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II. Effects of pH and Salts
TAIJI IMOTO, YASUTAKA DOI, KATSUYA HAYASHI, MASARU FUNATSU
1969 Volume 65 Issue 5 Pages
667-671
Published: May 25, 1969
Released on J-STAGE: November 18, 2008
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The effects of pH and salts on the activity of lysozyme [EC 3. 2. 1. 17] and the formation of the enzyme-substrate complex were studied spectrophotometrically and by chromatography on a column of an insoluble substrate, carboxymethyl (CM) chitin. The activity of lysozyme was strongly inhibited by neutral salts at high concentrations. In contrast, the formation of the enzyme-substrate complex measured by difference spectrophotometry and chromatography on CM-chitin column increased on increasing the salt concentration. The effect of salts (ions) on the activity depended upon their ionic radii. These effects of salts could not be explained merely by their influence on the ionization of amino acid side chains of the lysozyme molecule. The effects of salts on the complex formation and the activity, therefore, seemed to be mediated by a fine conformational change at the active site, resulting in an unsuitable arrangement of catalytic groups for the catalytic reaction and a strong interaction between the lysozyme molecule and the substrate.
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MITSUO NAKAGAWA, MITSURU UCHIYAMA
1969 Volume 65 Issue 5 Pages
673-677
Published: May 25, 1969
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Desaturation
in vitro of palmitic acid by a postmitochondrial supernatant of rat liver homogenates proceeded about 3 times faster than that of stearic acid. When mitochondria and carnitine were added to the system 15min after the start of incubation, palmitoleic acid produced from palmitic acid decreased more rapidly than oleic acid from stearic acid. However, the rate of oxidation of exogenous palmitoleic acid by isolated mitochondria was about the same with that of exogenous oleic acid. Incorporation of oleic acid into esterified lipids by the postmitochondrial supernatant was about 2 times faster than that of palmitoleic acid, and the release of palmitoleic acid from esterified lipids seemed to be more rapid than that of oleic acid. These findings suggested that the discrepancy between the efficiency of desaturation of the two saturated acids
in vitro and the ratio of the two mono-unsaturated acids found
in vivo was due to the difference in the rates of incorporation of the two unsaturated acids into esterified lipids.
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TAKAYUKI OZAWA
1969 Volume 65 Issue 5 Pages
679-691
Published: May 25, 1969
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1. At the point of re-establishment of controlled respiration which was released by the addition of AMP, a significant amount of modified AMP, “AMP”, was found in the mitochondrial suspension.
2. Distribution of
14C-nucleotides and esterified
32P in and outside of mitochondria proved that there was negligible accumulation of nucleotides by the mitochondria during oxidative phosphorylation.
3. Studies using 5'-adenylate deaminase showed that the conversion of AMP to “AMP” was a respiration-dependent process. The conversion proceeded in the absence of added phosphate, even though its rate was slow.
4. “AMP” was provisionally identified to be acyl-adenylate on thin layer chromatography by comparison of its
Rf value with that of synthetic reference compounds other than both 5'-AMP and 3', 5'-cyclic AMP.
5. Parallel to the accumulation of “AMP” during state 3 respiration, there was accumulation of hydroxamate-reacting material. It was concluded that “AMP” might be a high energy ester derivative of AMP, possibly containing a substrate derived material.
6. The nature of “AMP” was discussed in relation with the transfer of high energy bonds across the mitochondrial membrane.
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MICHIKO HIRAMARU, TSUNEKO UCHIDA, FUJIO EGAMI
1969 Volume 65 Issue 5 Pages
693-700
Published: May 25, 1969
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1. Three RNases were isolated from
Physarum polycephalum and designated RNase P
p-1, RNase P
p-2 and RNase P
p-3.
2. They are thermostable.
3. The approximate molecular weights estimated by get filtration of RNases P
p-1 and P
p-2 are 40, 000 and that of RNase P
p-3 is 10, 000.
4. The optimum pH values of RNases P
p-1, P
p-2 and P
p-3 are pH 6.7, pH 4.5 and pH 5.5, respectively.
5. RNase P
p-1, is a guanyloribonuclease [EC 2. 7. 7. 26].
6. RNase P
p-2 is a nonspecific RNase [EC 2. 7. 7. 17].
7. RNase P
p-3 may be classified as a nonspecific RNase [EC 2. 7. 7. 17], but it attacks purine nucleotide bonds preferentially.
