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Volume 66 (1969)
- Issue 6 Pages 751-
- Issue 5 Pages 573-
- Issue 4 Pages 431-
- Issue 3 Pages 289-
- Issue 2 Pages 123-
- Issue 1 Pages 11-
Volume 66, Issue 6
Displaying 1-17 of 17 articles from this issue
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MASAHIKO ONODERA, KOICHI YAGI1969 Volume 66 Issue 6 Pages 751-758
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. ATPase activity of heavy meromyosin [EC 3. 6. 1. 3, ATP phosphohydrolase] in the presence of CaCl2 was remarkably enhanced by the reaction with 2, 2'-dihydroxy-6, 6'-dinaphthyl disulfide.
2. A new absorption spectrum having a peak at 375mμ, which was identical with that shown by a mixture of 2, 2'-dihydroxy-6, 6'-dinaphthyl disulfide and cysteine, was obtained after the reaction. Decrease in the content of SH-groups corresponding to the amount of thionaphthyl groups bound to heavy meromyosin was also observed. It was concluded that there occurred thionaphthylation of SH-groups of heavy mero-myosin caused by a disulfide exchange reaction.
3. The maximum activation of ATPase was brought about by the binding of 2 moles of thionaphthol per mole of heavy meromyosin.
4. Tryptic digestion of the thionaphthyl heavy meromyosin rapidly decreased the ATPase activity towards the control level probably by releasing thionaphthyl groups from enzymatically active subfragment.View full abstractDownload PDF (540K) -
XI. Some Properties of Nitric Oxide ReductaseMACHIKO MIYATA, TERUO MATSUBARA, TAKESHI MORI1969 Volume 66 Issue 6 Pages 759-765
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Intact cells of Ps. denitrificans were found to reduce NO to N2O and the latter to N2 using lactate as the hydrogen donor. The cell-free extract reduced NO to N2O but not N2O to N2. These results of gas interconversion by intact cells or cell-free extract were nearly the same as the gas evolution from nitrite (1).
2. The optimal concentration of NO in the gas phase for the reducing activity of NO to N2O was 10 per cent and the optimal pH was 7.0-7.4.
3. Experiments on inhibitors of the reaction suggested that both heavy metal (s) and sulfhydryl groups were involved in the reduction of NO to N2O.
4. The addition of flavin nucleotide showed no effect on the NO reduction.
5. The reduction of NO to N2O was realized by the particulate fraction obtained from disrupted cells in the presence of lactate as the hydrogen donor.
6. Lactate, the leuco-forms of thionine, brilliant cresyl blue and methylene blue were effective electron donors in the NO reduction by the cell particles, whereas formate, malate, succinate, oxaloacetate, reduced dichlorophenolindophenol, NADH, NADH plus FMN and NADPH were found to be less or entirely not effective.View full abstractDownload PDF (384K) -
V. The Binding Site of PhosphateFUJIO SUZUKI, KEIHACHI FUKUNISHI, YOSHIRO TAKEDA1969 Volume 66 Issue 6 Pages 767-774
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical nature of the phosphate bond in the enzyme-P (the enzyme-phosphate complex), the activated intermediate of ATP citrate lyase [EC 4. 1. 3. 8], was investigated. The linkage between the enzyme and phosphate in the complex was more labile in acid than in alkali. The pH stability curve of the enzyme-P is very similar to that of aspartyl phosphate and differs from that of butyl thiophosphate.
The phosphorylated form of the enzyme was digested with pronase and the phosphopeptides were isolated by paper electrophoresis. When the phosphopeptides were treated with hydroxylamine, bound phosphates were liberated as Pi with concomitant formation of peptidylhydroxamates. After conversion of the peptidylhydroxamates to the dinitrophenyihydroxamate derivatives, Lossen rearrangement of the latter and the subsequent hydrolysis yielded α, γ-diaminobutyric acid, indicating the presence of glu-tamyl-γ-phosphate in the hydroxylamine-sensitive groups of the original protein. From these results, it is concluded that the binding site of phosphate in the phosphorylated intermediate of ATP citrate lyase is the γ-carboxyl groups of glutamic acid residues.View full abstractDownload PDF (575K) -
HIDEKAZU IWASAKI, SOHSUKE SHIDARA1969 Volume 66 Issue 6 Pages 775-781
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c-553 was crystallized from the soluble fraction of aerobically grown cells of Pseudonronas denitrifzcans. Its molecular weight was estimated to be 45, 000. It had two haem prosthetic groups per molecule; one of them showed reduced α-peak at about 551mμ and the other at about 555mμ. Their E0' (pH 7) values were estimated to be -0.09V and between 0 and -0.09V, respectively. The cytochrome was readily autooxidizable. The ferro-form reacted with CO, and the ferro- and ferri-forms reacted with KCN at pH 7.View full abstractDownload PDF (1468K) -
II. Preparation of Fluorescein-Thiohydantoin Amino Acids and Their Thin-layer ChromatographyHIROSHI KAWAUCHI, KATURA TUZIMURA, HIROSHI MAEDA, NAKAO ISHIDA1969 Volume 66 Issue 6 Pages 783-789
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acids were shown to react with fluorescein-isothiocyanate (FITC) to form fluorescein-thiocarbamyl amino acids (FTC-amino acids). Fluorescein-thiocarbamyl amino acids were converted into fluorescein-thiohydantoin amino acids (FTH-amino acids) under acidic condition.
