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Volume 63 (1968)
- Issue 6 Pages 689-
- Issue 5 Pages 561-
- Issue 4 Pages 409-
- Issue 3 Pages 279-
- Issue 2 Pages 139-
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Volume 63, Issue 3
Displaying 1-19 of 19 articles from this issue
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V. Evidence for a Phosphate Exchange Reaction between Adenosine Triphosphate and the “Reactive Myosin-phosphate Complex”HIROSHI NAKAMURA, YUJI TONOMURA1968 Volume 63 Issue 3 Pages 279-294
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn our previous work, it was shown that the initial reaction between myosin-ATPase [EC 3.6.1.3, ATP phosphohydrolase] and ATP produces one mole of “reactive myosin-phosphate complex” (EP) per 4×105g of myosin, which is TCA**-labile. However, there has been found so far no unequivocal evidence for a covalent bonding of phosphate to myosin in the complex. In the present work, therefore, a search was made for a phosphate exchange reaction between ATP and EP. The amount of TCA-labile 32Pi liberated was measured as a function of time after the reaction was started by adding 7-11P-labelled ATP to myosin in the ratio of 1 mole to 4×105g (the first, or control, experiment). In a subsequent experiment, 1 mole of non-labelled ATP was added to 4×105g of myosin, and at an appropriate time thereafter, a small amount of γ-32P-ATP, with about the same radioactivity as that of the ATP-32P used in the control experiment, was added to the myosin-ATP system (the second experiment). The 32Pi-liberation was followed after adding TCA to stop the reaction, and was compared with that in the control experiment. There are several lines of evidence either that the Michaelis complex (ES) is in equilibrium with myosin (E) and ATP (S), or that the amount of ES is negligibly small. Therefore, the time-course of TCA-labile 32Pi-liberation from the myosin-ATP system in the second experiment can be easily calculated from the time-course of the control experiment, if it is assumed that there is no phosphate-exchange reaction between EP and ATP. The difference between the experimental values and the ones calculated on this assumption can be attributed to the exchange of phosphate between EP and γ-32P-ATP.
Myosin was preincubated in 2.8M KCl and 10mM MgCl2 at pH 7.5 and 0°C, prior to the addition of ATP, and it was found that the initial reaction of the myosin-ATP system was not affected by preincubation lasting up to, at least, 3hr. The time-course of TCA-labile 32Pi liberation in the second experiment was insensitive to a variation in the amount of hot ATP (of constant radioactivity) from 0.005 to 0.05 moles per 4×105g of myosin, when this hot ATP was added to the system one minute after the addition of 1 mole of cold ATP per 4×105g of myosin.
Therefore, the amount contained in EP of an intermediate ex-changing P with the terminal P of ATP could be measured by the method mentioned above. In the presence of 2.8M KCl and 10mM MgCl2 and at pH 7.5 and 0°C, 15-20 per cent of the total EP were found to be P-exchangeable with ATP 1min after the addition of ATP to myosin at a molar ratio of 1:1. The content of phosphate-exchangeable intermediate in the total EP decreased with decreasing concentrations of KCl and MgCl2: it was 0-10 percent of EP in the presence of 1.08M KCl and 5mm MgCl2, and varied considerably from one preparation to another. It was not affected when the temperature was raised from 0 to 15°C, or when 0.1mm CaCl2 or 0.1mm EGTA was added to the reaction mixture.