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MICHIKO HIRAMARU, TSUNEKO UCHIDA, FUJIO EGAMI
1969 Volume 65 Issue 5 Pages
701-708
Published: May 25, 1969
Released on J-STAGE: November 18, 2008
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1. Two nucleases hydrolyzing both RNA and denatured DNA (Nucleases P
p-1, and P
p-2) and one ribonuclease (RNase P
p-4) were isolated from
Physarum polycephalum in addition to the three RNases (RNases P
p-1, P
p-2, and P
p-3, ) reported in the preceding paper (
1).
2. They are thermolabile.
3. The pH optima of nucleases P
p-1 and P
p-2 are pH 4.5, and that of RNase P
p-4 is pH 4.0.
4. Nuclease P
p-1 and nuclease P
p-2 hydrolyze denatured DNA as well as RNA, but not native DNA.
5. Nuclease P
p-1 hydrolyzes RNA forming nucleotides with terminal 3'-phosphate.
6. Nuclease P
p-2, hydrolyzes RNA to produce nucleotides with terminal 5'-phosphate. It has no base specificity.
7. RNase P
p-4 hydrolyzes RNA forming nucleotides bearing 5'-phosphate group with purine preference in regards to 3'-terminal nucleotides.
8. Nuclease P
p-2 and RNase P
p-4 are strongly activated by Zn
++
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I. Presence of Membrane Subunits and the Physical Properties
MITSUHIRO YANAGIDA, HARUHIKO NODA
1969 Volume 65 Issue 5 Pages
709-720
Published: May 25, 1969
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The isolation method of cell membrane of vegetative myxamoebae of
Dictyoslelium discoideum is described.
The isolated cell membrane was morphologically pure with the thickness of 70-90Å, and composed of 65-70% of dry weight of protein and 25-30% of lipid.
Isolated cell membrane was dissolved completely into structural units in alkaline pH of above 11 as well as in solutions which contained detergents, urea or acetic acid. The nature of the structural unit was investigated mainly with sedimentation and electron microscopy. Lipid-free protein fraction was also dissolved in 0.1M NaOH.
The structural unit was a lipoprotein particle of which the molecular weight was about 160, 000 and had a sedimentation coefficient of 7S. This particle seemed to be globular with a diameter of 70-100Å. Lipid-free protein fraction showed a single peak in sedimentation and electrophoresis. The sedimentation coefficient was 3.3S and the molecular weight was about 65, 000.
An intermediate filamentous structure, which was a linear aggregate of 7S subunits, appeared in the process of degrading the membrane into particles in an alkaline solution.
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II. Reassociation of Membrane Subunits
MITSUHIRO YANAGIDA, HARUHIKO NODA
1969 Volume 65 Issue 5 Pages
721-739
Published: May 25, 1969
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Reassociation of 7S lipoprotein subunits and 3.3S protein subunits obtained from the cell membrane of vegetative myxamoebae of
Dictyostelium discoideum was investigated with electron microscopy, turbidometry and sedimentation.
7S subunits reassociate into five kinds of aggregates;
i.e., unit membrane structure, thick membrane of about twice as thick as that of the unit membrane, filamentous structure, partial aggregates and random aggregates. The proportion of various aggre-gates depended on the conditions for reassociation. The rate of subunit reassociation, hydrogen ion concentration, temperature, metal ion concentration, the valence of metal ion and the presence of substances such as urea influenced the subunit interaction and the morphology of the product.
Lipid-free protein subunits of the membrane reassociated into membrane structure by going through stages of intermediate aggregates, filaments and then side-by-side aggregation of filaments.
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II. Dependence of the Rates of Formation of Phenol, Phenyl α-Glucoside and Maltotriose on the Substrate Concentration
HOZUMI YOSHIDA, KEITARO HIROMI, SÔZABURO ONO
1969 Volume 65 Issue 5 Pages
741-750
Published: May 25, 1969
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1. The hydrolysis of phenyl α-maltoside (φM) catalyzed by crystalline saccharifying α-amylase [EC 3. 2. 1. 1] of
Bacillus subtilis was studied at various substrate concentrations at 25°C and pH 5.4 in 0.02M acetate buffer by thin layer chromatography, phenol reagent method and modified Somogyi-Nelson's method. The rates of formation of phenol (φ),
vφ, phenyl α-glucoside (φG),
vφG, and maltotriose (MT), VMT, were deter-mined, and their dependencies on the substrate concentration were investigated. The effect of pH on the rates of formation of φ and φG was examined at a fixed substrate concentration.
