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Volume 78 (1975)
- Issue 6 Pages 1113-
- Issue 5 Pages 863-
- Issue 4 Pages 641-
- Issue 3 Pages 435-
- Issue 2 Pages 247-
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Volume 78, Issue 4
Displaying 1-26 of 26 articles from this issue
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Minoru KURODA, Fumio SAKIYAMA, Kozo NARITA1975 Volume 78 Issue 4 Pages 641-651
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA tryptophan residue in hen's egg-white lysozyme [EC 3. 2. 1. 17] was modified by ozone in an aqueous solution. One of the six tryptophan residues in the enzyme was oxidized to N'-formylkynurenine with concomitant loss of the enzymatic activity. Physiccchemical studies of this modified enzyme (OL-I) revealed that the ozonization of lysozyme in aqueous media resulted in little change of the gross molecular conformation. It was deduced that the modified tryptophan residue in OL-I was possibly located in position 62 (or 63) of the protein.View full abstractDownload PDF (742K) -
I. Preparation and Characteristics of an Affinity Adsorbent Containing Tryptic Peptides from Protamine as LigandsKen-ichi KASAI, Shin-ichi ISHII1975 Volume 78 Issue 4 Pages 653-662
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn adsorbent for the affinity chromatography of trypsin [EC 3. 4. 21. 4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di-and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was adsorbed at relatively low pH (7-4), but was not adsorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not adsorbed. The adsorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.View full abstractDownload PDF (807K) -
Nobuo SHIMAMOTO, Hideo FUKUTOME1975 Volume 78 Issue 4 Pages 663-671
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of substituents on the steady state and pre-steady state kinetics in α-chymotrypsin [EC 3. 4. 21. l]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-broma-, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased kcat for all substrates examined and greatly diminished the substrate activation for the activatable substrates. This suggests that substrate activation is brought about by binding of the substrate(s) to activator binding site(s).
The value of kcat decreased in accordance with increase of the σ- value of substituents. On the other hand, kcat/Km(app) showed an opposite σ- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The σ- dependence of kcat is apparently not consistent with the prediction derived from that of kCat/Km(app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.View full abstractDownload PDF (688K) -
Kazuyuki SUGAHARA, Susumu FUNAKOSHI, Ikuo FUNAKOSHI, Pertti AULA, Ikuo ...1975 Volume 78 Issue 4 Pages 673-678
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo major glycoasparagines (2-acetamido-N-(4'-L-aspartyl)-2-deoxy-β-D-glycosyIamines) were isolated from the urine of patients with aspartylglycosylaminuria (AGU). They were composed of equimolar amounts of sialic acid, galactose, glucosamine, and aspartic acid. They were isomeric with respect to the position of sialic acid attachment, since they produced the same glycoasparagine on incubation with the neuraminidase from Clostridium perfringens. The structure of the resulting sialic acidfree glycoasparagine was determined as β-Gal-(l→4)-β-GlcNAc-Asn based on the following findings. It produced galactose on incubation with β-galactosidase, and N-acetyllactosamine and aspartic acid on incubation with 4-L-aspartylglycosylamine amido hydrolase.View full abstractDownload PDF (1454K) -
Tadashi TAI, Katsuko YAMASHITA, Akira KOBATA1975 Volume 78 Issue 4 Pages 679-686
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA series of partially O-methylated N-methylglucosamines was synthesized by limited-time methylation of methyl-2-N-methylacetamido-2-deoxyglucopyranoside by Kuha's procedure, followed by acid hydrolysis. These partially O-methylated N-methylglucosamines were separated satisfactorily by gas chromatography on a column of OV-17 on Gas-chrom Q as ammo alditol acetates and identified from their mass spectra. For specific analysis of the methylated aminosugar derivatives, a mass fragmentographic method was established. Methylated aminosugars can be successfully determined in amounts as low as about 1 ng by this method.View full abstractDownload PDF (608K) -
Shun-ichiroh OGATA, Takashi MURAMATSU, Akira KOBATA1975 Volume 78 Issue 4 Pages 687-696
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing [3H]-labeled oligosaccharides, we found that the presence of at least two α-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligsaccharides to be retained by a concanavalin A-Sepharose column. This finding is also applicable to N-[I4C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which had been purified by paper electrophoresis, were further separated into two fractions by chromatoeraphy on a concanavalin A-Sepharose column.View full abstractDownload PDF (787K) -
Ikukatsu SUZUKI, Hitoshi INABA, Yukio NAITO, Takaharu MIZUTANI, Akira ...1975 Volume 78 Issue 4 Pages 697-703
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has previously been shown in our laboratory that the hypocalcemic substances purified from the thymus have a potent lymphocyte-increasing action in mice. Then, lymphocyte-increasing activity was examined with bovine parotid gland extracts, which showed a hypocalcemic activity as potent as that of the thymus extracts. The lymphocyte-increasing activity was assayed in the littermates of neonatal mice of Swiss-Webster strain; the materials used for this experiment were purified preparations and several fractions obtained from the parotid gland extracts in the course of purification. It was found that the potency of lymphocyte-increasing activity rose approximately in parallel with the rise in hypocalcemic activity with progress in purification.
