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Volume 67 (1970)
- Issue 6 Pages 753-
- Issue 5 Pages 611-
- Issue 4 Pages 507-
- Issue 3 Pages 333-
- Issue 2 Pages 157-
- Issue 1 Pages 1-
Volume 67, Issue 2
Displaying 1-16 of 16 articles from this issue
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TAKAYUKI OZAWA1970 Volume 67 Issue 2 Pages 157-174
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. In the phosphorylating electron transfer particle, incorporated 32P by oxidative phosphorylation into the bound adenine nucleotides was found mainly in ADP and partly in ATP, 92% and 8% of the total 32Pe, respectively. Both kinetic and enzymatic analyses demonstrated that the bound ATP was terminally labelled and the labelling was due mainly to 32Pi-ATP exchange reaction. The data indicated that the bound AMP was an earlier phosphoryl acceptor in the phosphorylation compartment.
2. Approximately 65% of esterified 32P was found to be tightly bound with the phosphorylation compartment, being eluted from the particle neither by incubation for 30min at 30°C, nor by the digestion of “head” and “stalk” of the particle with Nagarse, but by the existence of exogenous adenine nucleotides. In contrast to 32Pe among the bound nucleotides, 32Pe appeared mainly in the extra-particular ATP with 76% of the total 32Pe . The presence of fluoride increased the percentage up to 85%. A phosphoryl transfer between the bound and exogenous ADP was postulated.
3. Treatment of the particle with EDTA suppressed both the amount of the bound nucleotide and that of the incorporated 32P down to about 4%. The externally added AMP to EDTA particle increased the amount of 32P incorporated into the bound ADP up to three fold without concomitant increase in that into the bound ATP.
4. Fluoride suppressed 32P incorporation into the bound ATP but accelerated the incorporation into the bound pyridine nucleotides and flavins. Percentage of 32P-labelled ADP to the total esterified 32P was not influenced by fluoride. It is postulated that a complicate equilibrium of high energy phosphoryl transfer exists in the bound nucleotides.View full abstractDownload PDF (2130K) -
NOBUO KATO, KINJI OKABAYASHI, DEN'ICHI MIZUNO1970 Volume 67 Issue 2 Pages 175-184
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen exponentially growing cells of E. coil were treated with Mitomycin C (MC) or Panfuran (AF-5), the following results were obtained.
1. Simultaneous synthesis and degradation, i.e., a rapid turnover of RNA was observed in the presence of MC or AF-5, whereas DNA remained unaffected during the treatment.
2. The degraded RNA was shown to be the ribosomal RNA by methylated albumin kieselgur column chromatography.
3. The total activity of RNase I [EC 2. 7. 7. 16] in the treated cells was not different from that in the control cells. Ribosomes prepared from the drug treated cells were degraded at the same rate as those from control cells in the presence of such agent as EDTA, KCl or NaH2PO4.
4. By the treatment of E. coli with the drug, several new ribosomal particles appeared in the cell.View full abstractDownload PDF (698K) -
VIII. Modification of the Enzyme with Diazonium-1H-TetrazoleTAKAMICHI SHIMADA1970 Volume 67 Issue 2 Pages 185-198
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Myosin was treated with DHT (diazonium-lH-tctrazole) in the presence or absence of Mg-ATP. Results indicated that the SH-groups of myosin were modified by DHT.
2. To prevent reaction of the SH-groups of myosin with DHT, the groups were blocked reversibly by DGM through their conversion to disulfide bonds. The Ca2+-ATPase [EC 3. 6. 1. 3] activity of myosin disappeared on treatment with DGM (dithio-diglycolic acid dimethyl ester) for 50 hr and the amount of free SH-groups decreased to 2 moles per mole of myosin. The Ca2+-ATPase activity was restored to the original level by reversing the blocking reaction with excess β-ME or DTT.
3. DGM-treated myosin was treated with DHT, and then with β-ME or DTT. The Ca2+-ATPase activity decreased linearly with increase in the amount of monoazotyrosine in myosin, and complete inactivation occurred when approximately one mole of monoazo-tyrosine was formed per 4.0×105g of myosin. During treatment of DGM-treated myosin with DHT, the Ca2+-ATPase activity was protected to some extent by the presence of Mg-ATP.