In the presence of 1.1M KCl and 5mm MgC12 and at pH 7.5 and 0°C, the time-course of TCA-labile 32P i-liberation was followed, first, with a system in which 1 mole of hot ATP was added per 4×105g of myosin, and second, with the systems in which 1 mole of hot ATP per 4×105g of myosin was added various times after the addition of 1 mole of cold ATP. In this experiment a myosin preparation was used which showed no exchange reaction between EP and ATP under the above conditions. In this case, the initial rate of 32Pi-liberation was proportional to the amount of total E minus EPView full abstractDownload PDF (1167K) -
HIROKO TODA, IKUNOSHIN KATO, KOZO NARITA1968 Volume 63 Issue 3 Pages 295-301
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe essential calcium atom in Taka-amylase A [EC 3.2.1.1] could be removed reversibly by incubating the enzyme with EDTA at pH 8.0 and 50°C. Concomitantly, the masked sulfhydryl group became available to sulfhydryl reagents under the conditions for the removal of calcium. Modification of the resultant sulfhydryl group caused alteration in the state of the active site and affinity for calcium atom. One of the calcium-binding sites in Taka-amylase A is probably the sulfhydryl group of the sole cysteine residue. The sulfhydryl group seemed not to be the active site of the amylase, but likely to play a key role to maintain the active configuration by chelating with the essential calcium atom.View full abstractDownload PDF (538K) -
HIROKO TODA, KOZO NARITA1968 Volume 63 Issue 3 Pages 302-307
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acid composition of Bacillus subtilis saccharifying α-amylase [EC 3.2.1.1] was determined. The results suggested that the protein has consisted of approximately 331 amino acid residues. On the basis of the amino acid composition, the molecular weight of the amylase was estimated to be 41, 922. One cysteine residue was shown to be contained per mole of the enzyme as a masked state, which became reactive to the sulfhydryl reagents after inactivation with EDTA at 60°C and pH 8.0. Calcium and the sole sulfhydryl group seemed to be essential for the enzymatic activity. The peptide containing the essential sulfhydryl group was isolated from the peptic digest of the amylase and its constituent amino acids were preliminarily determined.View full abstractDownload PDF (398K) -
SATOSHI NAKAMURA, YASUYUKI OGURA1968 Volume 63 Issue 3 Pages 308-316
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The reaction of glucose oxidase [EC 1.1.3.4, β-D-glucose: oxygen oxidoreductase] obtained from Aspergillus niger was investigated by overall reaction kinetics as well as by the “stopped flow” and “rapid flow” methods. The experiments were carried out using D-glucose and 2-deoxy-D-glucose as substrates at pH 5.5 and at different temperatures (15°, 20°, 25°, and 30°).
2. The difference absorption spectrum of the enzyme (steady state minus reduced level), which was obtained by the “stopped flow” method, was the same as that of the oxidized minus reduced level. No ESR-signal was observed within 5 msec. after mixing the oxidized enzyme solution with D-glucose or the reduced form with molecular oxygen.
3. When D-glucose or 2-deoxy-D-glucose was used as substrate, the value of kmaxred, the rate constant for reduction of the FAD moiety of oxidized enzyme at a sufficient concentration of the substrate, was almost the same as that of respective Vm/e obtained by overall reaction kinetics, (Vm/e: the maximum velocity per unit enzyme concentration). The value of Km at 25°C for 2-deoxy-D-glucose was the same as that for D-glucose at the same temperature.
4. The following scheme for the action mechanism of the glucose oxidase of Aspergillus niger was proposed to account for the data obtained.
Eox+S_??_EoxS_??_Ered+P
Ered+O2_??_Eox+H2O2,
where Eox stands for the oxidized form of the enzyme, EoxS the enzyme (oxidized form)-substrate compound, Ered the reduced form of the enzyme, S the substrate (D-glucose or 2-deoxy-D-glucose) and P the product (δ-lactone).View full abstractDownload PDF (609K) -
SHOJI MIZUSHIMA1968 Volume 63 Issue 3 Pages 317-323
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane bound NADH2 dehydrogenase [EC 1.6.99.3] was extracted by a mild treatment with alkali and was purified in an almost homogeneous state. The enzyme contained FAD as a prosthetic group. The enzyme was specific for NADH2. 2, 6-Dichlorophenolindophenol and ferricyanide were effective electron acceptors, while the physiological acceptor remained unknown.
The purified dehydrogenase was recombined with the membrane fraction only when the alkaline membrane was treated with acidic buffer. The reconstructed system was, however, not similar to that in original protoplast membrane with respect to the extractability of the dehydrogenase.
A part of the dehydrogenase activity was not extracted from the membrane by the repetition of the treatment with alkali. This activity was much more sensitive to acidification than the extractable one, suggesting the existence of two kinds of NADH2 dehydrogenase in the membrane fraction.View full abstractDownload PDF (465K) -
SHOICHI HARIGAYA, YASUO OGAWA, HIDEO SUGITA1968 Volume 63 Issue 3 Pages 324-331
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A microsomal fraction which showed a considerable Ca-binding activity even in the absence of oxalate was prepared from rabbit red muscle, viz., soleus and semitendinosus muscle. The capacity of Cabinding reached 56mμmoles/mg protein which correspond to one third that of white muscle.
2. The Ca-uptake was not inhibited by various metabolic inhibitors, such as azide, oligomycin and 2, 4-dinitrophenol, which have been known to depress the Ca-uptake of mitochondria. It is very probable that the Ca-uptake of red muscle microsomes is due to the fragmented sarcoplasmic reticulum contained in this fraction.
3. This microsomal fraction inhibited the superprecipitation of myosin 13 and myofibrils and also the myofibrillar ATPase [EC 3.6.1.3] activity.
4. The properties of the ATPase activity of red muscle microsomes were different from those of white muscle in its response to Ca, GEDTA and metabolic inhibitors.