The final product, which was purified from the gland by isoelectric precipitation at pH 5.4, fractional precipitation with ammonium sulfate, chromatography on DEAE-cellulose, gel filtration on Sepharose 6B and preparative disc electrophoresis, gave a single band in polyacrylamide gel electrophoresis. Its intravenous injection in rabbits, in a dose of 10υg/kg, produced a significant lowering in serum calcium (percent decrease 10.24±1.06%) compared with control animals given an injection of physiological saline. Intraperitoneal injection of this purified product, in a dose of 0.5μg/ mouse, in the littermates of neonatal mice, also produced a significant increase (ratio of lymphocytes to polymorphs 2.47±0.07) in lymphocytes compared with control animals injected with physiological saline (L/P ratio 1.57±0.05). These facts suggest that this purified protein fraction inherently contains both activities, but the possibility cannot be ruled out of slight contamination by a substance having a high activity. On the other hand, a fraction having no activity for lowering serum calcium but which had activity for increasing the lymphocytes was obtained. This is the first paper to report the presence of lymphocyte-increasing substances in the bovine parotid gland and the purification of one of the substances from the gland.View full abstractDownload PDF (1380K) -
Nobuyuki ITANI, Seiki KURAMITSU, Kiyoshi IKEDA, Kozo HAMAGUCHI1975 Volume 78 Issue 4 Pages 705-711
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe difference spectra of hen and turkey egg-white lysozymes [EC 3. 2. 1. 17] produced by acidification were measured. The difference spectra of both lysozymes had peaks at 295 and 301nm which are characteristic of tryptophyl residues. The pH dependence curves of the extinction differences (Δε) at 301nm and 295nm for hen lysozyme were identical with the corresponding curves for turkey lysozyme. The pH dependence of Δε at 301nm was analyzed assuming that the extinction at 301nm is due to Tip 108 only, which interacts with the catalytic carboxyls, Glu 35 and Asp 52. The macroscopic pK values of Glu 35 and Asp 52 in both lysozymes thus determined were 6.0 and 3.3, respectively. These values were in excellent agreement with those determined by measuring the pH dependence of the circular dichroic band at 305nm (Kuramitsu et al. (1974) J. Biochem. 76, 671-683; (1975) J. Biochem. 77, 291-301). The pH dependence of Δε at 295nm could not be completely explained in terms of the electrostatic effects of the catalytic groups on Trp 108.View full abstractDownload PDF (511K) -
Hiroaki SUGITA, Koichi SEKIGUCHI1975 Volume 78 Issue 4 Pages 713-718
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe heterogeneity of arthropod hemocyanins was studied by polyacrylamide gel electrophoresis and immunochemical techniques. The spider (Argiope bruennichii), the scorpion (Heterometrus sp.), and the horseshoe crab (Tachypleus tridentatus) were found to have 4, 5, and 5 minimum functional units of hemocyanin, respectively, the apparent molecular weights of which were 79, 000, 81, 000, and 80, 000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.View full abstractDownload PDF (2785K) -
Effects of Ligands upon the Raman Spectra of Various C-type CytochromesTeizo KITAGAWA, Yoshimasa KYGOGOKU, Tetsutaro IIZUKA, Masao IKEDA-SAIT ...1975 Volume 78 Issue 4 Pages 719-728
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron was found around 1540cm-1. This ligand-sensitive Raman line indicated the transition from acidic (1545cm-1) to alkaline (1533 cm-1) forms with pK7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome b5 are histidines, most of the axial ligands of the four hemes in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical distortion of heme.View full abstractDownload PDF (782K) -
Kazuo OGAWA, Makoto OKUNO, Hideo MOHRI1975 Volume 78 Issue 4 Pages 729-737
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA comparison is made between dynein [flagellar ATPase; EC 3. 6. 1. 3], purified from sea urchin sperm flagella, and muscle myosin.
