The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 73, Issue 4
Displaying 1-23 of 23 articles from this issue
  • Yoshihiko IGARASHI, Takeyoshi IMAMURA, Minoru SUZUKI, Yuji MIYAZAWA
    1973 Volume 73 Issue 4 Pages 683-693
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
    JOURNAL FREE ACCESS
    Large amounts of 30, 50, and 70S ribosomes from Escherichia coli strain Q13 have been fractionated by sucrose density gradient sedimentation with a fixed angle rotor. The purity of each fractionated sample was satisfactory. The molecular weights of 30 and 50S subunits were measuredby means of light scattering and values of 77.5×104 and 148×104 were obtained, respectively. The dimensions of 70S ribosomes and those of the subunits were determinedfrom the values of intrinsic viscosity, radius of gyration, and partial specific volume. Measurements of the volume obtained by the former two methods gave values about 4 times larger than the volume calculated from partial specific volume and molecular weight. This suggests that the ribosomes arehydrated with a large amount of water and that they are swollen in solution. Experiments involving reassociation of the subunits revealed that fractionated subunits lose their ability to form 70S particles. A small RNA component obtained from 70S ribosomes on their dissociation to subunits played an important role in the reassociation of the subunits to 70S ribosomes. The component appeared to bea peptidyl (or aminoacyl)-tRNA. 30S subunit polymerized with itself to form dimer and trimer under someexperimental conditions. However, there was no evidence that 50S subunit forms a dimer.
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  • Shozo SUZUKI, Haruhiko NODA, Koscak MARUYAMA
    1973 Volume 73 Issue 4 Pages 695-703
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    When natural F-actin was incubated without the addition of ATP, its flow birefringence was easily lost. In this degeneration process, natural F-actin was conversted into three fractions designated as S2, P1, and P2. Characterization of these fractions was performed. P2 and P1 were identified as natural and denatured F-actin, respectively. S2 consisted of at least two kinds of proteins which were not identified. The apparent activation energy of the degeneration reaction was 7.3 kcal · Emole-1.
    ATP was incorporated into natural F-actin with a binding constant of about 5× 10-4M-1. This suggests that natural F-actin carries weakly bound ATP, in addition to ordinarily bound ATP, which protects it from degeneration.
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  • Masatomi HARADA, Masachika IRIE
    1973 Volume 73 Issue 4 Pages 705-716
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    1) In order to obtain the knowledge on the active site of ribonuclease from Aspergillus saitoi (RNase M)[EC 2. 7. 7. 17], chemical modifications of RNase M by various kinds of halo fatty acidsand iodoacetamide were studied.
    2) RNase M was inactivated most effectively by bromoacetate and iodoacetate at pH 4.0, and by 3-bromopropionate at pH 8.0 among the halo fatty acids tested. The rateof inactivation by iodoacetamide is higher at pH 8.0 and lower at pH 4.0 than by iodoacetate.
    3) The rate of inactivation of RNase M by iodoacetate was maximum at pH 3.5, and about one residue of carboxymethyl group was incorporated into RNase M with concomitant loss of enzymatic activity. The inactivation of RNase M by iodoacetate was protected in the presence of competitive inhibitors such as2'-AMP, indicating possible involvement of the amino acid residue to be carboxymethylated in the active site of the enzyme.
    4) Amino acid composition of carboxymethylated RNase M (CM RNase M) indicated that about one residue of carboxymethyl group was introduced into N3-position of histidine. The results were also confirmed by analyses of 14C-CM RNase M.
    5) In contrast to iodoacetate, iodoacetamide inactivated RNase M at pH 8.0 with the introduction of three carboxamidomethyl groups into the enzyme. Amino acid composition of carboxamidomethylated RNase M (CAM RNase M) indicated that two moles of carboxamidomethyl groups were introduced into N3-position of histidine residues.
    6) Spectrophotometric titration of phenolic groups and fluorescence spectrum of CM RNase M and CAM RNase M indicated that the gross conformation of the modified enzymes is notmuch different from that of the native enzyme. However, the structures of native RNase M and CAM RNase M differ slightly from CM RNase M by judging from the rate of reaction with dinitrofluorobenzene.
