrophoresis and equilibrium centrifugation. The protein was a glycoprotein having a molecular weight of 89, 500 and a carbohydrate content of 17%. The association constant (M-1) at 4°C was 1.1 × 108 and the number of binding sites per molecule was 0.8. Treatment with guanidine-HCI dissociated the protein into subunits having a molecular weight of 28, 400 (about one-third of that of the original molecule). SDS-gel electrophoresis showed that two of the three subunits were slightly larger than the other. The dissociation into subunits could also be accomplished by GEDTA treatment with concomitant loss of testosterone-binding activity. The activity and molecular size were reversibly restored by incubation with excess Ca2+.
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Itagaki, Eiji ; Arisawa, Masatoshi ; Hosoya, Toichiro ; 板垣, 英治
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Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) of reduced porcine thyro-globulin revealed more than
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ten distinct bands with molecular weights ranging from 19, 000 to 330, 000. When the thyroglobulin preparation was previously dialyzed against distilled water for 3 days at 4°C, the bands with large molecular weights disappeared, leaving only several small-sized components. These submolecular protein components, designated as Sub. I, Sub. II, Sub. III, and Sub. IV, were isolated by gel filtration on a Sephadex G-200 column in 0.1% SDS-0.04M Tris acetate buffer (pH 7.4) at 40°C, and the preparations were confirmed by sedimentation equilibrium analysis to be nearly homogenous in size. The weight average molecular weights determined by the sedimentation equilibrium method were 17, 500, 37, 400, 50, 300, and 70, 800 for Sub. I, Sub. II, Sub. III, and Sub. IV, respectively. Similar values were obtained by other methods, gel filtration and SDS-gel electrophoresis. Thus, the relative size of these components was very close to 1:2:3:4. The contents of amino acids and carbohydrates of these components were very similar to each other and also to entire thyroglobulin. The contents of iodoamino acids were also not greatly different between them, except that thyroxine and triiodothyronine seemed to be significantly high in the smallest protein, Sub. I. Immunochemical study also revealed a strong resemblance between each of them and thyroglobulin. Based on these results, together with other works, the structure and function of thyroglobulin are discussed.
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Itagaki, Eiji ; 板垣, 英治
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A steroid monooxygenase from cells of a fungus, Cylindrocarpon radicicola ATCC 11011, grown in the presence of progester
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one has been purified by affinity chromatography on a pregnenolone-Sepharose column. The obtained enzyme was gel electrophoretically homogeneous and exhibited a molecular weight of about 115, 000. SDS-gel electrophoresis revealed that the enzyme consisted of two equal-sized subunits with a molecular weight of 56, 000.Sedimentation equilibrium analysis at 20°C indicated that the enzyme protein behaved as a mixture of monomeric and dimeric subunit species. The enzyme contained one molecule of FAD in each subunit and exhibited absorption maxima at 375 and 440 nm.The monooxygenase catalyzed a Baeyer-Villiger type oxidation, i.e., oxygenative esterification of C21-20-ketosteroid to form an acetate ester of C19- 17β-hydroxysteroid with consumptions of NADPH and molecular oxygen. The enzyme displayed a wide substrate specificity toward C21-20-ketosteroids, while it strictly required NADPH as the external electron donor in a ratio of 1:1:1 for ketosteroid: NADPH: molecular oxygen. Kinetic study showed the enzyme to have very high affinity for progesterone.
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Itagaki, Eiji ; 板垣, 英治
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A steroid monooxygenase of Cylindrocarpon radicicola was found to catalyze oxygenative lactonization of 17α-etosteroid,
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androstenedione, to yield D-hmo-17α-oxasteroid, testololactone, i.e., the androstenedione monooxygenase reaction, in addition to catalyzing the progesterone monooxygenase reaction. The reaction product was identified by TLC, GLC, and mass spectrometry.The oxygenation proceeded with unitary stoichiometry for 17-ketosteroid, NADPH, and molecular oxygen, indicating that it is a typical monooxygenase reaction of the external electron donor type. The enzyme catalyzed successively the side chain cleavage reaction of 17α-hydroxy-20-ketosteroid to produce its 17-ketoerivative and the lactonization of the product.The effects of pH and of the concentration of substrate steroids on the androstenedione monooxygenase reaction were different from those on the progesterone onooxygenase reaction. Progesterone is a strong and competitive inhibitor of he lactonization of 17-ketosteroids. The steroid monooxygenase is concluded to ave the activities of both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids.
