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Volume 89 (1981)
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Volume 89, Issue 6
Displaying 1-36 of 36 articles from this issue
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Shigeru TAKETANI, Tokuzo NISHINO, Hirohiko KATSUKI1981 Volume 89 Issue 6 Pages 1667-1673
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSterol-ester hydrolase [EC 3. 1. 1. 13] from Saccharomyces cerevisiae grown aerobically was solubilized with 1% Tween 20 and purified about 700-fold by the protamine sulfate treatment, DEAE-cellulose-, Sepharose 6B- and DEAE-cellulose column chromatographies. The molecular weight of the enzyme was estimated to be 70, 000 by Sepharose 6B gel filtration. The enzyme activity showed two peaks of pH optimum at 4.4 and 6.8. Triton X-100 stimulated the activity at its low concentrations at both pH regions, but decreased the activity at its high concentrations at pH 6.8. The presence of Tween 20 or Tween 80 also stimulated the activity. These results were different from those in the previous report showing no stimulation of the crude enzyme by these detergents. The stimulation of the activity by phosphatidylcholine or low concentrations of lysophosphatidylcholine was similar to that by Triton X-100, and taurocholate was less effective than Triton X-100. The enzyme activity was inhibited by divalent cations such as Hg2+ and Cu2+View full abstractDownload PDF (470K) -
Yoshimi OHNO, Mitsuyo OKAZAKI, Ichiro HARA1981 Volume 89 Issue 6 Pages 1675-1680
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImmunologically pure lipoprotein fractions of human serum were subjected to high performance liquid chromatography (HPLC) on a combination system of aqueous gel permeation columns.
Each lipoprotein fraction appeared at a distinct elution volume as a single homogeneous peak.
Elution was carried out with 0.1M Tris-HCl buffer solution, pH 7.4, or 0.15M NaCl solution and no significant differences in elution volume and elution pattern were found.
The lipoprotein mixtures (total lipoprotein fraction d<1.210) of healthy and pathological subjects were subjected to HPLC and showed characteristic elution patterns which could be of diagnostic significance.View full abstractDownload PDF (905K) -
Tatsuo NAKAYAMA, Maki IRIKURA, Yumiko KUROGI, Hisayuki MATSUO1981 Volume 89 Issue 6 Pages 1681-1691
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus subtilis sporulating cells at stage III were fractionated into mother cell and forespore fractions by means of a lysozyme-detergent method. Three forms of DNA-dependent RNA polymerase enzymes, termed Mσ, Fσ, and Fδ, in addition to core enzyme (α2, β', and β) have been purified from the cell fractions. Enzymes Mσ and Fσ are present in the mother cell and forespore, respectively, and contain σ factor of 55, 000 daltons in addition to the core subunits. On the other hand, enzyme Fδ is present specifically in the forespore and contains δ1 factor of 28, 000 daltons instead of the a factor. The amount of RNA polymerase in the forespore is about twice that in the mother cell. The enzymes Mσ and Fσ also differed in their elution profiles from DEAE-cellulose columns and in their heat stabilities indicating that the two σ-containing holoenzyme forms may be different in their structural properties. The enzyme Fδ transcribed B. subtilis DNA about 1.6 times more actively than enzyme Fσ, and the enzymes Mσ and Fσ transcribed the DNA about 2.2 times more actively than did core enzyme.View full abstractDownload PDF (1888K) -
Yoshiyuki SAKAKI, Michitoshi NAKAMURA, Hisayuki NOMIYAMA, Yasuyuki TAK ...1981 Volume 89 Issue 6 Pages 1693-1698
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs a first step in studies on the structure and expression of rRNA gene of Physarum polycephalum, the cloning of the gene in Escherichia coli with plasmid vector DNAs was performed and 8 different kinds of clones containing 26 S, 19 S, and 5.8 S rRNA genes were obtained. Using these cloned fragments, the location of these rRNA genes was determined by the Southern hybridization and S l mapping method. Cloning of rDNA fragments made it possible to analyze the fine structure of the rDNA.View full abstractDownload PDF (991K) -
Nobuhiko IKARI, Hisao KATO, Teruko SUGO, Setsuro FUJII, Sadaaki IWANAG ...1981 Volume 89 Issue 6 Pages 1699-1709
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrevious studies from our laboratories (Sugo et al. (1980) Biochemistry 19, 3215-3220) have shown that bovine high-molecular-weight (HMW) kininogen remarkably accelerates the kaolin-mediated activation of Factor XII in the presence of prekallikrein, and that both fragment 1•2 and the light chain regions located in the COOH-terminal half of the kininogen molecule are essential for the activation. In the present study, we demonstrate that the accelerating effect of HMW kininogen is mediated through its adsorption on the kaolin surface through the fragment 1•2 region and its complex formation with prekallikrein through the light chain region. The evidence is as follows:
1. HMW kininogen radio-labeled with 125I was adsorbed on kaolin and the adsorption was inhibited by the prior treatment of kaolin with fragment 1•2, fragment 1•2-light chain, kinin-free protein or HMW kininogen, but not with kinin- and fragment 1•2-free protein, light chain or low-molecular-weight (LMW) kininogen.