    7) The binding constants of 2'-AMP with CAM RNase M and CM RNase M were about 1/1.5 and 1/10 as that with the native enzyme, respectively, indicating that CAM RNase M has a gross conformation moresimilar to RNase M than CM RNase M.
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  • Anadara satowi Molecular Weights and Oxygen Equilibria of Arca Hb I and II
    Shiro OHNOKI, Yoshitada MITOMI, Ryuichiro HATA, Kazuo SATAKE
    1973 Volume 73 Issue 4 Pages 717-725
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    The heterogeneity of hemoglobin from Arca was demonstrated by the salting-out curve, the sedimentation pattern, gel filtration, electrophoresis, and ion-exchange chromatography. The two main components (Hb I and II) were purified by DEAEcellulose and CM-cellulose and appeared homogeneous on ultracentrifugation and electrophoresis. The oxygen equilibria of these hemoglobins showed that neither had a Bohr effect in the neutral region and that they had less heme-heme interaction than human adult hemoglobin. The molecular weights of Hb I and II, determined by the sedimentation equilibrium method, were 34, 000 and 69, 000, respectively, and heme conents per molecule were 2 for Hb I and 4 for Hb II.Hb I and II each dissociated to a monomer on treatment with sodium dodecyl sulfate. From the above experiments it was concluded that Hb I has a dimer structure and Hb II, a tetramer structure.
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  • I. Mechanism of Circadian Increase in Liver Enzyme with Special Reference to Hormonal and Dietary Effects
    Mikio SUDA, Katsuya NAGAI, Hachiro NAKAGAWA
    1973 Volume 73 Issue 4 Pages 727-738
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    Phosphoenolpyruvate carboxykinase [EC 4. 1. 1. 32] activity in rat liver showed circadian variations with the highest activity in the evening (1600-1800h) and the lowest activity in the morning (0700-1000h).
    The circadian increase in enzyme activity was consistently observed through all seasons. The enzyme rhythm was found in rats of both sexes and with various body weights, though the amplitude varied with the season.
    The increase in enzyme activity in the evening was almost completely blocked by cycloheximide and was partially inhibited by actinomycin D.
    Neither adrenalectomy nor thyroidectomy affected the circadian rhythm. However, glucose was found to be an effective suppressor of the circadian increase in phosphoenolpyruvate carboxykinase activity, when given either by stomach tube or by intraperitioneal injection. Insulin was also effective in blocking the circadian. increase in enzyme activity. The mechanism of the circadian rhythm of phosphoenolpyruvate carboxykinase activity is discussed with special reference to the relationships between glucose, insulin, and food intake.
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  • Ross H. HALL, George MINTSIOULIS
    1973 Volume 73 Issue 4 Pages 739-748
    Published: April 25, 1973
    Released on J-STAGE: June 07, 2011
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    N6-(Δ2-isopentenyl) adenosine (i6A) and its analogs N6-isopentenyl, isopentyl, 4-hydroxy-3-methylbut-2-trans-enyl, and 3-hydroxy-3-methylbutyl, are competitive inhibitors of the deamination of adenosine by an adenosine amino-hydrolase [EC 3. 5. 4. 2] isolated from chicken bone marrow. The Ki values fall in the range 1-3×10-4. The same enzyme also catalyzes conversion of i6A and other N6-(alkyl substituted) analogs to inosine. The potent inhibitor of the aminohydrolase from calf intestinal mucosa, 6-amino-9-(1-hydroxy-2-octyl) purine, inhibits degradation of both adenosine and i6A by the bone marrow enzyme Ki 3.5×10-7 and 1.0×10-7, respectively.
    An assay using 8-14C-i6A as substrate was used toassay various tissues for this enzyme activity. The major site of occurrence of this enzyme is associated with the blood system. The enzyme activity was detected in chicken, pig, rabbit, and human samples.