4. H-Meromyosin prepared by tryptic digestion of azo-myosin had the same monoazo-tyrosine content as the parent myosin. All the monoazo-tyrosine was present in the f-subunit.
5. When a few moles of tyrosine residues per mole of myosin were modified with DHT, myosin became soluble even in water. One mole of tyrosine residue per mole of L-meromyosin fraction 1 reacted specifically with DHT, and filament formation in L-meromyosin fraction 1 was inhibited by this modification with DHT.View full abstractDownload PDF (2288K) -
Purification, Properties, and Identity with Microsomal NADPH-cytochrome c ReductaseHIROKO NISHIBAYASHI-YAMASHITA, RYO SATO1970 Volume 67 Issue 2 Pages 199-210
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA flavoprotein catalyzing the oxidation of NADPH by oxygen in the presence of vitamin K3 or other appropriate naphthoquinones as cofactor was solubilized and purified from rabbit liver microsomes. The most purified preparation was homogeneous in both sedimentation and electrophoretic analyses. Its molecular weight was determined to be 89, 000 and two moles of FAD were present per mole of protein. The enzymebound flavin was reducible not only by NADPH but also by NADH. In addition to the naphthoquinone-dependent NADPH oxidase activity, the purified enzyme catalyzed the NADPH-linked reductions of a variety of acceptors such as benzoquinones, ferricyanide, redox dyes and cytochrome c. Cytochrome b5 was also reduced slowly. NADH could act as a weak electron donor in the K3-dependent oxidase activity. The effects of pH, ionic strength and various inhibitors on both the K3-dependent NADPH oxidase and NADPH-cytochrome c reductase activities were compared. The results of these studies as well as the finding that both activities were purified in parallel established the identity of K3-dependent NADPH oxidase with microsomal NADPH-cytochrome c reductase [EC 1. 6. 2. 3]. Several kinetic parameters for the K3-dependent NADPH oxidase activity were determined. Finally, it was confirmed that in the oxidase reaction hydrogen peroxide was formed as the reduction product of oxygen.View full abstractDownload PDF (2086K) -
TOSHIO FUKUI, ATSUMI KAMOGAWA, ZIRO NIKUNI1970 Volume 67 Issue 2 Pages 211-218
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reaction of glyoxal with a-glucan phosphorylases [EC 2. 4. 1. 1] from potato and rabbit muscle was studied. Both phosphorylases were completely inactivated within an hour by incubating with approximately 1mM of glyoxal at pH 7.9 and at 30°C. The rate of reaction depended upon pH. Fall of one pH unit resulted in a decrease of the rate of inactivation to about one-tenth. The rate of inactivation by glyoxal was retarded by the presence of substrates; Pi, glucose 1-phosphate and starch. The muscle enzyme was also protected by the presence of its activator, AMP, against the inactivation by glyoxal. More protective effect was observed when both AMP and glucose 1-phosphate was present in the same reaction mixture than in the case of AMP alone. The potato enzyme was not protected by AMP. Less inactivation was attained with either formaldehyde and diacetyl. Glyoxylic acid was not effective. The complete inactivation occurred when approximately 12 moles of 14C-glyoxal were incorporated into the enzyme protein, while the incorporation continued almost linearly up to at least three hours in both enzymes.
