The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
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Volume 64, Issue 5
Displaying 1-22 of 22 articles from this issue
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  • Stepwise Solubilization of Chloroplast Lamellae by a Nonionic Detergent Nonidet P-40
    ISAO SHIBUYA, HIROKO HONDA, BUNJI MARUO
    1968 Volume 64 Issue 5 Pages 571-576
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    Chloroplast lamella preparations from spinach leaves were solubilized by a nonionic detergent, Nonidet P-40. Examination of turbidity, chlorophyll absorption spectra and the Hill reaction activity (dichlorophenol-indophenol photoreduction) revealed that the process of solubilization could be divided into four distinctive phases : Step I (at N/C ratios -Nonidet-chlorophyll ratio on molar basis- less than 4), II (N/C from 4 to 11), III (N/C from 11 to 60) and IV (N/C beyond 60). The turbidily decreased at Steps II and III with a plateau at N/C 11; the Hill reaction activity was lost at Step II, while the physical state of chlorophyll molecules as judged by the shift of absorption maximum changed in Steps III and IV. Circumstantial evidence indicated the separation of lipids from proteins mainly at Step IV. The preparation, obtained in a similar way but from chloroplasts washed with water and 1mM EDTA and purified by freezing, showed no Hill reaction and had only an insignificant amount of the components responsible for the turbidity changes in Step II.
    The results strongly suggest the lipoprotein subunit nature of the lamella membrane in which at least two types, intra- and intersubunit, hydrophobic association are present. The usefulness of the stepwise detergent treatment technique in membrane biochemistry is suggested.
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  • Oxidation-Reduction Potential-Dependent Adenosine Triphosphatase Activity of Chromatophores from Rhodospirillum rubrum
    YOSIFUMI HORIUTI, KATSUZO NISHIKAWA, TAKEKAZU HORIO
    1968 Volume 64 Issue 5 Pages 577-587
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    1. Chromatophores from Rhodospirillum rubrum hydrolyze ATP into ADP and P1 either in the light or in darkness; this activity is called ATPase activity.
    2. The ATPase activity in some, though not all, cases is inhibited by light. The light appears to be a competitive inhibitor against ATP. The light inhibition is neutralized by adding antimycin A; the maximum extent of neutralization is approximately 70%.
    3. In the presence of phenazine methosulfate (PMS), the ATPase activity is stimulated in the light, but hardly affected in darkness. The light appears to be a non-competitive “stimulant” against ATP.
    4. Oligomycin inhibits the ATPase activities in the light and in darkness; both activities are approximately 90% inhibited by 3.3 μg/ml of the inhibitor, which can inhibit the photosynthetic ATP formation completely.
    5. The ATPase activities in the light and in darkness are accelerated with increasing concentrations of 2, 6-dichlorophenol indophenol in the oxidized form (DCPI) up to 6.7×10-5M and retarded at higher concentrations. At 6.7×10-5M DCPI, both activities are almost the same in rate. The activities are significantly stimulated by 2×10-3M 2, 4-dinitrophenol (DNP) in the range of DCPI concentration lower than 6.7×10-5M but not above this concentration. In the presence of 6.7×10-4M DCPI, the ATPase activities in the light and in darkness are accelerated with increasing concentrations of ascorbate up to 5.0×10-4M and retarded at higher concentrations. The Eh value of the reaction mixture containing 6.7×10-4M DCPI and 5.0×10-4M ascorbate is approximately +0.15 volt.
    6. From these findings, it is concluded that the ATPase activity by chromatophores is to a great part exhibited by one of the coupling systems leading to ATP formation, and brought about depending upon the Eh value of the reaction mixture.
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  • Formation of the Crystalline Molecular Complex of Riboflavin and Adenine
    KIHACHIRO UEHARA, TADASHI MIZOGUCHI, SABURO HOSOMI, TAKAJI FUJIWARA, K ...