    An assay based on gas chromatography was used to monitor the levels in vivo of N6-(substituted) adenosine derivatives in the blood of a rabbit. i6A is cleared fromthe blood in about 4 hr after administration of a single dose (intravenous, 150 mg/kg) while the analog, N6-(3-chlorobut-2-enyl) adenosine, that is not a substrate for the enzyme, is clearedmuch more slowly. These results suggest that this enzyme activity contributes to the clearance of i6A from humoral blood.
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  • II. Preparation of Some p-Nitrophenyl 2-Halogenoacetylamino-2-deoxy-β-D-glucopyranosides and Their Susceptibility to Enzymic Hydrolysis
    Kazuhiko YAMAMOTO
    1973 Volume 73 Issue 4 Pages 749-753
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    Some of p-nitrophenyl 2-acylamino-2-deoxy-β-D-glucopyranosides with monofluoroacetyl, monochloroacetyl, monobromoacetyl, difluoroacetyl, dichloroacetyl, and trifluoroacetyl group as N-substituent were prepared in order to investigate the N-acyl specificity of Taka-N-acetyl-β-D-glucosaminidase [EC 3. 2. 1. 30].
    All of the substrate analogs prepared in this experiment, except the N-dichloroacetyl derivative, were hydrolyzed by this enzyme.
    Comparison of the hydrolytic rate ofeach substrate analog to the N-acetyl derivative led to the conclusion that monohalogen substitutionon the N-acetyl group of the substrate, even di-, and tri-substitution in the case of fluorine atom, is permissible in the N-acyl specificity and the specificity is predominantly controlled by the steric factor of the N-substituent of substrate.
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  • Yutaka UDA, Yasuo NAKAZAWA
    1973 Volume 73 Issue 4 Pages 755-761
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    1. Proteolipid was prepared from bovine brain white matter by a method involving fluff formation. The preparation was soluble in chloroform: methanol (2: 1 and 1: 1, v/v) but insoluble in acetone, ether, and ethanol.
    2. The lipid composition of the proteolipid was determined by stepwise hydrolysis and characterized by a large amount of sphingomyelin.
    3. Much of the lipid portion, containing most of the sphingomyelin, was removed by treating the proteolipid with a mixture of ethanol and ether. The residual portion was still soluble in chloroform: methanol (2: 1, v/v) and was found to have a specific affinity to sphingomyelin.
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  • Katsutada TAKAHASHI, Sôzaburo ONO
    1973 Volume 73 Issue 4 Pages 763-770
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    1. Calorimetric measurements were made on the heat change accompanying the mutarotation on D-galactose and D-mannose to evaluate quantitatively the anomeric stability of the two sugars in aqueous solution.
    2. It was found that in D-galactose the β-anomer is 1, 300±50 J mol-1 energetically more stable than the α-anomer, while in D-mannose the α-anomer is 1, 900±80 Jmol-1 more stable than the β-anomer at 25°C.
    3. From stereochemical considerations regarding D-mannose, D-galactose, D-glucose, and D-xylose, it was assumed that the hydroxyl group on C2 in the pyranose ring plays a major role in determining the preferred form of the anomeric pairs.
    4. By combining the data with those reported for the isomerization of eq-D-glucose to eq-D-mannose, the energies required for the conversion of a hydroxyl group on C2 of α-D-glucose and β-D-glucose in chair-1 form from equatorial to axial were estimatedto be 7, 950 J mol-1 and 10, 880 J mol-1, respectively.