2. In the parallel reactions forming φ and φG from φM,
vφ follows the Michaelis-Menten equation over the whole range of substrate concentration, s (0.12-120mM). On the other hand, the plot of
vφG
versus substrate concentration shows a sigmoidal curve at lower substrate concentrations (0.12-10mM), and the plot of
s2/vφG
versus s gives a straight line, although the rate obeys the Michaelis-Menten kinetics at higher substrate concentrations (11-120mM). The dependency of
vφG/
vφ on
s shows a saturation curve of the Michaelis-Menten type.
3. The optimum pH for the processes forming phenol and phenyl α-glucoside is between 5.3 and 5.4, and the pH values, which give one half the velocity at the opti-mum pH, are 4.0 and 6.75 for formation of phenol, and 3.7 and 7.05 for formation of phenyl α-glucoside.
4. The dependencies of
vφ and vφG on the substrate concentration can be explained neither by a simple mechanism of a single active center producing both phenol and phenyl α-glucoside nor by a mechanism of two active centers each forming a single set of products, phenol and maltose or phenyl α-glucoside and glucose. Besides an active site (active center), an activator site to which the substrate is bound is considered necessary for the formation of φG, while such an additional site is not necessary for the formation of phenol.
5. The apparent rate of formation of maltotriose, VMT, was found to be propor-tional to the product of
vφG and
vφ.
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SHIGERU NAKAYAMA, SHIZUKO AMAGASE, MOTOKO TAHARA, KEIKO OKUMURA
1969 Volume 65 Issue 5 Pages
751-754
Published: May 25, 1969
Released on J-STAGE: November 18, 2008
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A simple, rapid micro-method is described for identification and differentiation of amylases in a limited quantity of preparation. The method is a modification of the ‘oligosaccharide map’ procedure, involving chromatography of malto-oligosaccharides in one direction on a thin layer of microcrystalline cellulose (Avicel SF), incubation of the oligosaccharides with a small quantity of amylase preparation, separation of the hydrolytic products by chromatography in a second direction and location of the hydro-lytic products.
Partially purified amylase from the root of Japanese radish (
Raphanus sztivus L. var.
aconthiformis Makino) was analyzed by this method. β-Amylase [EC 3. 2. 1. 2] seemed to be the main component, with a glucose-forming amylase or glucoamylase [EC 3. 2. 1. 3] as a minor component.
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ATSUSHI SAKURAI, MIKI GOTO
1969 Volume 65 Issue 5 Pages
755-757
Published: May 25, 1969
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7-Amino-2-(1', 2'-dihydroxyäthyl)-5-hydroxy-3-methylthiothiothieno[3, 2-g]pteridin wurde synthetisiert und die Identität der synthetisierten Substanz mit dem Urothion festgestellt.
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TATSUYA SAMEJIMA, MASAKO KITA
1969 Volume 65 Issue 5 Pages
759-766
Published: May 25, 1969
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Optical rotatory dispersion and circular dichroism of solutions of horse ferri-myo-globin and its derivatives (ferro-, carbonyl-, oxy-, cyano-, azide-, apo- and reconstituted myoglobin) were measured over a wavelength range of 650 to 185mμ. All myoglobins except apo-myoglobin showed distinct positive Cotton effects and circular dichroic bands in the region of Soret absorptions. However, the crossover points of the Cotton effects were essentially identical with those of the positive maxima of the circular dichroic bands and the absorption maxima of the Soret bands. The magnitude of the Soret Cotton effect and circular dichroic band of carbonylmyoglobin was most prominent. In the near-ultraviolet region between 260-280mμ all proteins showed positive circular dichroic bands, which may be due to asymmetry of the aromatic amino acid groups. The intensities of these bands were enhanced giving a blue shift when the myoglobin was complexed with the ligand molecules. In the ultraviolet region intense negative circular dichroic bands near 222mμ and 207mμ were present in accord with the cor-responding Cotton effects having a trough at 233mμ and a peak near 200mμ. Helix contents, based on the 233mμ trough and the band intensity at 222mμ, were estimated for all the proteins. Within experimental error, horse ferrimyoglobin and its deriva-tives have almost the same helix content of about 70%, whereas apomyoglobin has less helix content, approximately 50%. This indicates that the addition of ligands to the heme group causes no drastic conformational change in the protein moiety, while the removal of heme has an effect on the diminution of the helix content. Reconstituted ferrimyoglobin regained essentially the same conformation as the native protein.