The amino acid composition of dynein was found to be statistically different from that of myosin. The same was true of their tryptic fragments retaining ATPase activity, i.e., Fragment A of dynein and heavy merornyosin. At low ionic strength, no superprecipitation took place when ATP was added to a mixture of dynein and actin, and stimulation of the Mg2+-ATPase activity of dynein remained below 50% even when a one-hundred-fold excess of actin was present. No viscosity drop was caused by adding ATP to a solution containing dynein and actin. Anti-myosin antiserum did not react with dynein, while anti-Fragment A antiserum formed no precipitin line against myosin. Furthermore, the amount of dynein that combined with F-actin was less than one-fifth of the amount of dynein that fully combined with microtubules. These results are consistent with the dissimilarity in enzymatic and other physicochemical properties of these two proteins.View full abstractDownload PDF (726K) -
Norio OOKUBO, Hitoshi UENO, Tatsuo OOI1975 Volume 78 Issue 4 Pages 739-747
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit skeletal tropomyosin was separated into two components, α and β, by CM cellulose column chromatography in the presence of urea. The two components are apparently different from TN-T, since, 1) upon addition of the components to F-actin solutions, they increase the degree of flow birefringence Δn, while TN-T does not, 2) the reduced mean residue elipticities [θ] at 220nm are about 2.5-fold higher than for TN-T, and they contain no proline. These features are similar to those of intact tropomyosin, but the two components are not identical for the following reasons; 1) leucine is the C-terminus of the β component and isoleucine is the C-terminus of the α component, 2) the β component has a lower helicity and a somewhat lower capacity to increase Δn of F-actin solutions than the a component, and 3) the β component has a higher content of glutamic acid and methionine than the α component. The two components can be crystallized into paracrystals in the presence of magnesium. Electron micrographs of the paracrystals of both components show a band pattern with 400 A periodicity.
Bovine cardiac tropomyosin migrates on SDS gels as two poorly resolved bands, which could be separated by CM cellulose column chromatography. The C-terminus of the slower moving component was leucine, and that of the faster moving component was isoleucine, corresponding to the β and α components of skeletal tropomyosin.View full abstractDownload PDF (2414K) -
Transition of the Phosphorylated Intermediate from an ADP-sensitive to an ADP-insensitive FromYoshihiro FUKUSHIMA, Yuji TONOMURA1975 Volume 78 Issue 4 Pages 749-755
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNa+-K+-Dependent ATPase [EC 3. 6. 1. 3] was preincubated with ATP in the presence of a high concentration of MgCl2, and the phosphorylated intermediate, EP, was formed by adding a high concentration of NaCl. The following results showed that EP was converted from an ADP-sensitive to an ADP-insensitive form by a single turnover of the ATPase reaction.
1. After initiating the reaction by adding NaCl, almost all the EP was at first sensitive to added ADP, but its sensitivity to ADP decreased with increase in the time interval between the additions of NaCl and of ADP.
2. Both in the presence and absence of KC1, the time course of the replacement of ADP-sensitive EP by ADP-insensitive EP coincided with the time course of the decomposition of EP after addition of EDTA.View full abstractDownload PDF (479K) -
Michihiro SUMIDA, Sadao SASAKI1975 Volume 78 Issue 4 Pages 757-762
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit antiserum was prepared against a partially purified Ca2+, Mg2+-dependent ATPase [EC 3. 6. 1. 3] of the SR isolated from chicken skeletal muscle. The γ-globulin fraction of antiserum contained antibodies which combined with the purified ATPase and the SR vesicles. Binding of the antibodies strongly inhibited active transport of Ca2+ ions into the SR, but not passive leakage of Ca2+ ions from the SR. The antibodies scarcely affected the ATPase activity.View full abstractDownload PDF (1237K) -
Takashi OSUMI, Toichi EBISUNO, Hidehiko NAKANO, Hirohiko KATSUKI1975 Volume 78 Issue 4 Pages 763-772
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a cell-free extract of Rhodospirillum rubrum, studies were made of the condensation reaction between propionyl-CoA and glyoxylate. When [14C] propionate was incubated with the extract in the presence of glyoxylate, ATP, CoA, Mg2+, and Mn2+, radioactivity was incorporated into several compounds. Two of the main products were characterized as citramalate (CMA) and erythro-β-meihylmalate (erythro-MMA) on the basis of their behavior compared with authentic samples of CMA and erythro-MMA in the following three analyses: (i) paper chromatography using two solvent systems, (ii) radio-gas chromatography of their methyl esters, and (iii) chemical conversion to readily crystallizable derivatives, that is, citramalyl chloralide for CMA, and thymine for MMA. The CMA was thought to be of L(+)-form based on the results of optical resolution with brucine and also its susceptibility to L(+)-citramalate lyase of Clostridium tetanomorphum.
