The Journal of Biochemistry

Mechanism of Cell Division

I. Changes in Pyridine Nucleotide and DHNH-Cytochrome c Reductase in Tetrahymena geleii W. during the Course of Synchronous Culture

TOMOYA KAMIYA, TAIJO TAKAHASHI

1961Volume 50Issue 4 Pages 277-283
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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Changes in oxidized pyridine nucleotide, nicotinamide sensitivity of cell division, and DPNH-cytochrome c reductase activity were studied in synchronously dividing cultures of Tetrahymena geleii W using enzymatic and chemical methods. In addition, the effects of temperature, nicotinamide and other pyridine derivatives were observed on these parameters.
The level of ox. PN increased after the heat treatment and decreased after cell division. This increase returned to the original value after exposure to 0.05M NA. This concentration of NA inhibited completely the synchronous division of Tetrahymena. NA sensitivity curve showed its maximum within the first 30 minutes after the heat treatment. DPNH-cytochrome reductase activity was irreversibly inhibited by the heat treatment and competitively inhibited by NA in vitro.
The results of these and other experiments indicate that there may he a direct relationship among various phenomena, such as increase of ox. PN, change in NA sensi-tivity of cell division, DCR activity and cell division in synchronized cells of Tetrahymena.
We wish to thank Miss S. Kikuchi for her tech-nical assistance in a certain phase of the present work and Mr. Sonoda for his help in the enzymatic determinations.
This research was supported by a Grant-in-Aid of Scientific Research from the Ministry of Education of Japan.

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Studies on the Gamma Ray Irraditation on the Protein

II. The ESA Absorption of Proteins, Amino Acids and Peptides

YASUO IMAI

1961Volume 50Issue 4 Pages 284-292
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1. Investigations on the ESR absorption spectra of γ-ray irradiated bovine and human serum albumin, fibrin and silk fibroin were made.
2. The free radicals in the irradiated proteins are associated with their cystine or cysteine content, the higher their content of these sulphur-containing amino acids, the more resemble their ESR patterns that of cystine or cysteine.
3. Silk fibre provided a doublet quite similar to that of glycyl-glycyl-glycine. Such an orientation-dependent pattern would be explained as an unpaired electron localized on the oxygen atom hydrogen-bonded to NH group of the adjacent polypeptide chain.
4. When irradiation was made in the presence of oxygen, the ESR signals of proteins are remarkably different from those irradiated in vacuo, the former being attributable to peroxide radicals.
5. The proteins irradiated in the presence of water could barely provide a noticeable ESR signal.
6. When irradiated albumin, or cysteine were dissolved into the solution of native albumin, the titrable -SH group decreased, probably due to reaction of sulphhydryl radicals with the -SH group.
From these results some considerations were made on the free radical formations in the irradiated protein.
The author wishes to thank to Prof. A. Inouye for his valuable discussion, and Dr. H. Honda. for his kind permission to use the ESR spectrometer (V-4500) and Mr. S. Toyoda and Dr. A. Hirai for their valuable assistance to performm this experiment.

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The Spectrophotometric Determination as Trinitrophenyl Derivatives of Amino Acids and Peptides Resolved on Paper by Electrophoresis and Chromatography

TOMOTAKA SHINODA, KAZUO SATAKE

1961Volume 50Issue 4 Pages 293-298
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1. Amino acid and peptide resolved on paper by electrophoresis and chromatography (0.01 to 0.03μmoles per cm².) could be detected as yellow spots by TNP-ation with TNBS.
2. Each spot so colored with TNBS (0.01 to 0.5μmoles) could be determined within an error of ±3 per cent by dipping the cut spot into TNBS solution bufferized at pH 8 to complete the TNP-ation, then acidification, and spectrophotometry of the resulting supernatant.
3. Using this TNBS technique, amino acid compositions of bovine insulin, globins α and β were determined.
4. This method could also be used with success for the quantitative determination of peptides, as molar extinction coefficients of their TNP-derivatives were of similar magnitude (1.05×10⁴).
The authors are indebted to Miss M. Yoshida for her technical assistance.

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Biochemical Studies on Sulfate-Reducing Bacteria

X. Adenosine-5'-Phosphosulfate Reductase

MAKOTO ISHIMOTO, DAISABURO FUJIMOTO

1961Volume 50Issue 4 Pages 299-304
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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Adenosine-5'-phosphosulfate reductase was purified by 40 times from the extract of Desulfovibrio desulfricans. This preparation catalyzed the reduction of APS to sulfite using methylviologen as an electron carrier. It can also reduce guanosine-5'-phosphosulfate, cytidine-5'-phosphosulfate and uridine-5'-phos-phosulfate. AMP inhibited the APS reduction but SH reagent such as arsenite or p-chloro-mercuribenzoate or metal chelating agents had no effect. Reversibility of APS reductase was also confirmed.
The authors thank Prof. F. Egami for his valu-able advice and Dr. Shimojim a for preparation of S35-sulfite. The expences of this work were aided partly by the Scientific Research Funds from the Ministry of Education.