Glyoxal-modified potato phosphorylase exhibited little increase in the absorbance around 330mμ, and sedimented as a single and symmetrical boundary with the same sedimentation velocity as the native enzyme upon ultracentrifugation. Total amino acid analysis of the fully inactivated enzyme revealed loss of lysine and arginine residues. Determination of arginine content showed that arginine residues were modified when the potato enzyme was completely inactivated.View full abstractDownload PDF (1235K) -
Existence of Lipid InhibitorsAKIHIKO KAWAGUCHI1970 Volume 67 Issue 2 Pages 219-227
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was demonstrated that the incorporation of radioactivity from 1-14C-acetate into the nonsaponifiable fraction in S. cerevisiae was depressed by the addition of ergosterol to the growth medium under strictly anaerobic conditions. These results suggested that ergosterol in the culture medium might control its own synthesis. Unexpectedly, with cell-free extracts of this organism, ergosterol had no effect on the incorporation of radioactivity from 1-14C-acetate into the nonsaponifiable fraction. However, the acidic lipids extracted from this organism were found to inhibit 14C-incorporation into the nonsaponifiable fraction from 1-14C-acetate as well as from 3-14C-3-hydroxy-3-methyl-glutaryl-CoA, but not from 2-14C-mevalonate. The results suggested that acidic lipids inhibited the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate. The inhibitors were purified by column chromatography followed by thin-layer chromatography. Four compounds which had inhibitory activities were found on the thin-layer plate. One of the inhibitors was purified 1, 000 fold on the basis of its inhibitory activity. The possible role of these inhibitors in the control of ergosterol synthesis in yeast is discussed.View full abstractDownload PDF (631K) -
XII Gas Production from Amines and NitriteTERUO MATSUBARA1970 Volume 67 Issue 2 Pages 229-235
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Nitrogen was formed from dimethyl-p-phenylenediamine (DMPD) and nitrite (DMPD-nitrite system) by Pseudomonas denitrifrcans. The formation of N2 in the DMPD-nitrite system was not inhibited by 10-2M NaN3 which perfectly suppressed the formation of N2 in the normal denitrification of the lactate-nitrite system. In the crude cell extract, the activity of N2 formation in the lactate-nitrite system was lost entirely, while N2 formation in the DMPD-nitrite system remained intact. DMPD inhibited the reduction of N2O to N2 NO reacted with DMPD nonenzymatically, producing N2
2. From hydroxylamine and nitrite the intact cells produced N2O, and in the concomitant presence of lactate as hydrogen donor, produced N2O and NO instead. When a lower concentration of hydroxylamine was used, it was utilized only a little as a nitrogen source but it acted as an inhibitor, where N2O, N2 together with a small amount of NO were produced from nitrite and lactate.
3. The formation of N2 from N2O was strongly suppressed in the presence of both NO and NH2OH. The reduction of NO to N2O, the second enzymatic step of nitrite reduction, was also sensitive to hydroxylamine
4. A possible scheme of the process of gas formation from nitrite under various conditions is presented.View full abstractDownload PDF (435K) -
MASAO TAKEUCHI, AKIRA TSUGITA1970 Volume 67 Issue 2 Pages 237-248
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nuclear fraction was isolated from B. subtilis. It is composed of DNA, RNA, and protein in the ratio of 1:0.2-0.3:0.8-0.9. The physicochemical properties of the nuclear fraction were also studied.
The nuclear fraction was dissociated by a CsCl density gradient centrifugation into DNA with a portion of RNA, free RNA, protein with another portion of RNA, and free protein. The RNA fractions thus separated had different sedimentation coefficients.
The amino acid composition of the protein in the nuclear fraction was analyzed. From the analysis, the protein was found to be either a neutral or an acidic protein, i.e., different from the nuclear protein in animal cells (histone), which have been known to be basic.
When the nuclear protein was mixed with the nuclear DNA, a protein-DNA complex was formed. The recombined complex behaved in the same manner as the original nuclear fraction with regard to a melting profile and Sephadex column chromatography.View full abstractDownload PDF (880K) -
TSUNEO OMURA, SACHIKO TAKESUE1970 Volume 67 Issue 2 Pages 249-257
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A new procedure for the purification of cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes is described. This procedure is a preparative application of a small-scale procedure reported earlier, and is suitable for purifying both enzymes simultaneously starting from several hundred grams or more of rat liver. The method is applicable to liver microsomes of other animals.
2. The recovery of cytochrome b5 and NADPH-cytochrome c reductase from microsomes was 35-45%, and the homogeneity of purified preparations was confirmed by sedimentation analysis and disc electrophoresis. The high purity of the preparations obtained by the previous small-scale procedure was also confirmed.