When the reaction was carried out with lower concentrations of the enzyme, only MMA was accumulated. However, when the reaction was allowed to proceed further after addition of higher concentrations of the enzyme and of excess semicarbazide to prevent further condensation, the amount of accumulated MMA was decreased and CMA was formed instead. Furthermore, the time course of MMA and CMA formation exhibited a pattern typical of a precursor-product relationship. From these results, it was concluded that MMA was formed by α-condensation between propionyl-CoA and glyoxylate, and that CMA was derived from MMA, possibly from its CoA derivative.View full abstractDownload PDF (869K) -
Yoshihiro MIYAKE, Yusuke NAKAMURA, Naoko TAKAYAMA, Kihachiro HORIIKE1975 Volume 78 Issue 4 Pages 773-783
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics of α-NADH-dichlorophenolindophenol (DCPIP) and α-NADH-cytochrome c reductase reactions of rat liver microsomes showed that the reactions proceeded by a ping-pong mechanism, and that the oxidation of α-NADH was the rate-determining reaction. The DCPIP-reducing activity with α-NADH in the presence of ADP was about 1% of that with β-NADH. ADP inhibited the α-NADH-DCPIP reductase reaction in a competitive manner with respect to α-NADH and a value of 1.2mM for the inhibition constant was obtained. ADP also inhibited cytochrome b5 reduction with α-NADH. More than 90% of cytochrome b5 was reduced under conditions where 90% of the α-NADH-DCPIP reductase activity was suppressed with ADP. The reduction of DCPIP with α-NADH preceded that of cytochrome b5, but the reductions partly overlapped. From these results, a diversed electron flow from α-NADH to cytochrome b5 and electron sharing between cytochrome b5 and DCPIP were indicated. α-NAD+ also inhibited the α-NADH-DCPIP reductase reaction. Analyses of the inhibition indicated that two types of α-NADH-DCPIP reductase reaction existed, one of which was resistant to α-NAD+ inhibition. In contrast to the reoxidation of β-NADH-reduced cytochrome b5, the process was largely monophasic when cytochrome b5, was reduced with β-NADH.View full abstractDownload PDF (872K) -
III. Spectral Characterization of Cytochrome P-450Yuzo YOSHIDA, Hiroshi KUMAOKA1975 Volume 78 Issue 4 Pages 785-794
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA carbon monoxide-binding pigment which shows an absorption peak at about 450nm in the reduced carbon monoxide difference spectrum was purified from the microsomal fraction of yeast grown anaerobically. The spectral characteristics of the pigment were practically identical with those of cytochrome P-450 of hepatic microsomes, especially from polycyclic hydrocarbon-induced animals.
The pigment was denatured to P-420, and bound with ethyl isocyanide in the reduced state. Although Type I spectral change was not evident, the pigment showed Type II and modified Type II spectral changes upon binding with some organic compounds, as in the case of hepatic cytochrome P-450.
These observations clearly indicate that the carbon monoxide-binding pigment of yeast microsomes may be designated as cytochrome P-450 of yeast.View full abstractDownload PDF (748K) -
Tatsuo ERABI, Tomihiko HIGUTI, Tomisaburo KAKUNO, Jinpei YAMASHITA, Ma ...1975 Volume 78 Issue 4 Pages 795-801
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRedox components bound with chromatophores of Rhodospirillum rubrum, and pure samples of ubiquinone-10 and rhodoquinone were studied polarographically at 24°.