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Itagaki, Eiji ; Iwaya, Teturou ; 板垣, 英治
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An NAD+-linked 17β-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATC
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C 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58, 600 by gel filtration and polyacrylamide gel electropho-resis. SDS-gel electrophoresis gave Mr=26, 000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17β-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax=85 μmol/min/mg; Km for the steroid =9.5 μM; Km for NAD+=198 μM at pH 10.0) and for the reduction of androstenedione (Vmax=1.8 μmol/min/mg; Km for the steroid=24 μM; Km for NADH=6.8 μM at pH 7.0). In the purified enzyme preparation, no activity of 3α-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, Δ5-3-ketosteroid-4, 5-isomerase, or steroid ring A-Δ-dehydrogenase was detected. Among several steroids tested, only 17β-hydroxysteroids such as testosterone, estradiol-17β, and 11β-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17α-3H] testosterone.
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Morii, Shingo ; Sawamoto, Shizue ; Yamauchi, Yuhji ; Miyamoto, Masahiko ; Iwami, Masafumi ; Itagaki, Eiji ; 板垣, 英治
概要:
Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C17- and C20-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA
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fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1, 650 nucleotides long, starts with a TTG colon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60, 133. Thus, the molecular mass of the holoenzyme is 60, 919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.
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板垣, 英治 ; 石田, 啓 ; Itagaki, Eiji ; Ishida, Hajime
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金沢大学理工学域
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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Itagaki, Eiji ; Matsushita, Hiroyuki ; Hatta, Takashi ; 板垣, 英治
概要:
金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />3-Ketosteroid-Δ1-dehydrogenase from Nocardia corallina catalyzes transhydrogen
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ation of 3-keto-4-ene-steroid to 3-keto-1,4-diene-steroid e.g., progesterone to 1,4-androstadiene-3,17-dione. The reaction proceeded linearly at first and then soon slowed down owing to equilibration. The turnover number of this reaction was of the same magnitude as that of the dehydrogenation of 3-keto-4-ene-steroid. The pH optimum was 8.4, which is lower than that of the dehydrogenase reaction. The enzyme has a wide specificity for hydrogen acceptor steroids. The K(m)' and K(max)' values for these steroids and the values of the corresponding 3-keto-4-ene-steroids were compared. Kinetic studies of the steroid transhydrogenase reaction demonstrated a typical ping-pong mechanism. The enzyme oxidized 1,2-tritiated progesterone and transferred the tritium atoms to the reaction product, 4-androstene-3,17-dione, and water. Transhydrogenation in D2O resulted in the incorporation of a deuterium atom into the C2-position of 4-androstene-3,17-dione. The results indicate that the enzyme catalyzes C1,C2-trans axial abstraction of hydrogen atoms from progesterone, transfer of the 1α-hydrogen to the C1-position of 1,4-androstadiene-3,17-dione and release of the 2β-hydrogen to water. Reaction schemes based on the experimental results are proposed. The enzyme also catalyzes the reduction of 3-keto-1,4-diene-steroids with reduced benzoyl viologen.
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Matsushita, Hiroyuki ; Itagaki, Eiji ; 板垣, 英治
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金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />The variation with pH of kinetic parameters was examined for 3-ketosteroid-Δ1-
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dehydrogenase from Nocardia corallina. The V(max)/K(m) profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the inactivated enzyme revealed that the inactivation arises from modification of a histidine residue. Studies with [14C]diethylpyrocarbonate provided Support for the idea that the 1-2 essential histidine residues are essential for the catalytic activity of the enzyme. Dye-sensitized photooxidation led to 50% inactivation of the enzyme with the decomposition of two histidine residues. This inactivation was also prevented by androstadienedione. Dancyl chloride caused a loss of the enzyme activity. Modifiers of glutamic acid, aspartic acid, cysteine, and lysine did not affect the enzyme activity. Butanedione and phenylglyoxal in the presence of borate rapidly inactivated the enzyme, indicating that arginine residues also have a crucial function in the active site. The data described support the previously proposed mechanism of β-oxidation of 3-ketosteroid.
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Suzuki, Kenzi ; Mizuguchi, Mitsuo ; Gomi, Tomoharu ; Itagaki, Eiji ; 板垣, 英治
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金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivate
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d by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatagraphy as an apoprotein form and characterized. It could bind FAD with the same K(d) value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. V(max) for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P, putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.