Amino acid fluorescein-thiohydantoins were successfully separated by thin-layer chromatography.
These results are discussed in connection with the application for micro-analysis of amino acids, peptides and proteins.View full abstractDownload PDF (452K) -
KYOKO TORII, YASUYUKI OGURA, JINYA OTSUKA, AKIRA TASAKI1969 Volume 66 Issue 6 Pages 791-797
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe magnetic susceptibilities of horse erythrocyte catalase [EC 1. 11. 1. 6] and its derivatives were measured in the range from room temperature to 4.2°K. With high spin compounds, such as free catalase and the fluoride compound, the temperature dependence of the magnetic susceptibility was calculated assuming that the fine structure of the ground state of the iron ion can be expressed by a spin Hamiltonian, DS2z. In this calculation, the paramagnetic saturation effect was taken into consideration. The values of the parameter, D, of the spin Hamiltonian were estimated to be 12cm-1 for free catalase and 9cm-1 for the fluoride compound. The values of E were determined to be 0.3cm-1 for free catalase and 0.23cm-1 for the fluoride compound, using the values of E/D previously determined from the EPR signals. From these values, the energy differences of the dε orbitals, Ey-Ex, were calculated to be 700cm-1 for free catalase and 500cm-1 for the fluoride compound. With low spin compounds, such as the azide and cyanide compounds, the values of the effective numbers of the Bohr magneton, neff, were estimated to be 2.63 for the azide compound and 2.39 for the cyanide compound from measurements in an extremely low temperature range. These large apparent values of neff may be due to the contribution of a small amount of high spin compound remaining in the test sample.View full abstractDownload PDF (419K) -
III. Stimulation of 14C Incorporation from 14CO2 and 14C-Pyruvate into Glucose by Triamcinolone in the Presence of Ethanol in Rat Liver SlicesTETSUO UETE1969 Volume 66 Issue 6 Pages 799-804
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The effect of triamcinolone on the incorporation of 14C from 14CO2 and 14C-pyruvate into glucose and on the incorporation of pyruvate carbon into fatty acids and CO2 in rat liver slices was studied in the presence of sufficient amount of ethanol which inhibits gluconeogenesis.
2. Ethanol is known to inhibit gluconeogenesis and the oxidation of pyruvate, glucose, and various amino acids in the liver by increasing the generation of NADH2. In fact, ethanol inhibition to the 14C incorporation from 14CO2 and 14C-pyruvate into glucose in rat liver slices was observed in the present investigation. The incorporation of 14C from 14C-pyruvate into fatty acids and CO2 was also inhibited by ethanol.
3. Triamcinolone stimulated the 14C incorporation from 14CO2 and 14C-pyruvate into glucose, but not the 14C incorporation into CO2 and fatty acids.