In a mixture of ethanol and water (4:1, v/v) at pH7, ubiquinone-10 and rhodoquinone had half-wave potentials (E1/2) of+43mV and-63mV, respectively. For both quinones, values of the electron transfer number (n) were 2, and plots of E1/2 versus pH formed straight lines with slopes of-30mV/pH in the neutral pH range; thus, values of the proton transfer number (n-a) were estimated to be 1 for both quinones.
When bound with chromatophores, ubiquinone-10 and rhodoquinone had E1/2 values of +50mV (n=2) and -30mV (n=2), respectively, at pH7. Values of (n-a) were estimated to be 1 for ubiquinone-10 and 2 for rhodoquinone.
A component (POC-170) thought to be one of the active center bacteriochlorophylls (Liac-890) was characterized; it has E1/2 value of -170mV at pH7 and its oxidation-reduction is possibly brought about by dehydrogenation-hydrogenation.
Conceivably, the oxidation-reduction sites of ubiquinone-10, rhodoquinone and POC_170 partly, if not all, exist on the surface of chromatophore membrane or project outside the membrane, because of their accessibility to the polarographic electrode.View full abstractDownload PDF (531K) -
Yoshiko KASHIWABARA, Hiroko NAKAGAWA, Genji MATSUKI, Ryo SATO1975 Volume 78 Issue 4 Pages 803-810
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome c reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.View full abstractDownload PDF (655K) -
Takeshi MATSUMOTO, Takahiro UCHIDA1975 Volume 78 Issue 4 Pages 811-815
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChanges in the activity of adenylate cyclase [EC 4. 6. 1. 1] of serum-stimulated hamster BHK cells in culture were investigated in relation to changes in the intracellular level of adenosine 3', 5'-cyclic monophosphate (cyclic AMP). Addition of calf serum to quiescent cultures decreases the activity of adenylate cyclase, followed by cellular DNA synthesis. Cyclic AMP levels drop in parallel with the decrease in adenylate cyclase activity. Kinetic analysis of cyclic AMP phosphodiesterase [EC 3. 1. 4. 17] revealed that BHK cells contain two forms of the enzyme, one with a Km of 0.77 μM and the other with a Km of 17.0/μM. The activity of these enzymes is not affected by the addition of serum to tissue culture cells. The findings indicate that the decrease in adenylate cyclase activity is directly responsible for the decrease in cyclic AMP levels that is observed immediately after serum addition. Low levels of cyclic AMP continue for several hours during serum treatment, followed by a transient increase in cyclic AMP in the early stages of cellular DNA synthesis, at which time the activity of cyclic AMP phosphodiesterase with a low Km shows a slight decrease.View full abstractDownload PDF (393K) -
Toshihiro ITOH, Hiroshi KANEKO1975 Volume 78 Issue 4 Pages 817-820
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe occurrence of a novel phospholipid, pyrophosphatidic acid, in the lipid extracts of yeasts (23 species), bacteria (E. coli), algae (chlorella), mammalia (human, rabbit, guinea pig, and mouse), insect (cockroach), fish (carp), mollusc (clam), and spermatophyta (spinach) was investigated. Pyrophosphatidic acid was found exclusively in the lipid extracts of several kinds of yeast species, but not in other normal living species (animals, plants, and microorganisms) so far investigated. All of the yeast species containing this lipid belong to the asporogenous yeasts (Cryptococcus neoformans CBS-132, Cryptococcus laurentii Z 6-5, Rhodotorula glutinis H 3-9-1, Rhodotorula rubra AY-2, Kloeckera apiculata KK-3, and Trichosporon cutaneum KC 4-3), and ballistosporogenous yeast (Sporobolomyces salmonicolor WF 174). In contrast, no detectable amount of pyrophosphatidic acid was found in the cellular lipids of ascosporoeenous veasts.View full abstractDownload PDF (1245K) -
Hiroshi HATANAKA, Fujio EGAMI, Takeshi KATO, Toshiharu NAGATSU1975 Volume 78 Issue 4 Pages 821-824
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine adrenal dopamine β-hydroxylase [EC 1. 14. 17. 1] was considerably inhibited by ascorbate 2-sulfate. The inhibition was competitive with regard to ascorbate. The Ki value was 3.44mM. The possibility that ascorbate 2-sulfate may play a regulatory role in the biosynthesis of norepinephrine is suggested.