    1968 Volume 64 Issue 5 Pages 589-593
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    The riboflavin-adenine complex was obtained from dimethyl sulfoxide as red-yellowish plate-like crystals. It was confirmed by spectroscopic, analytical, chromatographic and X-ray methods that the complex crystals contained strictly equimolar amounts of the two components.
    The structure of this complex has been studied by X-ray diffraction analysis. Some physical properties of this complex are presented.
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  • Substrate Specificity of Ribonuclease from Aspergillus saitoi
    MASAOKI IMAZAWA, MASAGHIKA IRIE, TYUNOSIN UKITA
    1968 Volume 64 Issue 5 Pages 595-602
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    1. The substrate specificity of Ribonuclease M [EC 2. 7. 7. 17, Ribonucleate nucleotido-2'-transferase (cyclizing)], which was obtained from Aspergillus saitoi, was studied in detail using several kinds of RNA's and sixteen diribonucleoside monophosphates (XpY's, where X or Y indicates one of the four main ribonucleoside residues), as the substrates.
    2. The Km value of this enzyme for the XpY's differed depending on the nucleoside residues, X or Y. With different nucleoside residues the order of the Km values was: uridine>guanosine>cytidine>adenosine. On the other hand, the maximum velocity (Vmax) for XpY was lowest when either of X or Y was adenosine.
    3. It was found that, when mixtures of several XpY's were used as substrates of this enzyme, the XpY's giving the smallest Km value with the enzyme were split firstest.
    4. A spectrophotometric procedure was developed to follow the enzymatic transesterification of the substrates, XpY's, with purine nucleoside as X.
    5. Similar kinetic studies were made on ribonuclease T2 [EC 2. 7. 7. 17] from Aspergillus orizae.
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  • Oligonucleotide Studies
    VI. Large Scale Fractionation of Di- and Trinucleotides Obtained from Pancreatic Ribonuclease Digests
    SHOHEI AOYAGI, YASUO INOUE
    1968 Volume 64 Issue 5 Pages 603-611
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    Four dinucleotides and eight trinucleotides were obtained on a preparative scale from the pancreatic RNase [EC 2. 7. 7. 16] digestion of commercially available yeast RNA and its subsequent chromatographic separation with DEAE-Sephadex A-25 followed by AG 1-×2 (or Dowex 1-×2). The AG 1-×2 (or Dowex 1-×2) column employed in the separation of the di- and trinucleotide fractions according to base sequence showed satisfactory results at pH 2. 2. Since a pair of the sequence isomers ApGpCp and GpApCp were eluted in a single peak, these isomers were separated on a DEAE-cellulose column (formate form). Present chromatographic procedures are suited to the large scale preparation of pure di- and trinucleotides which is a prerequisite to further study of oligonucleotides. Optical rotatory dispersion (ORD) and circular dichroic spectra of ApGpCp and GpApCp*** were measured at pH 7 to show their dependence on the base sequence. The results of fractionation of the tetranucleotides obtained from digestion of RNA with RNase T1, followed by pancreatic RNase digestion, are also included in the Appendix. The spectrophotometric constants were determined for these tetranucleotides; ApApApCp, ApApApGp and ApAp ApUp.
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  • Structure of Lysozyme
    XIV. Acid-Base Titration of Lysozyme
    RYOHEI SAKAKIBARA, Kozo HAMAGUCHI
    1968 Volume 64 Issue 5 Pages 613-618
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    Hydrogen-ion titration of hen egg-white lysozyme [EC 3. 2. 1. 17] was performed in 0.2M KCl and in a mixed solvent of 5M guanidine hydrochloride and 1.2M urea, at 25°C. The number of carboxyl groups titratable was found to be eight in 0.2M KCl and eleven in the denaturing solvent. A theoretical titration curve which satisfactorily fits the experimental data of the native lysozyme, was obtained by assuming that, of the eight carboxyl groups, six have the pKint, values of 4.3 and the remaining two have the corresponding values of 3. 5 and 6. 0, respectively, and that the intrinsic pK's of α-amino and histidyl groups are normal. The assumption of the presence of the carboxyl groups with the pKint values of 3. 5 and 6. 0 is based on other available evidence. It was found that the ionization of the lysyl groups was normal, whereas the ionization behavior of the tyrosyl groups was complicated.