4. Methylene blue, which is a redox dye capable of oxidizing NADH2, restored all these ethanol inhibitions. Triamcinolone counteracted the ethanol inhibition of 14C incorporation from 14CO2 and 14C-pyruvate into glucose, but triamcinolone did not affect the ethanol inhibition of 14C incorporation from 14C-pyruvate into CO2 and fatty acids. Therefore, it is unlikely that the effect of adrenal cortical hormones to stimulate gluconeogenesis is primarily on the NADH2/NAD system.View full abstractDownload PDF (447K) -
XX. Fluorescence of Stilbene Dyes Adsorbed on Hydrophobic Regions of Protein MoleculesOSAMU TAKENAKA, KAZUO SHIBATA1969 Volume 66 Issue 6 Pages 805-814
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions of a fluorescent dye, Na-4, 4'-(bis-[2-chloro-4-diethanolamino-1, 3, 5-triazyl-(6)])-diaminostilbene-2, 2'-disulfonate (TAS) with proteins and poly-amino acids were studied by measuring absorption and fluorescence spectra of TAS and the rate of photo-isomerization in the presence and absence of proteins, and adsorption of TAS on hydrophobic regions of proteins was deduced from the following results. 1) Both absorption and fluorescence spectra of TAS are changed by the presence of protein. 2) The quantum yield of the fluorescence of TAS is increased by the presence of protein. 3) The rate of photo-isomerization of TAS from the trans to the cis form is decreased by the effect of protein. 4) The fluorescence excitation spectra of mixtures of TAS and proteins show a band near 280mμ, indicating energy transfer from aromatic amino acid residues to TAS molecules. 5) Na-4, 4'-diaminostilbene-2, 2'-disulfonate, a fluorescent stilbene, similar to TAS but without the two triazine rings of TAS does not undergo such fluorescence and absorption changes on the addition of proteins. The hydrophobic regions in insulin, lysozyme [EC 3. 2. 1. 17], ribonuclease [EC 2. 7. 7. 16], chymotrypsinogen and α-chymotrypsin [EC 3. 4. 4. 5] molecules were studied by means of the fluorescence enhancement of TAS and, in the case of insulin, the presence of two hydrophobic regions, one between A and B chains and the other in the heptapeptide (B23 Cly to B29 Lys) of the B chain was deduced; the hydrophobic interaction of TAS with the native insulin molecule was remarkably decreased by separation of the insulin molecule into the A and B chains, whereas tryptic digestion did not much affect the interaction. The interactions of TAS with other proteins with larger molec-ular sizes were increased by alkali-denaturation or by cleavage of disulfide bonds. These data were interpreted as due to exposure of the hydrophobic regions buried in the interior of protein molecules.View full abstractDownload PDF (682K) -
XXI. Chemical Accessibility of Histidine and Tyrosine Residues in Insulin as Examined with Diazonium-1-H-tetrazoleTAKAO SUZUKI, OSAMU TAKENAKA, KAZUO SHIBATA1969 Volume 66 Issue 6 Pages 815-821
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDiazonium-1-H-tetrazole (DHT) explored previously for discrimination of various states of histidine and tyrosine residues in proteins was applied to zinc insulin, zinc-free insulin and its oxidized A and B chains, and the following facts were revealed by photometric and amino acid analyses of their reaction products. One of the two histidine residues in zinc insulin and zinc-free insulin was bisazotized with DHT and the other one residue remained in the monoazotized stage. A similar experiment on the oxidized B chain containing these two histidine residues showed also the presence of a less reactive histidine residue which is hardly bisazotized. This phenomenon was, therefore, interpreted as due to an effect of neighboring amino acid residues in the B chain. The reactivity of the less reactive residue in the B chain was not appreciably changed by alkali denaturation. The biscoupling curve plotted against DHT concen-tration for zinc insulin rose less steeply as compared with the curve obtained for zinc-free insulin. Coordination of zinc ion to one of the histidine residues or folding of the insulin structure by this coordination was postulated to interpret this result. Three of the total four tyrosine residues in zinc insulin or in zinc-free insulin were bisazotized with DHT, and the other one residue remained in the monoazotized stage. The reaction curve obtained for the two tyrosine residues in the oxidized A chain rose more gradually and the curve for the two tyrosine residues in oxidized B chain rose more steeply as compared to the curve obtained for zinc or zinc-free insulin. An appreciable change in the native insulin structure by cleavage of the disulfide bonds was deduced from these data.View full abstractDownload PDF (476K) -
KATSUHIDE YUTANI, ASAKO YUTANI, TOSHIZO ISEMURA1969 Volume 66 Issue 6 Pages 823-829
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rate and extent of the renaturation increased with the increase of the con-centration of reduced Taka-amylase A [EC 3. 2. 1. 1] (1×10-4 to 1.5×10-2%). The most efficient renaturation was found to occur at the protein concentration of 1.3×10-2%. The decrease of renaturation in low concentration of the enzyme (1×10-3%) was not due to an adsorption of the enzyme on glassware.