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Suzuki, Kenzi ; Gomi, Tomoharu ; Kaidoh, Toshio ; Itagaki, Eiji ; 板垣, 英治
概要:
金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzed the fo
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rmation of catechol from substrate analogues such as o-nitro-, o-amino-, o-iodo-, o-bromo-, and o-chloro-phenol by removing the ortho-substituted groups.They are converted into nitrite, ammonia, and halide ions, respectively. Kinetic parameters of these reactions were determined by spectrophotometric and polarographic methods. Hydroxylation of o-nitro- or o-iodophenol proceeds with the unusual stoichiometry of 2 : 1 : 1 for consumed NADH, O2-uptake, and catechol formed. Other ortho-substituted phenols examined also gave the same results. Like salicylate, these substrates perturb the absorption spectrum of salicylate hydroxylase in the visible region, indicating the formation of enzyme·substrate complexes. Titration experiments with ortho-substituted phenols gave the dissociation constants of the complexes. The complexes were quantitatively reduced with NADH or dithionite without detectable formation of the intermediates. The fact that one atom of 18O2 was incorporated into the produced catechol in hydroxylation of o-nitrophenol indicates that the reaction is of monooxygenase nature. It is concluded that salicylate hydroxylase cleaves the C-N and C-X bonds of ortho-substituted phenols.
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Hatta, Takashi ; Wakabayashi, Teruko ; Itagaki, Eiji ; 板垣, 英治
概要:
金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />The inducible 3-keto-5α-steroid-Δ4-dehydrogenase of Nocardia corallina was pur
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ified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of M(r) 60,000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5α-steroid at the 4- and 5-position, e.g. the conversion of 5α-androst-1-ene-3,17-dione to 1,4-androstadiene-3,17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5α-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4β, 5α-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5α-steroids but not 3β-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5α-steroid and 3-keto-1,4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-Δ1-dehydrogenase.
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Suzuki, Kenzi ; Gomi, Tomoharu ; Itagaki, Eiji ; 板垣, 英治
概要:
金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />Salicylate hydroxylase [EC 1.14.13.1] from Pseudomonas putida catalyzes the hy
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droxylation of salicylate, and also o-aminophenol, o-nitrophenol, and o-halogenophenols, to catechol. The reactions with these o-substituted phenols comprise oxygenative deamination, denitration, and dehalogenation, respectively. The reaction try, as to NADH oxidized, oxygen consumed, and catechol formed, is 2 : 1 : 1, respectively. The mechanisms for the deiodination and oxygenation of o-iodophenol were investigated in detail by the use of I+-trapping reagents such as DL-methionine, 2-chlorodimedone, and L-tyrosine. The addition of the traps did not change the molar ratio of catechol formed to NADH oxidized, nor iodinated traps produced were in the incubation mixture. The results suggest that I+ was not produced on the deiodination in the hydroxylation of o-iodophenol. On the other hand, L-ascorbate, L-epinephrine, and phenylhydrazine increased the molar ratio. o-Phenylenediamine decreased it, being converted to phenazine. This suggests that o-benzoquinone is formed in the oxidation of o-iodophenol as a nascent product. The quinone was detected spectrophotometrically by means of the stopped-flow method. Kinetic analysis of the reactions revealed that o-benzoquinone is reduced nonenzymatically to catechol by a second molecule of NADH. A mechanism of elimination for the ortho-substituted groups of substrate phenols by the enzyme is proposed and discussed.
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板垣, 英治
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板垣, 英治
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| 44.
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板垣, 英治
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| 45.
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板垣, 英治
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| 46.
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板垣, 英治 ; 本康, 宏史
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| 47.
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板垣, 英治
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| 48.
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板垣, 英治
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| 49.
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板垣, 英治
概要:
石川県では猛毒のフグの卵巣を糠漬けや粕漬けにし た「珍味中の珍味」が生産・販売されています。「フ グの子糠漬け」は近年，テレビや雑誌などで広く紹介 されてご存じの方も多いことと思います。この「フグ の子糠漬け」の生産は石川県白山市美川の7戸
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の生産 者によって行われています。
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| 50.
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板垣, 英治
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| 51.
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板垣, 英治
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| 52.
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赤祖父, 一知 ; 板垣, 英治
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| 53.
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板垣, 英治
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| 54.
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板垣, 英治
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| 55.
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| 56.
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| 57.
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Fujii, Chifumi ; Morii, Shingo ; Miyoshi, Toshio ; Iwami, Masafumi ; Itagaki, Eiji ; 板垣, 英治
概要:
金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />The gene encoding 3-ketosteroid-Δ1-dehydrogenase from Rhodococcus rhodochrous
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was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Δ1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Δ1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.
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| 58.
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Fujii, Chifumi ; Morii, Shingo ; Kadode, Michiaki ; Sawamoto, Shizue ; Iwami, Masafumi ; Itagaki, Eiji ; 板垣, 英治
概要:
金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />Tetranitromethane treatment of 3-ketosteroid-Δ1-dehydrogenase of Rhodococcus r
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hodechrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0.014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.