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Studies on Laccases of Lacquer Trees

II. Copper Exchange Studies of Rhus vernicifera Laccase Using Radioactive Copper

TSUNEO OMURA

1961Volume 50Issue 4 Pages 305-311
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1. The exchange reaction of laccase copper with ionic copper under varying conditions was studied using radioactive copper as the tracer.
2. The copper atom of oxidized laccase did not exchange with cupric ion in the medium. The copper-protein linkage of oxidized laccase must be very stable.
3. The copper atom of reduced laccase exchanged with cuprous ion rapidly, but it can not be replaced by the cupric ion in the medium.
4. The copper atom of laccase exchanged with ionic copper while the enzyme was catalyzing the oxidation of hydroquinone, and the extent of exchange increased with the amounts of hydroquinone oxidized, but the velocity of exchange was very slow compared with the rapid exchange reaction between reduced laccase and cuprous ion.
The author wishes to express his hearty gratitude to the late Prof. A. Nishimura for his interest and encouragement during this investigation, and to Prof. Y. Ogura, University of Tokyo, for his valua-ble advices. Thanks are also due to Mr. K. Kita-mura for his co-operation in this work, and to Saito Co. Ltd. for the kind supply of lacquer latex.

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Chemical Activities of Azotobacter agile Adsorbed on a Resin

TSUTOMU HATTORI, CHOSEKI FURUSAKA

1961Volume 50Issue 4 Pages 312-315
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1) Cells of A. agil were adsorbed on a resin, Dowex-1 of the chloride form (adsorb-ed cells). As in the case of E. coll, adsorbed cells of A. agile oxidized glucose or succinate more slowly than the cells suspended freely in a solution (‘A effect’). In the case of glucose oxidations activity-pH curve with adsorbed cells was similar to that with free cells, and pH value for a maximum of the former was higher by one unit than that of the latter (‘B effect’).
2) The lag time, observed in induced oxidation of succinate with free, glucose-grown cells, disappeared when the cells were adsorbed on the resin (‘C effect’). In succinate oxidation with desorbed, glucosegrown cells the lag time also disappeared. A nucleotides fraction obtained from hot-water extract of glucose-grown cells showed an effect on the lag time of desorbed cells to prolong conspiciously, but the same extract obtained from succinate-grown cells did not exhibit such an effect. Possible mechanism involved in the ‘C effect’ was disccussed.
Authors thanks are due to Prof. T. Uemura and Dr. T. Suto for their valuable suggestions, and to Miss T. Konno for her skilful technical assist-ance.

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Microbiological and Enzymatic Studies on the Isoleucine Isomers

I. Utilization of the Stereoisomers of Isoleucine by Lactic Acid Bacteria

SHIGEKI YAMADA

1961Volume 50Issue 4 Pages 316-324
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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Microbiological studies on the utilization of isoleucine isomers by lactic acid bacteria were carried out and the results obtained are summarized as follows:
1. Str. faecalis R and Leuc. mesenteroides P-60 are unable to utilize L-alloisoleucine, D-isoleucine and D-alloisoleucine both in the presence or absence of vitamin Bg compounds.
2. Among the three organisms studied, only Lact. arabinosus 17-5 is capable of utilizing all four isoleucine isomers in the presence of vitamin B6 compounds, but not in the absence of this vitamin.
3. By lowering the initial pH of the medium, the growth of Lact. arabinosus 17-5 to isoleucine isomers is accelerated.
4. Excessive amounts of leucine inhibit the utilization of isoleucine isomers by Lact. arabinosus 17-5.
5. It is considered taat discrepancy between the results obtained by the author and previous reports concerning the utilization of D-isoleucine and D-alloisoleucine is due to the difference of conditions of cultures.
6. Pyridoxamine is most effective for the utilization of the stereoisomers of isoleucine by Lact. arabinosus 17-5.
7. The strain cultured successively in the medium containing one of the other isomers in place of L-isoleucine is able to utilize isoleucine isomers more rapidly.
The author wishes to thank Prof. S. Akabori and Assist. Prof. Y. Izumi, Osaka University, for their interest and helpful advice throughout this work. The author is also greatly indebted to Mr. T. Hira-bayashi, Representative Director of the Tanabe Seiyaku Co. Ltd., Dr. M. Fujisawa, Research Executive, Dr. N. Sugimoto, Director of Osaka Research Laboratory, and Dr. I. Chibata, Chief, and the staffs of the Biochemical Section of the Laboratory for continued encouragement and assistance.