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  • Akiharu WATANABE, Kazuhisa TAKETA
    1973 Volume 73 Issue 4 Pages 771-779
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    The activity of liver glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate NADP+ oxidoreductase, EC 1.1.1.49](G6PD) increased in rats following a single dose of CCl4. From the time course of the increase in enzyme activity, the rate constants for synthesis and degradation of G6PD in vivo were calculated to be increased 4.3- and 1.2-fold, respectively, over the initial rates. A corresponding increase in antigenic activity was found by titration of the induced enzyme with a specific antibody prepared to rat liver G6PD. Pulse labeling of the dehydrogenase protein in vivo with [14C]-leucine demonstrated that the maximum increase of 2.9-fold in the rate of G6PD synthesis was attained 24 hr after CCl4 liver injury in spite of inhibited incorporation of the labeled amino acid into soluble hepatic proteins and albumin. Cycloheximide effectively blocked the increased synthesis of the enzyme induced by CCl4 intoxication, whereas actinomycin D, at a dose sufficient to inhibit most RNA synthesis in the liver, had no effect on either the enzyme activity or the rate of enzyme synthesis in the liver after CCl4 injury. These results indicate that the increased production of liver G6PD induced by CCl4 is due to an altered mechanism of posttranscriptional regulation of the enzyme synthesis, probably at the level of translation. The apparent half-life of G6PD during the period of CCl4-mediated induction of the enzyme was estimated to be reduced to 17 hr from the control value of 20hr, and this agreed well with value calculated from kinetic data. Thus the turnover rate of enzyme protein in the damaged liver seems to be slightly increased. That the enhanced synthesis of liver G6PD induced by CCl4 is caused by hepatic injury and is not the consequence of liver regeneration following hepatic cell necrosis is discussed.
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  • II. Preparations with High Specific Activity Obtained Using Aminoethyl Cellulose Chromatography
    Toshiko NAKAO, Makoto NAKAO, Fumiko NAGAI, Koichi KAWAI, YOKO FUJIHIRA ...
    1973 Volume 73 Issue 4 Pages 781-791
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    The microsome fraction from pig brain was treated with NaI and extracted with Lubrol. The extract was treated batchwise with aminoethyl-cellulose or phosphocellulose. Then, the extracts were charged to an AE-cellulose column, equilibrated with 14 mM veronal-HCI buffer, pH 7.1 and eluted with NaCl plus ATP at pH 6.6. Three peaks of activity were eluted and named α, β, and γ. The specific activities of the three peaks were very high (up to 7000 μmoles Pi liberation/mg protein/hr), and the activities were inhibited completely by ouabain. The labilities of the three peaks differed. On sodium dodecylsulfate polyacrylamide electrophoresis they gave a main band with an apparent molecular weight of 100, 000.
    The molecular weights of native active ATPase [EC 3. 6. 1. 3] in the α and γ peaks were both determined as 480, 000 using gel chromatography on Sephadex G-200 and Sepharose 6B. The specific gravities of the α and γ peak fractions were 1.085 and 1.115, respectively, determined by the method of Mizuno et al. Their protein-. lipid ratios were roughly 1: 6 and 1: 2 respectively, although the peak partly overlapped that of free Lubrol. These findings suggest that this enzyme contains only one subunit, with a molecular weight of 100, 000, in an active molecule with a molecular weight of 500, 000. The amount of phosphorylated protein formed from 32P-ATP (using a preparation with a specific activity of 5, 600) was 7. 8 μmoles/g protein, which corresponded to a molecular weight of 125, 000 per mole of phosphate. This finding also supports the view that the enzyme contained only one subunit, although the existence of small subunits with, for example, a molecular weight of 10, 000, was not disproved completely.
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  • Makoto MATSUMOTO, Yasuo SUZUKI
    1973 Volume 73 Issue 4 Pages 793-802
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    1. Enzymatic acylation of lysophospholipids was studied in a cultured cell line, FL cells, derived from human amniotic membrane. Acylation with radioactive oleic acid of lysophosphatidylcholine, which was prepared from egg yolk phosphatidylcholine by means of snake venom, in the presence of ATP, CoA, and Mg2+ occurred more actively with FL cell homogenate than with bovine erythrocyte hemolysate. Lysophosphatidylethanolamine was acylated at a slightly lower rate than lysophosphatidylcholine by FL cells.
    2. Lysoplasmalogens (1-alkenyl-glycerophosphorylcholine and 1-alkenyl-glycerophosphorylethanolamine) were also quite highly acylated by FL cells. The rate of acylation of the former was higher than that of the latter.
    3. In addition, the venom of a Japanese snake, Trimeresurus flavoviridis, was found to contain phospholipase A2 activity which specifically attacks the 2-position of glycerophospholipid; phospholipase A1 activity, which hydrolyzes the 1-position of the substrate, was practically negligible.