Another copper-containing oxidase, squash ascorbate oxidase [EC 1. 10. 3. 3], was not inhibited by the same compound at a concentration of 150mM.View full abstractDownload PDF (270K) -
Susumu OI1975 Volume 78 Issue 4 Pages 825-834
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat liver rhodanese [EC 2. 8. 1. 1] purified by ammonium sulfate fractionation, CM-cellulose and Sephadex G-200 chromatography yielded two active fractions (I & II). Their molecular weights were estimated to be 1.75×104 (I) and 1.26×104 (II) by the gel filtration method.
Kinetic studies revealed that Fraction I rat liver rhodanese catalyzes thiocyanate formation from thiosulfate and cyanide by a double displacement mechanism. Carboxylic acids such as DL-isocitric, citric, malic, pyruvic, and oxaloacetic acid were competitive inhibitors with respect to thiosulfate, whereas fumaric, succinic, and α-ketoglutaric acids were noncompetitive inhibitors with respect to thiosulfate.
Incubation of mitochondria with sulfate and α-ketoglutaric acid caused a significant rlpcrensp in rhodanese activitv.View full abstractDownload PDF (1431K) -
Haruhito TSUGE, Osamu NATSUAKI, Kazuji OHASHI1975 Volume 78 Issue 4 Pages 835-843
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucose oxidase [&-D-glucose: oxygen 1-oxidoreductase, EC 1. 1. 3. 4] from Aspergillus niger was purified by DEAE-cellulose chromatography and Sephadex G-200 gel filtration using phosphate buffer, pH 7.0.
1. The preparation has a specific activity of 172 μmoles oxygen consumed/min/mg of enzyme at 30° and pH 5.6, and is essentially catalase-free. The constituents of the enzyme are protein (74.0±2.8%), neutral sugar (16. 4±0.3%), amino sugar (2.4±0.5%), and 2 moles of iron per 160, 000 daltons, in addition to FAD.
2. Optical data for the newly purified holoenzyme show that the ratio A280/A450 is 11.1, and ε for enzyme-bound FAD at 450nm is 1.52×l04.
3. The holoenzyme (molecular weight, 160, 000) consists of two identical subunits with molecular weights of 79, 000±4, 000, and the molecular weight of the apoenzyme seems to be identical with that of the subunit, as shown by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-200.
4. FHD (flavin-hypoxanthine dinucleotide) has coenzymatic activity equal to that of FAD.View full abstractDownload PDF (1333K) -
Takayasu DATE, Koichi SUZUKI, Kazutomo IMAHORI1975 Volume 78 Issue 4 Pages 845-858
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA-dependent RNA polymerase [EC 2. 7. 7. 6] was purified from the thermophile Thermits thermophilus HB8 and some properties were investigated.
1. Two kinds of the enzyme (enzymes A and B) were found and separated from each other by phosphocellulose column chromatography. Enzyme A could utilize various DNA's as templates, while enzyme B was active only when alternating copolymer of deoxyadenylic and deoxythymidylic acids (poly d (A-T)) was used as a template. The specific activities of enzymes A and B at 65° were 30, 000 and 16, 000, respectively, which are markedly higher than the value obtained with E. coli enzyme.
2. The enzyme A was extremely thermostable. The activity did not decrease even after incubation for 3hr at 70°. The maximal rate of RNA synthesis was found at 70°.
3. Both polymerases were resistant to rifampicin but were markedly sensitive to streptolydigin.
4. Four kinds of subunits were found in the enzymes by sodium dodecylsulfate (SDS)- gel electrophoresis; subunit I (M. W. 42, 000), II (M. W. 58, 000), III (M. W. 140, 000), and IV (M. W. 180, 000). The subunit compositions of enzymes A and B were 2I-H-III-IV and 2I-III-IV, respectively. A part of enzyme B contained 1 mole of extra polypeptide (named X) of molecular weight 100, 000.View full abstractDownload PDF (1842K) -
Setsuro EBASHI, Teruhiko TOYO-OKA, Yoshiaki NONOMURA1975 Volume 78 Issue 4 Pages 859-861
Published: October 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNative tropomyosin from the gizzard, was separated into troponin and tropomyosin. The mode of action of the troponin-tropomyosin system of gizzard was shown to be distinctly differenf from that of skeletal muscle.View full abstractDownload PDF (770K)
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