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FUMIO SAWADA
1969 Volume 65 Issue 5 Pages
767-776
Published: May 25, 1969
Released on J-STAGE: November 18, 2008
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Bovine pancreatic ribonuclease [EC 2. 7. 7. 16] (RNase) was inactivated by irradia-tion with ultraviolet light of 330-365mμ in the presence of a substrate analogue, 4-thiouridine-2'(3')-phosphate (4-thiouridylic acid), while a little or no inactivation was found in absence of the nucleotide and in the presence of a nucleoside, 4-thiouridine. The inactivation was inhibited by an excess amount of uridylic acid. The inactivation was notable at pH 5.6 and was feeble at pH's 3.6 and 7.6, and the degree of the inactivation was in paralell with the degree of the interaction between the enzyme and the nucleotide. It was deduced from these results that the complexing between RNase and 4-thiouridylic acid is an essential step for the photosensitized inactivation of the enzyme. Oxygen was required for the inactivation.
A photoproduct of RNase irradiated in the presence of 4-thiouridylic acid was isolated. Chemical analyses of this product revealed that histidyl, phenylalanyl and valyl residues at the active site were intact after the irradiation, but one tyrosyl residue was modified. The spectrophotometric titration of the irradiated protein suggested that the native conformation was retained.
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I. Covalent and Non-Covalent Binding of Fluorescein-Isothiocyanate and Fluorescein to Proteins
HIROSHI MAEDA, NAKAO ISHIDA, HIROSHI KAWAUCHI, KATURA TUZIMURA
1969 Volume 65 Issue 5 Pages
777-783
Published: May 25, 1969
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1. The reaction of fluorescein-isothiocyanate (FITC) with proteins was studied under various conditions.
2. It was found that the α-amino groups of the N-terminal amino acids were the prime target of the reaction (at pH<9.5) of FITC with neocarzinostatin and insulin although the ε-amino groups became reactive at higher pH.
3. Egg white lysozyme [EC 3. 2. 1. 17] and bovine serum albumin were found to bind with FITC even at low pH, while ovomucoid and neocarzinostatin did not bind below pH 7.
4. Trifluoroacetic acid treatment of fluorescein-thiocarbamylated insulin (FTC-insulin) resulted in the release of terminal FTH-amino acids (Phe and Gly) only. These FTH-amino acids were identified for the first time.
5. Non-covalent binding of this fluorochrome to proteins was studied with fluoro-scein. The results indicated non-covalent dye-binding of the fluorescein with albumin and Iysozyme [EC 3. 2. 1. 17].
6. Above results showed that FITC is a useful reagent but that non-covalent dye-binding should be born in mind when used with some proteins.
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KEIJI NAKAMURA, AYAKO MATSUSHIMA
1969 Volume 65 Issue 5 Pages
785-792
Published: May 25, 1969
Released on J-STAGE: November 18, 2008
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The states of amino acid residues in porcine and bovine trypsins were compared by spectrophotometric titration of tyrosine residues and by using several reagents: β-naphthoquinone-4, 6-disulfonic acid (NQDS) and monochlorotrifluoroquinone (CFQ) for amino groups, H
2O
2-dioxane for tryptophan, glyoxal for arginine, diazo-l-H-tetra-zole (DHT) for histidine and tyrosine, and cyanuric fluoride for tyrosine. Almost all the amino groups in these trypsins [EC 3. 4. 4. 4] were modified uniformly with NQDS and 5-6 amino groups of bovine trypsin and 4-5 amino groups of porcine trypsin were modified with CFQ. Glyoxal modified two of the total four arginine residues in porcine trypsin and one of the two arginine residues in bovine trypsin. Three of the four histidine residues in porcine trypsin and two of the three histidine residues in bovine trypsin were bisazotized with DHT. Three of the four tryptophan residues in bovine trypsin and all the four tryptophan residues in porcine trypsin were oxidized with H
2O
2-dioxane. Of the eight tyrosine residues in porcine trypsin three residues had values of p
K=10.2,
m (the order of the sigmoid ionization curve)=1.0, three values of p
K=10.5,
m=1.0 and two of p
K=12.2,
m=3.0. The three residues with a p
K value of 10.2 were further classified in terms of their reactivity with CyF into two more reactive and one less reactive residues. The two residues with the highest p
K value of 12.2 were not bisazotized while the other six residues were completely bisazotized. These data were compared with the previous observations that the ten tyrosine residues in bovine trypsin were of two types: six which ionized rapidly and reacted with CyF and four which ionized slowly and did not react with CyF. The difference in stability between bovine and porcine trypsins is discussed on the basis of these data, and the participation of a serine residue in the activity of porcine trypsin is postulated.