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  • Terumi SAITO, Kenkichi TOMITA
    1973 Volume 73 Issue 4 Pages 803-810
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    Malic enzyme [EC 1. 1. 1.40] was purified 270-fold to apparent homogeneity from the hepatic cytosol of rats fed on a high carbohydrate diet containing desiccated thyroid. On gel electrophoresis in the presence of sodium dodecyl sulfate, the purified enzyme was dissociated into a smaller unit of about one fourth the original size in accord with the ultracentrifugal results obtained with the alkaline degraded enzyme.
    On Ouchterlony double diffusion tests, the rabbit antiserum prepared against the purified rat hepatic malic enzyme gave a single precipitin line with the purified enzyme, but two lines, major and minor, with crude hepatic extract. The main precipitin line was continuous with that of the purified enzyme and also with a single line formed by crude extract of adipose tissue reacting with the antibody. The minor line joined with a single precipitin line formed by crude heart extract and the antibody. These results imply that there are two immunologically different types of soluble malic enzyme in rat tissues, A (adipose tissue)-and H (heart)-types. Hepatic cytosol contained both types, and the amount of its A-type protein was apparently influenced by hormonal and nutritional stimuli.
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  • Zeljko KUCAN, Robert W. CHAMBERS
    1973 Volume 73 Issue 4 Pages 811-819
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    L-Tyrosine: tRNA ligase (AMP)[EC 6. 1. 1. 1] has been purifeed te homogeneity from a genetically well-characterized, haploid strain of yeast, Saccharomyces cerevisiae α S288C. The kinetic constants for tRNATyr are: Km=3.5×10-7; kcat=365 min-1. Under dissociating conditions, the enzyme gives a single band on discontinuous gel electrophoresis, corresponding to a molecular weight=31, 500. Under non-dissociating conditions, where the activity of the enzyme is retained, the molecular weight estimated by gel filtration is 116, 000. Though tentative, these data indicate this enzyme may be composed of four subunits.
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  • I. The Preparation and Properties of the Products of Bisulfite Ion-catalyzed Reaction of Amino Acids and Peptides with Cytosine Derivatives
    Irina V. BONI, Edward I. BUDOWSKY
    1973 Volume 73 Issue 4 Pages 821-830
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    1. N4-substituted cytosine derivatives are formed in good yield in aqueous solutions containing bisulfite ions and cytosine, cytidine or cytidine 5'-phosphate, on the one hand, and glycine, glycyl-glycine, or poly-L-lysine, on the other hand. The products are models of the cross-links arising within nucleoproteins on treatment with nucleophilic reagents, and, probably, those arising after photohydratation of cytosine residues.
    2. The effects of temperature, reagent concentrations, and pH on the reaction kinetics were studied. The maximum rate of transamination and minimum deamination are observed at pH 7.2-7.4. Changes of temperature between 6 and 25°C do not affect the ratio of the rates of the two reactions.
    3. The compounds obtained are stable under conditions of alkaline and acidic degradation of polyribonucleotides. Complete cleavage of the N-glycosidic bonds with 6 N HC1 (110°C, 96 hr) or 70% HClO4 (100°C, 1 hr) is accompanied by less than 10-20% cleavage of the bond between the pyrimidine nucleus C4-position and the amino acid residue amino group.
    4. With the polylysine derivative of cytidine, it was demonstrated that the N-C4 bond can be cleaved by nucleophilic reagents such as O-methylhydroxylamine and bisulfite-O-methylhydroxylamine mixture.
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  • III. Purification and Proteolytic Specificity of Alkaline Proteinase C from Pronase
    Yoshiko NARAHASHI, Kaoru YODA
    1973 Volume 73 Issue 4 Pages 831-841
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    The purification of DFP-sensitive alkaline proteinase C from pronase is described. When the proteolytic specificity of alkaline proteinase C was investigated using the reduced, carboxymethylated phenylalanyl chain of insulin, angiotensin II, and oxytocin as substrates, nine splittings in the first substrate and two splittings in the second and third substrates were detected, respectively.