5. The rate of Ca-binding of red muscle microsomes was determined by the double beam spectrophotometric method. In view of this result together with the yield and the Ca-binding capacity of the microsomal fraction, the role of the sarcoplasmic reticulum in the con-traction-relaxation cycle was discussed. It was suggested that the sarco-plasmic reticulum of red muscle might play essentially the same role as that in white muscle.View full abstractDownload PDF (548K) -
ICHIO HONJO, HIDENARI TAKASAN, KAZUE OZAWA1968 Volume 63 Issue 3 Pages 332-340
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe present results show the isolation method of mitochondria with high respiratory control from guinea pig pancreas which is comparable to those prepared from other tissues.
Care was mainly taken to separate the brown pellets as quickly as possible, which were precipitated from pancreas homogenates between 2000×g for 7min and 6000×g for 10min.
The mitochondria) fractions prepared by this method consisted primarily of mitochondria with high phosphorylating efficiencies and were much less contaminated by non-mitochondrial particles, especially zymogen, than those obtained by other procedures.
The quality of the mitochondrial preparation is considered acceptable as a starting material for biochemical work.View full abstractDownload PDF (3567K) -
TATEO YAMANAKA, KAZUO OKUNUKI1968 Volume 63 Issue 3 Pages 341-346
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpectral and enzymatic properties of cytochrome c-555 obtained from Chlorobium thiosulphatophilum were studied in comparison with those of algal and nonsulphur purple bacterial c-type cytochromes. Chlorobium cytochrome c-555 had an asymmetric α-peak, the molar extinction coefficient of the peak was considerably low, and the ratio of Aγ(reduced)/ Aα(reduced) was very high. The cytochrome reacted fairly rapidly with Pseudomonas cytochrome oxidase [EC 1.9.3.2]. In these respects, the cytochrome c-555 was very similar to algal c-type cytochromes (the f-type cytochromes) but different from nonsulphur purple bacterial c-type cytochromes (the c2-type cytochromes).View full abstractDownload PDF (391K) -
VII. Occurrence of Mannose-6 Phosphate in Corbicula GlycolipidOSAMU ITASAKA1968 Volume 63 Issue 3 Pages 347-350
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phosphate component of a corbicula glycolipid, GL-3, was isolated from an acid hydrolyzate of the glycolipid by column chromatography on Dowex-1, formate form, and was identified as mannose-6 phosphate by gas chromatography after treatment with alkaline phos-phatase [EC 3.1.3. 1, E. coli] and by periodate oxidation. No other phosphorylated sugar was detected.View full abstractDownload PDF (231K) -
CHING TSANG HOU, YASUKIYO UMEMURA, MICHINORI NAKAMURA, SABURO FUNAHASH ...1968 Volume 63 Issue 3 Pages 351-360
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSteryl glucoside was synthesized using a particulate enzyme preparation from immature soybean seeds. The identification of this steryl glucoside is described. Some properties, and the subcellular localization of the enzyme are also reported. UDP-glucose and TDP-glucose served as glucosyl donors; the latter was 40% as effective as the former as a glucosyl donor. None of the other nucleotide glucoses tested nor UDP-glucuronic acid was active as a glycosyl donor.View full abstractDownload PDF (707K) -
ATSUMI KAMOGAWA, TOSHIO FUKUI, ZIRO NIKUNI1968 Volume 63 Issue 3 Pages 361-369
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-Glucan phosphorylase [EC 2.4.1.1] was isolated in crystalline state from potato tubers. The crystalline material showed apparent homo-geneity, and sedimented at 6.5S upon ultracentrifugation. The amino acid composition of the potato enzyme was rather similar to that of the muscle enzyme. We conclude from experiments with extensively purified glucose- l-phosphate and enzyme preparation that α-glucan phosphorylase cannot catalyze the “de novo” synthesis of polysaccharide from glucose-1-phosphate in the complete absence of primer. The effects of adding α-, β-, and gluco-amylases to the reaction mixture, and other features of this enzyme were also studied.View full abstractDownload PDF (2645K) -
YOSHIO IMAI, RYO SATO1968 Volume 63 Issue 3 Pages 370-379
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The Soret region of the difference spectrum appearing on addition of ethyl isocyanide to liver microsomes in the presence of NADPH and oxygen was found to consist of three absorption bands of different origin; i.e., the peak at 434mμ arising from the reagent interaction with oxidized P-450 and those at 430 and 455mμ due to the two different states of the ethyl isocyanide compound of reduced P-450. The contribution of the 430mμ peak was much smaller than that of the 455mμ peak.