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Microbiological and Enzymatic Studies on the Isoleucine Isomers

II. Microbiological Determination of L-Isoleucine and Total Isoleucine Isomers

SHIGEKI YAMADA, HIROSHI ITO, SETSUKO FURUTSU, ICHIRO CHIBATA

1961Volume 50Issue 4 Pages 325-329
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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The microbiological determination of L-isoleucine and the total amount of isoleucine isomers by empoying the adapted strain of Lact. arabinosus 17-5 is described.
This assay method is found to be very convenient and satisfactory for the determination of total isoleucine isomers in amino acids mixtures and this method also can be applied to the assay of the proportion contaminated with allo-form in synthetic isoleucine preparations.
The authors wish to thank Prof. S. Akabori and Assist. Prof. Y. Izumi, Osaka University, for their interest and helpful advice throughout this work. The authors are also greatly indebted to Mr. T. Hira-bayashi, Representative Director of the Tanabe Seiyaku Co. Ltd., Dr. M. Fujisawa, Research Executive and Dr. N. Sugimoto. Director of Osaka Research Laboratory, for continued encouragement and assistance.

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Studies of H β-Proteinase of Habu Snake Venom, with Special Reference to Substrate Specificity and Inhibitory Effect of Serum

HIROO MAENO, SUSUMU MITSUHASHI

1961Volume 50Issue 4 Pages 330-336
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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Normal sera obtained from horse and rabbit show a strong inhibitory effect on S proteinase and especially on trypsin, but their effect on Hβ-proteinase from Habu venom iss far weaker. Of the proteins tested, Hβ-proteinase is able to hydrolyze casein most rapidly, but fails to hydrolyze all synthetic substrates investigated.
N-terminal amino acids resulting from the hydrolysis of whale insulin by Hβ-pro teinase are glutamic acid and valine in phenylalanyl chain, and glutamic acid in glycyl chain, respectively.
The fact that Hβ-proteinase can cause serious pathological lesions in spite of its high substrate specificity may be explained partly by the less inhibitory effect of sera on Hβ-proteinase, which inhibit strongly the activity of proteinases such as trypsin and S-proteinase.
We are greatly indebted to Prof. R. Sato, of the Institute for Protein Research, Osaka University, for his invaluable advice and discussion.

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The Presence of Adenylyl 2, 3-Diphosphoglyceric Acid in Pig Blood

TAKASHI HASHIMOTO

1961Volume 50Issue 4 Pages 337-344
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1. A new adenine nucleotide was isolated from pig blood.
2. Spectrophotometric, chromatographic, chemical and enzymatic studies indicate that it is adenylyl 2, 3-diphosphoglyceric acid.
The auther wishes to thank Prof. H. Yoshika-wa for his kind helpfulness and encouragement and to Dr. M. Nakao, Dr. M. Tatiban a and Dr. Y. Ishii for their helpfulness and discussions through-out the period of this work. The auther also wishes to thank the member of the Department of Biochemist-ry, Faculty of Medicine, Gunma University, for their kind helpfulness for the preparation of some portion of the nucleotide, to Dr. H. Chiba for a kind supply of crystalline phosphoglyceric acid mutase and Dr, T. Suzuki for his snake venorn giver.

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Studies on Hemoglobin

VI. Amino Acid Compositions of the Fractionated Bovine Globin α and β

SIGERU SASAKAWA

1961Volume 50Issue 4 Pages 345-351
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1. The amino acid compositions of bovine globin α and β were determined by two different methods: (a) dinitrophenylation of amino acids, and resolution and spectrophotometry of these yellow spots; (b) direct resolution of amino acids, and assay as their trinitrophenyl derivatives.
2. Bovine globin α and β had the following composition:
α-Gly 10, Ala 18, Val 10, Leu 16, Ileu 0, Pro 5, Phe 8, Tyr 3, Try 1, Ser 9, Thr 7, CysH 1, Met 1, Arg 3, His 7, Lys 10, Asp 11, Glu 7, Total 127, molecular weight 13, 700.
β-Gly 12, Ala 19, Val 16, Leu 19, Ileu 0, Pro 7, Phe 8, Tyr 2, Try 2, Ser 7, Thr 8, CysH 1, Met 2, Arg 5. His 7, Lys 11, Asp 15, Glu 10 Total 150, molecular weight 16, 400.
3. By comparing the N-terminals liberated from intact and from completely acetylated bovine globin, the arginyl and lysyl residues were presumed to present as the following sequences.
α-Arg-Val, -Vat, -Phe. Lys-Gly (3), -Ser, -Thr, -Ala (2), -Val, -Leu, His-Lys-OH.α β-Arg-Glu, -Ala, -Leu, -Phe. Lys-Gly (3), -Ala (3), -Val (3), -Leu (2).
The author wishes to express her gratitude to Prof. K. Satake for his interest and discussion, and also to Mr. Y. Kimura, Miss M. Yoshida and Miss Y. Nagatsuka for their assistance in the experiments.