    In the reduced, carboxymethylated phenylalanyl chain of insulin, more than 30% hydrolysis of the bonds Leu (11)-Val (12), Tyr (16)-Leu (17), and Phe (25)-Tyr (26) was found. Additional cleavages (less than 20%) of the bonds Phe (1)-Val (2), Gln (4)-His (5), Leu (15)-Tyr (16), Leu (17)-Val (18), and Phe (24)-Phe-(25) were also noted. Hydrolysis of angiotensin II and oxytocin was observed at Tyr (4)-Ile (5) in the former and Leu (8)-GlyNH2 (9) in the latter substrate. The specificity of Streptomyces griseus alkaline proteinase C was compared with those of other DFP-sensitive alkaline proteinases.
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  • Masaru SAGAI, Makoto ISHIMOTO
    1973 Volume 73 Issue 4 Pages 843-859
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    1) Enzymatic reduction of adenosine 1N-oxide by an extract of Escherichia coli was followed by measuring decrease of absorbance of the compound at 233 mμ and that of dithionite at 317mμ in the presence of benzylviologen and sodium dithionite added as an electron donor system.
    2) The enzyme was purified 23 fold from the crude extract by isoelectric precipitation, ammonium sulfate precipitation, DEAE-and phospho-cellulose chromatographies and Sephadex G-200 gel filtration.
    3) Three protein peaks with enzyme activity were found on Sephadex filtration of a preparation obtained using a slightly modified purification process. Their molecular weights were 640, 000 (PI), 320, 000 (PII), and 160, 000 (PIII). The purified preparation was identified with PIII.
    4) The properties of the purified preparation (PIII) were investigated. The optimum pH was 5.0-5.5, the isoelectric point was pH 4.6, and the Km, was 2×10-4M, but substrate inhibition was observed at concentrations over 0.3mM. The main product was identified as adenosine by paper chromatography. N-Oxide or a-and γ-picoline, nicotinic acid trimethylamine, and nitrite were reduced. One mole of hydrogen was consumed per mole of N-oxide in a system coupled with hydrogenase. FAD, FMN, or cytochrome c served as electron donors, but NADH or NADPH did not. The reaction was stimulated by Fe2+ or Mn2+ and inhibited by α, α'-dipyridyl or o-phenanthroline.
    5) A PII preparation was obtained, by Sephadex gel filtration from cells after induction by growth in the presence of trimethylamine N-oxide. The properties of PII were essentially similar to those of PHI. A preparatian of PII' with less abilityto reduce trimethylamine N-oxide was obtained from another active peak obtained by Sephadex filtration. Nitrite reductase [EC 1.7. 99.3] was eluted in a different position from the enzyme reducing N-oxide.
    6) Bands of activity obtained by disc electrophoresis on polyacrylamide gel were negatively stained with reduced benzylviologen and N-oxides. One band from normal cells and three bands from cells grown in the presence of trimethylamine N-oxide were found and attributed to PIII, PII, and PII'. The same bands were negatively stained with the N-oxide of adenosine and trimethylamine.
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  • II. Diglycyl-L-phenylalanyl-L-phenylalanyl (and L-tyrosyl)-glycines: Sensitive Synthetic Substrates for Pepsin
    Hiroo YONEZAWA, Shigeyuki TERADA, Nobuo IZUMIYA
    1973 Volume 73 Issue 4 Pages 861-870
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    A study was carried out to find a sensitive synthetic substrate for pepsin [EC3. 4. 4. 1] which is soluble in acidic buffer and shows no transpeptidation reaction. Several oligopeptides, Glym-Tyr2-Glyn (m=1, 2, 3, and 4; n=1, 2, and 3) and Gly2-X-Y-Gly (X and Y, Tyr or Phe), soluble in acidic buffer, were synthesized and subjected to the action of pepsin. Analyses of an incubation mixture by paper chromatography and by an amino acid analyzer proved that the substrates are hydrolyzed at the peptide bond linking the two aromatic amino acid residues with no indication of any transpeptidation reaction. Among the series of the substrates, Gly2-Phe-Phe-(and Tyr)-Gly were most susceptible to hydrolysis, and are recommended as convenient synthetic substrates for pepsin.