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  • Kinetic Studies on Taka-amylase A-Catalyzed Reaction by Continuous Spectrophotometric Method
    Hydrolysis of p-Nitrophenyl α-Maltoside and its Inhibition by Phenyl α-Maltoside and Anomers of Some Mono- and Disaccharides
    NOBUYUKI SUETSUGU, KEITARO HIROMI, MASANOSUKE TAKAGI, SÔZABURO O ...
    1968 Volume 64 Issue 5 Pages 619-624
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    Taka-amylase A [EC 3. 2. 1. 1]-catalyzed hydrolysis of p-nitrophenyl α-maltoside was studied at pH 5. 3 and 25°C by a continuous spectro-photometric method.
    The inhibition of this reaction by anomers of maltose, glucose, mannose and xylose was studied to elucidate the effect of the structural change at C1 position of the saccharides upon the affinity to the enzyme. It was found that the binding affinity of α-maltose was larger than that of β-maltose, whereas the binding affinity of the monosaccharides was larger in β-form than in α-form.
    Phenyl α-maltoside was found to act as a competitive inhibitor to the hydrolysis of p-nitrophenyl α-maltoside, with an inhibitor constant equal to the Michaelis constant for the hydrolysis of phenyl α-maltoside.
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  • Glycolipids from Rat Kidney
    JUN'ICHI KAWANAMI
    1968 Volume 64 Issue 5 Pages 625-633
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    1. Several glycolipids were isolated from rat kidney and characterized.
    2. Minced kidney was dehydrated with acetone and the residue was extracted with chloroform-methanol. The extracts were treated with mild alkali and after removal of the released fatty acids with acetone, the alkali-stable lipids were fractionated on silicic acid, Florisil and DEAE-cellulose columns.
    3. The main glycolipids were identified as ceramide monohexoside, cerebroside sulfuric acid ester, and a globoside similar to cytolipin R, while the minor glycolipids were ceramide dihexoside, ceramide trihexoside and a hematoside.
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  • Effect of Erythromycin on Polylysine Synthesis Directed by Polyadenylic Acid in an Escherichia coli Cell-free System
    KENTARO TANAKA, HIROSHI TERAOKA
    1968 Volume 64 Issue 5 Pages 635-648
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    1. The effect of erythromycin on polyadenylic acid (poly A) directed polylysine synthesis was examined in cell-free extracts from E. coli K-12.
    2. The inhibition by erythromycin of 14C-lysine incorporation into trichloroacetic acid-tungstate (W-TCA) insoluble materials was dependent on the amount of ribosomes in the system.
    3. Changes in amount of 105, 000×g supernatant fraction and E. coli transfer RNA (tRNA) showed no influence on the inhibition of 14C-lysine incorporation by erythromycin.
    4. Paper chromatographic analysis of the reaction products demonstrated that increasing additions of erythromycin caused decreased synthesis of highly polymerized polylysine and at the same time resulted in the accumulation of di- and trilysine.
    5. In the presence of an excessive amount of erythromycin, only di- and trilysine were observed as main products on paper chromatogram.
    6. The amounts of these peptides were increased by addition of E. coli tRNA. From 14C-lysyl-tRNA, more than 30% of lysine was incorporated into di- and trilysine in the presence of large amounts of nonradioactive lysine and erythromycin.
    7. These results are interpreted to indicate that erythromycin interacts with ribosomes and this interaction causes a loss of the activity of ribosomes to synthesize highly polymerized polylysine but leaves their ability to form small lysine peptides such as di- and trilysine still intact.