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Stero-Bile Acids and Bile Sterols

XL. Biosynthesis of Pentahydroxycoprostane

TARO KAZUNO, KYUICHIRO OKUDA

1961Volume 50Issue 4 Pages 352-354
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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Cholesterol [4-C¹⁴] was injected intraperitoneally into 5 toads and several radioactive bile sterols were isolated from their bile, one of them being identified withpentahydroxycoprostane.

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Studies on the Metabolism of Rat Ascites Tumor with Nitrogen Mustard Sensitive and Resistant Strains

V. P³²-Incorporation into Ribonucleic Acid in Vitro

YOSHIAKI MIURA, SISTER RAPHAELLE, HISASHI KATAYAMA

1961Volume 50Issue 4 Pages 355-361
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1. The composition of the incubation medium for the P³²-incorporation into ribo-nucleotides of rat ascites tumor was studied. The relative specific activity increased about ten times when sera or ascites fluid was added to the incubation medium. The addition of adenine derivatives apparantly increased the incorporation but evidence was lacking as this was merely based on the increasing exchange reaction.
2. The addition of nitrogen mustard to the incubation medium inhibited the P³²-incorporation into four ribonucleotides. The change of nucleotides ratio in susceptible cells was obserbed. The ratio of purines to pyrimidines or that of (G+C):(A+U) was modified by the addition of nitrogen mustard in the incubation medium of the sensitive strain.
We are indepted to Dr. T. Yoshida and his staff in the Sasaki Institute for the tumor supply. This investigation has been supported by grants from the ministry of Education for Cancer Chemo-therapy investigation group.

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Studies on the Metabolism of Rat Ascites Tumor with Nitrogen Mustard Sensitive and Resistant Strains

VI. Inhibition of the in Vitro Incorporation of C¹⁴-Orotic Acid into Nucleic Acids by Nitrogen Mustard

YOSHIAKI MIURA, AKIKO MORIYAMA

1961Volume 50Issue 4 Pages 362-366
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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1. The addition of 10^-5M of nitrogen mustard to the incubation media of rat ascites hepatoma cells or homogenates was accompanied by an increase in the uptake of C¹⁴-orotic acid into RNA and DNA. But in the incubation of the isolated nuclei, the incorporation was inhibited by the same concentration of nitrogen mustard.
2. Increase in the concentration of nitrogen mustard (10^-4M) in the incubation media led to an inhibition of the incorporation. The extent of the inhibition was much less in the case of the nitrogen mustard resistant cells (AH 7974) than in sensitive cells (AH 130). However, this difference was not observed when the cells were once homogenated.
3. The resistant cells apparantly have some barrier system on the surface of the cells for the penetration of nitrogen mustard into the inside of the cells. However direct evidence was lacking for the existence of this barrier system at the present stage of investigation.
It is a pleasure to thank Prof. T. Yoshid a and his staff of the Sasaki Institute for their valuable advice. It is also a pleasure to acknowledge grants from the Ministry of Education.

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Inorganic Polyphosphate in Spinach Leaves

SHIGETOH MIYACHI

1961Volume 50Issue 4 Pages 367-371
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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Labile phosphorous compounds were separated from the acid-insoluble fraction of spinach leaves. The nucleic acid fractions obtained from the leaves by the method of Ogur and Rosen also contain a labile phosphorous compound(s) which can be separated from the nucleic acids by Ba-precipitation. The labile compounds thus separated gives a positive metachromatic reaction with toluidine blue, and shows no mobility on the paperchromatogram, indicating that the compound(s) is a long-chained polyphosphates.
The author wishes to thank Prof. H. Tamiya and Prof. A. Takamiya for their guidance and encouragement in this work.

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Preparation of p-Nitrophenyl S³⁵-Sulfate

NORIKO TAKAHASHI, FUJIO EGAMI

1961Volume 50Issue 4 Pages 372-373
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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Ascorbic Acid Synthesis with the Reconstructed Enzyme System

KAZUO YAMADA, KANTARO SUZUKI, YOSHITAKE MANO, NORIO SHIMAZONO

1961Volume 50Issue 4 Pages 374-375
Published: October 25, 1961
Released on J-STAGE: November 18, 2008

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