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  • Yusuke WATAYA, Hikoya HAYATSU, Yutaka KAWAZOE
    1973 Volume 73 Issue 4 Pages 871-877
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    A new and potentially useful method for labeling nucleic acid was developed.Cysteine catalyzes hydrogen-deuterium exchange at position 5 of uridine 5'-phosphate under mild conditions. The pD dependence of the catalysis by cysteine, as well as that by 2-mercaptoethylamine showed a maximum at pD 8.9. Typical rates of the exchange at 37°C, determined by NMR spectroscopy were as follows: Reagent and its concentration/pD/the apparent rate constant (hr-1); cysteine, 0.5 m/9.0/2.29×10-2;cysteine, 1.0 M/9.0/6.05×10-2; 2-mercaptoethylamine, 0.5 M/9.1/1.26×10-2; 2-mercaptoethanol, 0.5 M/9.5/1.51×10-3. N-Acetylcysteine, S-methylcysteine, or a mixture of the two agents was essentially ineffective in catalysis of the exchange. The amino group of cysteine is important in the catalysis since cysteine was much more effective than 2-mercaptoethanol as catalyst with 3-methyluridine at pD 10.0 where the SH-group of both compounds is largely dissociated. Based on these data, together with the known roles of a sulfur nucleophile and of amine in the bisulfite-catalyzed hydrogen isotope exchange of uridine (Y. Wataya and H. Hayatsu, Biochemistry, 11, 3583 (1972)), a reaction mechanism is proposed in which the mercapto anion adds across the 5, 6-double bond of the undissociated uracil moiety forming a 5, 6-dihydrouridine-6-mercapto compound and the NH2-group of cysteine abstracts the hydrogenat the 5-position intramolecularly. Studies on the effect of added amines and on the dependence of the exchange rate on the cysteine concentration suggested that protonated amines, either of added amine or of cysteine itself, accelerate the overall reaction. The acceleration is probably due to promotion of the addition of the SH to uridine. The implications of these findings in relation to the mechanism of the thymidylate synthetase reaction are discussed. This catalysis by cysteine appears to be the mildest method so far known for labeling uracil.
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  • II. Factors Stimulating the Activity of RNA Polymerase II
    Shunji NATORI, Kazuyuki TAKEUCHI, Kyuichiro TAKAHASHI, Den'ichi MIZUNO
    1973 Volume 73 Issue 4 Pages 879-888
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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    Two factors stimulating RNA polymerase II [EC 2. 7. 7. 6] of Ehrlich ascites tumor cells were isolated. Both factors showed significant template specificity. One of them, S-I, stimulated RNA synthesis on Ehrlich ascites tumor DNA, rat hepatoma DNA and poly dAT but not on DNA of calf thymus or Φ80. The other factor, S-II, stimulated RNA synthesis only when Ehrlich ascites tumor DNA or rat hepatoma DNA was used as template and had no effect using poly dAT or calf thymus or Φ80 DNA. Neither factor stimulated RNA synthesis by E. coli RNA polymerase on DNA of Ehrlich ascites tumor cells. Both factors were found to have affinity for Ehrlich ascites tumor DNA. These factors were effective when added in the initiation stage of transcription. When these factors were preincubated with DNA and then removed, their readdition to the DNA no longer stimulated RNA synthesis. The stimulatory activities of the two factors could not be separated from their ribonuclease H activities.
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  • Shozo KAJIYAMA, Yoshiaki NOSOH
    1973 Volume 73 Issue 4 Pages 889-891
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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  • Susumu ANDO, Kazuo KON, Miyoko ISOBE, Tamio YAMAKAWA
    1973 Volume 73 Issue 4 Pages 893-895
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
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  • A Possible Regulatory Mechanism of Collagenase Activity in Vivo
    Shigeto ABE, Yutaka NAGAI
    1973 Volume 73 Issue 4 Pages 897-900
    Published: April 25, 1973
    Released on J-STAGE: January 25, 2011
    JOURNAL FREE ACCESS
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