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  • Studies on DNA Synthesis in Silkworm Pupae Infected with Nuclear Polyhedrosis Virus
    KONOSHIN ONODERA, MICHIO HIMENO, FUKUMI SAKAI, ISAO MORISHIMA
    1968 Volume 64 Issue 5 Pages 649-655
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    DNA synthesis was studied in silkworm pupae after infection with nuclear polyhedrosis virus. Infection resulted in a marked change in the rate of DNA synthesis in the pupal bodies. The change was found to be due to the apparent DNA polymerase activity. The activity did not increase until 20 hr after infection, and then increased logarithmically from 24 and 48 hr. Sixty hours after infection, the activity was twenty times that in normal pupae, and then began to decrease. Actinomycin D and chloramphenicol inhibited the development and increase in enzyme activity. Partially purified DNA polymerase [EC 2. 7. 7. 7] was prepared from extracts of infected pupae. It required all four 5'-deoxyribonucleoside triphosphates, Mg++ and native or thermally denatured DNA as primer.
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  • Biosynthesis of Pteridines in the Skin of the Tadpole, Rana catesbeiana
    KATSURA SUGIURA, MIKI GOTO
    1968 Volume 64 Issue 5 Pages 657-666
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    Biosynthesis of biopterin in the skin of the tadpole, Rana catesbeiana was investigated with guanosine-14 C (U), neopterin-8a-14C, 7, 8-dihydro-neopterin-8a-14C, neopterin-3'-phosphate-8a-14C and 7, 8-dihydro-2-amino-4-hydroxypteridine-8a-14C. It was found that after incubating any of these compounds with tadpole skins radioactive biopterin could be isolated. Neopterin-3'-phosphate-8a-14C and 7, 8-dihydroneopterin were both incorporated into biopterin more effectively than neopterin. Thus, it was postulated that dihydroneopterin-3'-phosphate or its pyrophosphate might be a direct precursor of biopterin.
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  • Studies on Chemically Modified Cytochrome c
    I. The Acetylated Cytochrome c
    KEISHIRO WADA, KAZUO OKUNUKI
    1968 Volume 64 Issue 5 Pages 667-681
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    When cytochrome c purified from bovine heart muscle was acetylated with acetic anhydride under specific conditions, the acetylated cytochrome c preparations were obtained, in which 1, 2 and 3 acetyl groups had incorporated to the ε-amino groups of lysine residues. They were purified chromatographically to homogeneous states. With these preparations it was found that the enzymatic activity of cytochrome c in the cytochrome oxidase [EC 1. 9. 3. 1] reaction decreased gradually and linearly with the decrease in its basicity. The acetylation of 6 ε-amino groups in cytochrome c molecule led the protein to complete loss of the activity. From the amino acid analysis of these preparations of the acetylated cytochrome c, it was found that the 22 nd lysine residue from the amino terminus of the protein was most reactive toward acetic anhydride.
    When cytochrome c was treated with a large excess amount of acetic anhydride in the absence of sodium acetate, not only its ε-amino group of lysine residue but also the hydroxyl group of tyrosine residue was acetylated completely. With this preparation, a drastic change was observed in the visible and ultraviolet absorption spectra of the oxidized form, while that of the reduced form remained unchanged. The heme of this preparation combined with CO and oxygen. It was suggested that the heme moiety of this preparation had less interaction with protein moiety than that of the native one.
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  • The Amino Acid Sequences, the Peptide-Carbohydrate Linkages, and the Composition and Size of the Polysaccharide Units of the Glycopeptides from α1-Acid Glycoprotein of Human Plasma of Human Plasma
    TAKASHI YAMAUCHI, MAYUMI MAKING, IKUO YAMASHINA
    1968 Volume 64 Issue 5 Pages 683-698
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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    1. α1-Acid glycoprotein (α1-AGP) was digested with pronase and the digest was fractionated by chromatography on DEAE-cellulose, which gave six well separated glycopeptide fractions in good yield. Five out of the six glycopeptide fractions were also homogeneous on high-voltage paper electrophoresis. The glycopeptides were designated GP-I to GP-VI in the order in which they were eluted from the DEAE-cellulose column.