3. Purified NADPH-cytochrome c reductase was reducible by either NADPH or NADII, and had a very weak N-ADH-linked reductase activity.View full abstractDownload PDF (1402K) -
SACHIKO TAKESUE, TSUNEO OMURA1970 Volume 67 Issue 2 Pages 259-266
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. NADH-cytochrome b5 reductase of rat liver microsomes was efficiently solubilized by incubation with the lysosomal fraction of the same tissue. Examination of optimum conditions for the solubilization suggested the participation of a lysosomal enzyme (or enzymes) in the release of the reductase from microsomal particles. Available evidence denied the principal role of Naja naja venom in the solubilization of the reductase reported by previous workers.
2. Solubilization of microsomal enzymes by lysosomal digestion showed a peculiar specificity. While most of NADH-cytochrome b5 reductase [EC 1. 6. 2. 2] was easily made soluble by the treatment, NADPH-cytochrome c reductase and cytochrome b5 were solubilized to much lesser extents. Only a small portion of microsomal protein was solubilized, and phospholipid was not released detectably from microsomes.
3. In contrast to lysosomes, digestion with trypsin [EC 3. 4. 4. 4], Nagarse, and other proteinases failed to solubilize NADH-cytochrome b5 reductase, whereas these proteinases easily solubilized NADPH-cytochrome c reductase and cytochrome b5 from microsomes. The nature of the effective solubilizing factor in lysosomes is still to be elucidated.
4. NADH-cytochrome b5 reductase activity was considerably stimulated when the reductase was solubilized from microsomes by lysosomal digestion. NADH-ferricyanide reductase activity showed no stimulation by the treatment. However, these two activities were released in parallel from microsomes, and seem to be associated with the same enzyme.View full abstractDownload PDF (643K) -
SACHIKO TAKESUE, TSUNEO OMURA1970 Volume 67 Issue 2 Pages 267-276
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A new method for the purification of NADH-cytochrome b5 reductase [EC 1. 6. 2. 2] from rat liver microsomes is presented. Solubilization of the reductase was achieved by incubating liver microsomes with lysosomes of the same tissue, and external addition of solubilizing agents was not needed. The method is suitable For a large scale preparation of the purified reductase starting from l kg or more of liver.
2. The purification ratio of the reductase activity was 600 to 700. The overall yield from microsomes was about 40%. The properties of the purified enzyme were almost identical with those reported by previous workers for “snake venom-solubilized” enzyme.
3. The purified enzyme preparations obtained by this method still contained two protein components with identical enzymatic activities and very similar physicochemical properties. These two components were immunologically indistinguishable. Examination of solubilization treatment suggested that these two NADH-cytochrome b5 reductase components had been derived from a single enzyme present in microsomes, and long incubation with lysosomes was responsible for the appearance of two components.
4. NADH-ferricyanide reductase activity of liver microsomes could be mostly explained by NADH-cytochrome b5 reductase. The reduction of neotetrazolium by microsomes was, however, dependent on the interaction of the reductase with cytochrome b5 in microsomal membranes as in the case of microsomal NADH-cytochrome c reductase.View full abstractDownload PDF (2321K) -
HARUO SUZUKI, YASUYUKI OGURA1970 Volume 67 Issue 2 Pages 277-289
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The rates of oxidation and reduction of FMN and protoheme, which are the prosthetic groups of L(+)-lactate dehydrogenase [EC 1. 1. 2. 3, L-lactate: ferricytochrome c oxidoreductase], were estimated spectrophotometrically by the “stopped flow method.” The rates of reduction of these prosthetic groups were measured also with the “continuous flow method” by mixing the oxidized enzyme with a solution of the substrate under anaerobic conditions. The values obtained were compared with that of the rate of the overall reaction.
2. Although no formation of a semiquinone of the bound FMN was observed in the steady state at a sufficient concentration of ferricyanide, it was found that the semiquinone of the bound FMN can be produced very rapidly when the enzyme solution (oxidized form) is mixed with the substrate in the absence of electron acceptor under anaerobic conditions. It was suggested that the enzyme having the semiquinone form of the bound FMN is an active intermediate which appears during the reaction.