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HARUKI YAMAGUCHI, TOKUJI IKENAKA, YOSHIO MATSUSHIMA
1969 Volume 65 Issue 5 Pages
793-798
Published: May 25, 1969
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The homogeneity of a glycopeptide obtained from Taka-amylase A [EC 5. 2. 1. 1], was reinvestigated by column chromatography on Dowcx 50×2 and high voltage paper electrophoresis. About 90 per cent of the glycopeptide was found to be homogeneous and to consist of one mole each of aspartic acid, ser ne and ammonia, 2 moles of N-acetylglucosamine and 6 moles of mannose.
Sequential periodate oxidation of the purified homogeneous glycopcptide indicated that its carbohydrate sequence is Man-Man-G1eNAc-G1cNAc-NH.
_??_
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YASUO KAGAWA, TOSHIKO TAKAOKA, HAJIM KATSUTA
1969 Volume 65 Issue 5 Pages
799-808
Published: May 25, 1969
Released on J-STAGE: November 18, 2008
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1. Mitochondrial lipids of mouse fibroblasts of the substrain Lp3, one of the sub-lines of L-929 which had been cultured in the lipid-free and protein-free synthetic medium DM-120 for 9 years, were analyzed.
2. Essential fatty acids were not detected but monounsaturated fatty acids were found to have increased in the mitochondria. The presence of coenzyme Q, inositol, and vaccenic acid was confirmed.
3. By the addition of linoleic acid into DM-120, considerable changes were induced in the fatty acid composition of each phospholipid fraction, whereas the proportional ratios among phospholipids were little influenced.
4. Mitochondrial functions, such as oxidative phosphorylation
in situ, ATPase [EC 3. 6. 1. 3] activity and electron transport by cytochromes, were kept unchanged in the lipid-free synthetic medium. The oxidative phosphorylation in the isolated mitochon-dria, however, was found unstable, suggesting that it awaits further investigation.
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TADASHI INAGAMI, ABRAHAM PATCHORNIK, SHELDON S. YORK
1969 Volume 65 Issue 5 Pages
809-819
Published: May 25, 1969
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Derivatives of N-acetyl-L-tyrosine anilide with substituents,
p-CH
3,
p-CH
3O,
m-CH
3O,
p-Cl and
m-G, on the aniline ring, were synthesized as substrates of chymotrypsin [EC 3. 4. 4. 5]. It was confirmed that the acylation of the enzyme was the rate limiting step for these anilide substrates. It was, therefore, possible to study the electronic effect of the substituents on the catalysis of the acylation. The ρ-σ
- plot indicated participation of an acidic group of chymotrypsin in the proton transfer to the substrate molecule. The deuterium isotope effect was observed to depend on the σ- value of the substituent. Such dependence was found to be compatible with a mechanism in which the proton transfer occurs rapidly before the rate limiting step, but not with a mechanism in which the proton transfer occurs simultaneously with the acylation. The pH profile of the catalysis was studied. The temperature dependence of the rate constant of catalysis revealed that the heat of activation changes with the σ
- value more steeply than the free energy of activation. This steeper change is compensated for by a change in the entropy of activation in the reverse direction.
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MASAHIRO IWAMOTO, YASHUSHI ABIKO
1969 Volume 65 Issue 5 Pages
821-823
Published: May 25, 1969
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KYOKO TORII, YASUYUKI OGURA
1969 Volume 65 Issue 5 Pages
825-827
Published: May 25, 1969
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YAKAKO TOMITA, TATUO OZAWA, ISAO TOMITA
1969 Volume 65 Issue 5 Pages
829-830
Published: May 25, 1969
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KOICHI SUZUKI, TOSHIO ANDO
1969 Volume 65 Issue 5 Pages
831-834
Published: May 25, 1969
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AKIRA KOTAKI, MAKOTO NAOI, MINORU HARADA, KUNIO YACI
1969 Volume 65 Issue 5 Pages
835-837
Published: May 25, 1969
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KEN KADOTA, TOKU KANASEKI
1969 Volume 65 Issue 5 Pages
839-842
Published: May 25, 1969
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HAJIME HIRATA, SAKUZO FUKUI, SHINJI ISHIKAWA
1969 Volume 65 Issue 5 Pages
843-847
Published: May 25, 1969
Released on J-STAGE: November 18, 2008
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