    2. Amino acid sequences of individual glycopeptides were deter-mined using proteolytic enzymes, mainly carboxypeptidases A and B [EC 3. 4. 2. 1 and 3. 4. 2. 2] and chemical degradations including dinitrophenylation, periodate oxidation, alkali treatment and the Edman procedure. The peptide sequences were found to be: GP-I, Asp-Lys; GP-II, Thr-Pro-Asp-Lys; GP-III, Asp-Lys; GP-IV, Thr-Asp-Lys; GP-V, Asp-Thr-(Ser, Ala)-Gly; GP-VI, Glu-(Ser)-Asp-Thr-(Gly). The sequences of GP-V and GP-VI were only tentative.
    3. The linkage between the amino acid and polysaccharide unit in GP-I, GP-II, GP-III and GP-IV were determined using β-aspartylglycosylamine amide hydrolase (“Amidase”). In GP-II, enzymatic breakdown of the linkage took place during incubation of the sialic-acid-free glycopeptide with the carboxypeptidase B preparation. The linkages in GP-V and GP-VI could not be determined because “Amidase” could not be used effectively due to the complex structures of these glycopeptides.
    4. All the glycopeptides were found to contain sialic acid, glucosamine and neutral sugars. The ratios of sialic acid to either glucosamine or neutral sugars increased approximately in the order of elution i.e. GP-I to GP-VI. In GP-I the ratio of sialic acid/glucosamine was 0.47 whilst in GP-VI it was 0.66. The ratio of neutral sugars to glucosamine did not differ markedly. Analysis of neutral sugar components by gasliquid chromatography showed that the ratios of fucose or galactose to mannose increased from GP-I to GP-VI. GP-I differed considerably from the other glycopeptides in that it contained no fucose and had a considerably lower galactose/mannose ratio. On the basis of these analytical results, tentative assignment of molar sugar composition for each polysaccharide unit was presented and it was concluded that there were at least six different polysaccharide units present in α1-AGP, all of which appeared to have some degree of heterogeneity and which could not be fractionated further by the present procedures.
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  • Action of Ribonuclease T1 on Smooth-surfaced Vesicles of Liver Microsomes: Evidence for the Presence of Membrane Ribonucleic Acid
    AKIO ITO, RYO SATO
    1968 Volume 64 Issue 5 Pages 699-701
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • Studies on the Hydrolytic Enzyme Models
    JUNZO NOGUCHI, SEIICHI TOKURA, TAKASHI KOMAI, HIROYUKI YAMAMOTO, AKIO ...
    1968 Volume 64 Issue 5 Pages 703-707
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • Circular Dichroism of Potato α-Glucan Phosphorylase
    ATSUMI KAMOGAWA, TOSHIO FUKUI, ZIRO NIKUNI
    1968 Volume 64 Issue 5 Pages 709-711
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • On the C-Terminal Amino Acid of Bacterial α-Amylase
    HISAKO KOJIMA, KIN-ICHI SUGAE
    1968 Volume 64 Issue 5 Pages 713-715
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • An Attempt to Estimate the Type of Metabolic Turnover of Actin by Continuous Administration of Acid Hydrolyzate of 14C-Labelled Chiorella Protein
    RYUHEI FUNABIKI, MAKOTO KANDATSU
    1968 Volume 64 Issue 5 Pages 717-718
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • Acyl-Adenylate Formation Coupled with Mitochondrial Respiration
    TAKAYUKI OZAWA
    1968 Volume 64 Issue 5 Pages 719-721
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • Presence of two New Hemoproteins in Mammalian Mitochondria
    NOBUHIRO SATO, BUNJI HAGIHARA
    1968 Volume 64 Issue 5 Pages 723-726
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • Primary Structure of Rabbit Liver Cytochrome b5
    AKIRA TSUGITA, MIDORI KOBAYASHI, TOSHIMASA KAJIHARA, BUNJI HAGIHARA
    1968 Volume 64 Issue 5 Pages 727-730
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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  • Reversible Change in Physical State of Troponin Induced by Calcium Ion
    TAKEYUKI WAKABAYASHI, SETSURO EBASHI
    1968 Volume 64 Issue 5 Pages 731-732
    Published: November 25, 1968
    Released on J-STAGE: June 30, 2008
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