3. Assuming that an electron acceptor can react directly with the reduced protoheme but not with the reduced FMN, a mechanism of this enzyme reaction was proposed. On the basis of the kinetic data, it was concluded that the rate-limiting step in the overall reaction is the hydrogen transfer from the substrate attached to the enzyme to the bound FMN, and that the intramolecular electron transfer from the reduced FMN (FMNH2 or FMNH.) to the oxidized protoheme is very rapid.View full abstractDownload PDF (956K) -
HARUO SUZUKI, YASUYUKI OGURA1970 Volume 67 Issue 2 Pages 291-295
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of the pH value on the rate of the overall reaction catalyzed by L(+)-lactate dehydrogenase [EC 1. 1. 2. 3] was studied spectrophotometrically using ferricyanide as electron acceptor. The rates of reduction of the FMN and protoheme of the enzyme with L-lactate at various pH values were measured by the “stopped flow method” using ferricyanide as acceptor. From the relationships between the kinetic parameters and the pH value, the acidic pK-values were found to be 5.3 and 6.0 and the alkaline ones to be 8.8 and 9.7. It was concluded that two kinds of ionizable groups in the enzymeunit are essential for activity, that shifts of the pK-values occur on binding of the substrate to the enzyme-unit, and that in the overall reaction the hydrogen transfer reaction from the substrate to the bound FMMN is inhibited by the association and dissociation of protons of the essential groups.View full abstractDownload PDF (357K) -
SHINJI ISHIKAWA1970 Volume 67 Issue 2 Pages 297-312
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A system for oxidative phosphorylation was reconstituted from three components of Micrococcus lysodeikticus: membrane fragments, coupling factor cF and coupling factor Ft, or from membrane fragments and either one of these coupling factor.
2. ATPase [EC 3. 6. 1. 3] activity which was sensitive to the cation translocating uncoupler, gramicidin A was also reconstituted with this system. It was suggested that the reconstitution of this gramicidin sensitive ATPase indicated the partial or total reconstitution of energy transfer starting from ATP. The effects of other antibiotics affecting ion transport across the inner mitochondrial membrane were also tested on the reconstituted phosphorylation and ATPase systems.
3. The effects of trypsin [EC 3. 4. 4. 4] on the isolated coupling factors and on the reconstituted system were examined. Only the isolated factors were highly susceptible to trypsin, whereas the reconstituted system was resistant in the presence of divalent cations.
4. The results were compared with those on reconstituted mitochondrial and chloroplast energy transfer systems, and with other ATPase systems of the bacterial membrane.View full abstractDownload PDF (1179K) -
HISAO KATO, SHIGEHARU NAGASAWA, TOMOJI SUZUKI1970 Volume 67 Issue 2 Pages 313-323
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe possible numbers of sulfhydryl groups and disulfide bridges in bovine kininogen-II*, which is a precursor protein of physiologically active peptide, bradykinin or kallidin, have been evaluated by means of 14C-monoiodoacetic acid before or after the reduction with β-mercaptoethanol and by means of amino acid analysis after reduction and alkylation. The figures were in good agreement and no sulfhydryl groups were detectable even in the presence of 8M urea, and suggest that all half cystine residues participate in the formation of disulfide bridges. Approximately seven disulfide bonds were determined per molecule, assuming a molecular weight of about 50, 000.
Immunological properties of intact kininogen-II and its modified proteins were examined with antikininogen-II antibody prepared from immunized rabbit serum. In immunoelectrophoresis, two precipitin lines of the kininogen-II against the antibody were observed, suggesting tendency with an antigenic heterogeneity or microheterogeneity of the intact protein. On immunodiffusion, both native and kinin-free-kininogen reacted identically with the antikininogen antibody. However, no cross-reaction was found between the S-carboxymethylated protein and the antibody, indicating complete abolishment of the antigenic activity by the destruction of disulfide bonds. Kininfree-kininogen cross-reacted positively with the antikininogen antibody and this suggests that the kinin moiety in the polypeptide chain of kininogen molecule does not participate in formation of the antibody as a determinant group.
Treatment of bovine kininogen-II with cyanogen bromide produced a fragment containing kinin peptide sequence. The BrCN-fragment was deduced to have following amino acid sequence:
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-HomoserView full abstractDownload PDF (1545K) -
SHIN-ICHIRO EJIRI, HIDEHARU TAIRA, KENSUKE SHIMURA1970 Volume 67 Issue 2 Pages 329-331
Published: February 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (234K)
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