2. The apparent dissociation constant of the ethyl isocyanide compound of the reduced hemoprotein, determined from the 455mμ peak, was smaller than the value expected from the affinity of the reagent for reduced P-450 and that of oxygen estimated from the aniline hydroxylase [EC 1.14.1.1] activity. The value was in agreement with that for the ethyl isocyanide interaction with oxidized P-450.
3. It was concluded that the reduced hemoprotein existed mainly in the state showing the 455mμ peak in the steady state established in the presence of NADPH, oxygen and ethyl isocyanide. Under these conditions, the reduction of microsomal P-450 by NADPH seemed to produce preferentially the “455 state” of the hemoprotein. The ethyl isocyanide bound to the heme seemed to be replaced rapidly by oxygen, when the 455 state was converted to the “430 state.”View full abstractDownload PDF (729K) -
YOSHIO IMAI, RYO SATO1968 Volume 63 Issue 3 Pages 380-389
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an appropriate concentration range, ethyl isocyanide activated, rather than inhibited, the aniline hydroxylase [EC 1.14.1.1] activity of rabbit liver microsomes. The dependence of the activity on the ethyl isocyanide concentration suggested that the reagent acted as both an activator and an inhibitor on the hydroxylase system. From the effect of oxygen tension on the activity in the presence of ethyl isocyanide, it was concluded that the inhibitory effect was due to the competition of the reagent with oxygen for the heme of reduced P-450. Assuming that the inhibitory effect could be neglected at infinite oxygen tension, the “ideal” activity under such an extreme condition was estimated from the activity dependence on the oxygen tension. The ideal activity, representing only the stimulatory effect, increased as the ethyl isocyanide concentration was increased. From this dependence, the ethyl isocyanide concentration giving half maximal stimulation could be determined. This value was affected by the aniline concentration, indicating that aniline interfered with the stimulatory effect of ethyl isocyanide. These and other observations suggested that the stimulatory effect resulted from a change in reactivity of oxidized P-450 caused by its combination with ethyl isocyanide. The effects of ethyl isocyanide on aniline hydroxylation were also affected by the hydrogen ion concentration. At pH 6.5 only the inhibitory effect was observable, but the stimulatory effect emerged and increased progressively as the pH was increased up to 8.0. The contribution of the two effects was also different depending on the source of liver microsomes and the nature of the hydroxylatable substrate.View full abstractDownload PDF (760K) -
VI. Structure of Hydroxy Acids and the Degradation by Escherichia coli K-12MICHINAO MIZUGAKI, MASANORI YONAHA, MITSURU UCHIYAMA, SEIICHI OKUI1968 Volume 63 Issue 3 Pages 390-394
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSE. coli has been shown to have a specific mode of the oxidative breakdown of hydroxy acids. The structual requirements leading to the interruption of metabolism was investigated.
5-Hydroxytetradecanoic acid δ-lactone, 10-hydroxytetradecanoic acid and mixtures of 10- and 9-hydroxyoctadecanoic acids were synthesized and employed as substrates.
9-Hydroxyoctadecanoic acid was completely oxidized after transient accumulation of 7-hydroxyhexadecanoic and 5-hydroxytetradecanoic acids. 10-Hydroxyoctadecanoic acid was converted by E. coli K-12 to 8-hydroxyhexadecanoic, 6-hydroxytetradecanoic and 4-hydroxydodec-anoic acids, further degradation was negligible. l0-Hydroxytetradecanoic acid was broken down leading to accumulation of 8-hydroxydodecanoic and 6-hydroxydecanoic acids in the culture medium.
On the basis of the results summerized in Fig. 8, it can be concluded that C2 cleavages of hydroxy fatty acids by E. coli cease when the total chain length of a hydroxy acid becomes twelve to ten, and, at the same time, the hydroxy group is on the sixth, fifth or forth carbon atom of the acid.View full abstractDownload PDF (321K) -
YOSHIO UENO, IKUKO UENO, KIYOHISA MIZUMOTO, TAKASHI TATSUNO1968 Volume 63 Issue 3 Pages 395-397
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (166K) -
ETSUKO MARUYAMA, KATSUKO KOJIMA, TANEAKI HIGASHI, YUKIYA SAKAMOTO1968 Volume 63 Issue 3 Pages 398-399
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (109K) -
AKIO SHIRAI, AKIRA ICHIHARA, TAKUMA TAKEMOTO, TOMOZO ABURAYA1968 Volume 63 Issue 3 Pages 400-402
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (153K) -
KOICHI SUZUKI, TOSHIO ANDO1968 Volume 63 Issue 3 Pages 403-405
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
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RYUICHI KATO, AKIRA TAKANAKA1968 Volume 63 Issue 3 Pages 406-408
Published: March 25, 1968
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (167K)
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