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| 59.
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Yabuuchi, Tsuyoshi ; Suzuki, Kenzi ; Sato, Takao ; Ohnishi, Kuniharu ; Itagaki, Eiji ; Morimoto, Yukio ; 板垣, 英治
概要:
金沢大学自然科学研究科 <br />金沢大学理工研究域自然システム学系<br />Apo-salicylate hydroxylase from Pseudomonas putida S-1 has been crystallized b
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y the dialysis method, using ammonium sulfate as the precipitant. The crystals belong to hexagonal space group P62 or P64 with unit cell dimensions of a = b = 142.8 Å and c = 63.8 Å, and diffract X-rays at higher than 3.5 Å resolution. A heavy-atom derivative has been prepared by soaking a crystal in an ammonium sulfate solution containing p-chloromercuriphenylsulfonate.
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| 60.
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板垣, 英治
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| 61.
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板垣, 英治
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| 62.
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板垣, 英治
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| 63.
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板垣, 英治
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| 64.
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板垣, 英治
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| 65.
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板垣, 英治
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| 66.
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板垣, 英治
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| 67.
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板垣, 英治
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| 68.
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板垣, 英治
概要:
金沢大学名誉教授<br />報告書の一部 : 4章
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| 69.
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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板垣, 英治
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| 77.
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板垣, 英治 ; 寺西, 一栄
概要:
Benkiti Ohno, a technician who made mechanical dolls, wrote about mechanical dolls who had metal-coiled springs in his notebook "Ittousi Kyuroku". In this article, we described the structures and working mechanisms of his dolls. These
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included the 'tea-cup serving doll' and the 'Sanbasou-festival doll.' We discovered how original the specified structures of the machines were, which are different from those ofYorinao Hosokawa's tea-cup serving dolls. The dresses of the Sanbasou-festival doll showed that the upper clothing was made of the linen cloth, Linum usitatissimum. This is a characteristically Japanese cloth called "Sanbon Roh". The "Hakama" skirt was also made of a very beautiful and valuable silk cloth embroidered flowers and grass. Other figures, which were related to artillery and bullets that were described in "Ittousi Kyuroku", were copies of the real life and large size working pieces used at the Kanazawa Clan's Suzumi artillery production factory in 1856. This article describes Benkiti's activities in Kanazawa in the second half of the 19th century.
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| 78.
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板垣, 英治
概要:
The oldest colored picture scroll depiction of the Tatumi irrigation canal was recently discovered. This scroll is estimated to have been made around 1800. The scroll also clearly depicts the Kaga Clan's Tutisimizu gunpowder making factory
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as well as a three-stage stone wall with sluice gates in the Sai River. The stone wall was constructed soon after the Kanazawa earthquake in 1799. The picture scroll shows all the configurations of the Tatumi irrigation canal at the beginning of the 19th century.
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板垣, 英治
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| 80.
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板垣, 英治
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| 81.
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板垣, 英治
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| 82.
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| 83.
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板垣, 英治
概要:
For defense against possible attack by foreign warships, the Kaga clan initiated a massive building program which saw the construction of numerous fortresses along the coast of the Kaga, Noto, and Etuchu areas in 1850, the third year of
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the Kaei era (1848 to 1854). Tamon Kanaya and three other persons, the chief overseers of the program, examined the coast of the Noto peninsula in order to determine the most suitable locations for the construction of fortresses. After eleven days of travel, six places were chosen at an official meeting in Kanazawa. These were the seaside villages of Motoyosi, Ohono, Kurosima, Wajima, Ushitu and Fushiki. In each of these fortresses, four to six arrow firing cannons were arranged and stored in arsenals alongside gunpowder and bullets. After opening the Suzumi molding factory in 1854, the first year of the Ansei era (1854 to 1860), modern cannons were produced, and then arranged in each fortress for the purpose of reinforcing their defensive power.
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| 84.
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板垣, 英治
概要:
In the mid-1800s, the Kaga Clan constructed seventeen fortresses in the Kaga, Noto, and Etuchu areas along the Sea of Japan coast. This study describes the location of each fortress, their size and structural composition, and also the
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kinds of, and the numbers of, weapons arranged within each fortress. In the Kaei period (from 1848 to 1854), old type cannons were arranged within the fortresses. However, in the Ansei period (from 1854 to 1860), these older cannons were replaced by new western type cannons in order to strengthen the fortresses` defensive capabilities against possible attack by foreign ships. This is the first research paper to describe in detail these mid-nineteenth century fortresses and the related weaponry of the Kaga Clan.
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