The measurement of CD spectrum suggests that the reformation of helical structure occurs at the first stage in renaturation process. The changes in ultraviolet difference spectrum and the reappearence of enzymatic activity were found in the succeeding stage. When a concentration of the enzyme was again diluted in this stage, the rate of reactivation was dropped to the degree of renaturation at low enzyme concentration. The results show that the interaction among the polypeptide chains accelerates the rate of renaturation and it plays a role in the second stage of renaturation.View full abstractDownload PDF (483K) -
II. Purification and Spectral Properties of Cytochromes c-550 and c-554KEIZABURO MIKI, KAZUO OKUNUKI1969 Volume 66 Issue 6 Pages 831-843
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo c-type cytochromes were isolated from Bacillus subtilis K, a gram-positive bacterium, and purified by column chromatography on DEAE-cellulose and ion exchange resins. Cytochrome c-550 showed spectral properties similar to those of mammalian cytochrome c; it had absorption maxima at 528, 407, and 279mμ in the oxidized form, and at 550, 520, 414, 316, and 279mμ in the reduced form.
Cytochrome c-554 showed absorption maxima at 523, 409, and 280mμ in the oxidized form, and at 554, 521, 417, 316, and 280mμ in the reduced form. The α-peak of this cytochrome associated with a bump at 550mμ, and split into two peaks at 546mμ and 552mμ at liquid nitrogen temperature. CD*** spectra of the cytochrome showed that the chromophore responsible for the α-peak might be under the influence of anisotropic environments, and showed a large change in the ellipticity occurred in the Soret region with oxidation and reduction of the hemoprotein. Effects of pH, urea, guanidine-HCl, and SDS*** on the absorption spectra of cytochrome c-554 were studied. The absorption spectra of the ferricytochrome with cyanide, azide, and imidazole were also investigated.View full abstractDownload PDF (836K) -
III. Physicochemical and Enzymatic Properties of Cytochromes c-550 and c-554KEIZABURO MIKI, KAZUO OKUNUKI1969 Volume 66 Issue 6 Pages 845-854
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhysicochemical and enzymatic properties were determined with highly purified preparations of cytochromes c-550 and c-554 derived from Bacillus subtilis K.
Cytochrome c-550 possessed isoelectric point at pH 8.65 at 2°C and E0'** of 0.21 volt at pH 7.0 and 21°C. Molecular weight was determined to be 12, 500 by gel filtration method and 13, 000 on the basis of heme c content. Cytochrome c-550 scarcely reacted with bovine and yeast cytochromes a, but it reacted rapidly with Pseudomonas cytochrome oxidase [EC 1. 9. 3. 2] and Bacillus ETP**. It was reduced with NADH in the presence of ETP.
Cytochrome c-554 possessed one molecule of heme c in one molecule, although its α-peak at 554mμ was asymmetric, accompanied by a bump at 550mμ. Isoelectric point and E0' of the cytochrome were determined to be at pH 4.44 and 2°C and -0.08 volt at pH 7.0 and 21°C, respectively. Molecular weight of cytochrome c-554 was determined to be 14, 000 by gel filtration and amino acid analysis. Cytochrome c-554 reacted rapidly with Pseudomanas cytochrome oxidase, but scarcely reacted with bovine cytochrome a.
Both the hemoproteins were contained in the ETP and their function as an electron carrier was also investigated.View full abstractDownload PDF (1368K) -
TOSHIHIKO KOTAKE, SUNAO YACHIKU, FUMIO WADA1969 Volume 66 Issue 6 Pages 855-861
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucose-6-phosphatase [EC 3. 1. 3. 9] activity of kidney microsomes was increased by injection of parathyroid hormone into rats. This increase appears to be due to a specific and direct action of parathyroid hormone because it was not observed in the liver and the effect of parathyroid hormone was independent of adrenocortical function.
The increase in enzyme activity on injection of the hormone may be due to both increase in enzyme and a functional change of the microsomal membrane, and is de-pendent on RNA and protein syntheses.View full abstractDownload PDF (516K) -
KOZO OHTSUKI, CHIA LIEN LIU, HIROYUKI HATANO1969 Volume 66 Issue 6 Pages 863-865
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (208K) -
TAKIKO AKINO, MINORU AMANO, HIROMI MITSUI1969 Volume 66 Issue 6 Pages 867-869
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (169K) -
FUMIO WADA, HARUMI SHIMAKAWA1969 Volume 66 Issue 6 Pages 871-872
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (117K) -
SAKUZO FUKUI1969 Volume 66 Issue 6 Pages 873-876
Published: December 25, 1969
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (269K)
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