2. The complex formation of HMW kininogen with prekallikrein in bovine plasma or in the purified system was examined by gel-filtration on a column of Sephacryl S-200. In bovine plasma, prekallikrein was eluted in the same fraction as HMW kininogen, showing an apparent molecular weight of 250, 000, whereas purified prekallikrein was eluted in the fraction corresponding to an apparent molecular weight of 100, 000. When purified prekallikrein was mixed with purified HMW kininogen in a mol ratio of 1 to 2, all prekallikrein was found to be associated with HMW kininogen. Furthermore, purified prekallikrein mixed with kininogen derivatives, such as kinin- and fragment 1•2-free protein, fragment 1•2-light chain or light chain, was eluted in the higher molecular weight fraction. LMW kininogen did not form a complex with prekallikrein. Using the same technique, it was shown that kinin- and fragment 1•2-free protein forms a complex not only with prekallikrein but also with kallikrein.View full abstractDownload PDF (780K) -
Kiyoshi NOSE, Atsuko TANAKA, Hiroshi OKAMOTO1981 Volume 89 Issue 6 Pages 1711-1719
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTranscriptional activity of globin genes in inducible and uninducible Friend leukemic cells was studied by two different approaches in an attempt to identify the biochemical basis for the loss of inducibility.
(1) Nuclei were isolated, and transcripts synthesized endogenously were purified by the Hg-UTP/sulfhydryl-Sepharose method. Globin mRNA sequences were quantitated by hybridization with [32P]globin cDNA. The results showed that the transcription of globin genes was stimulated in DMSO-treated inducible cell nuclei, whereas there was no activation of globin gene transcription in nuclei of uninducible variants cultured with DMSO.
(2) Nuclei were prepared from inducible and uninducible Friend cells cultured in the presence or absence of DMSO, and were then subjected to limited digestion with DNase I. DNA was isolated from the nuclei after about 10% of total DNA had become acid-soluble, and was hybridized with globin cDNA. Globin genes either in inducible or in uninducible Friend cells were specifically susceptible to DNase I irrespective of the addition of inducer.
From these studies, it was concluded that globin genes of uninducible Friend cells retained the “active conformation” but were not actively transcribed even in the presence of inducers.View full abstractDownload PDF (635K) -
Kazufumi KURODA, Makoto KAGEYAMA1981 Volume 89 Issue 6 Pages 1721-1736
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPseudomonas aeruginosa strain PAF41 was found to produce a new F-type pyocin, pyocin F3, the action spectrum of which was different from those of previously reported pyocins F1 and F2. These three F-type pyocins were compared with respect to their structure and biological properties. These pyocins were almost the same with regard to the structure and the dimensions, and have similar amino acid compositions and S values. The particle weights of these pyocins were also suggested to be similar. Analyses of subunit proteins by SDS-polyacrylamide slab gel electrophoresis showed that these pyocins were composed of 5 major (bands 1, 2, 3, 4, and 6) and 2 minor (bands 5 and 7) subunit proteins and that no difference in the mobilities of these subunit proteins could be detected among the pyocins except that of the second major subunit protein (band 4), which did differ.
Pyocins F1, F2, and F3 were immunologically cross-reactive, and carried common antigens as well as specific ones. It was shown that band 6 was a common antigen among the three pyocins and that band 4 was antigenically different in pyocins F1 and F3 by immunological reaction after protein blotting. Electron microscopic observation of pyocin particles treated with anti-sera revealed that the common antigens were located on the rod part and the specific ones were on the fiber part.
Pyocin F3 was neutralized by both anti-F3 and anti-F1 sera showing apparent first order rate kinetics, whereas the neutralization for pyocin F1 by these sera did not show such kinetics, but a considerable increment of pyocin F1 activity was observed when small amounts of the sera were added. This increment seemed to be due to the antibodies common to pyocins F1, F2, and F3.
A phage, which had a flexuous rod-like tail, was found to be immunologically cross-reactive with the three pyocins and was named KF1.View full abstractDownload PDF (6071K) -
Seiichi SHIBATA, Kazuo MIURA1981 Volume 89 Issue 6 Pages 1737-1749
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new glycopeptide was isolated from the glomerular basement membrane (GBM) of normal rats.
Unlike already known glycopeptides, this glycopeptide has biological activity (nephritogenic activity) to induce glomerulonephritis when injected once into the footpads of homologous animals.
A close relationship was found between the nephritogenic activity and the non-dialyzable glucose content of this nephritogenic glycopeptide. Thus, the nephritogenic activity can be assessed quantitatively by estimating the content of “non-dialyzable glucose.”
Chemical purification of the nephritogenic glycopeptide involved the selective removal of inactive glycopeptide containing galactose, mannose, and N-acetyl-glucosamine (but no glucose). Trichloroacetic acid (TCA) treatment was a simple but highly effective procedure for selective removal of this inactive glycopeptide.
The non-reducing terminus of the nephritogenic glycopeptide is α-D-gluco-pyranoside, and the glycopeptide reacts specifically with concanavalin A, even in the crude state.
We propose that the nephritogenic glycopeptide is not an artifact produced during exhaustive proteolytic digestion, but a natural substance having a fixed molecular shape, even in the crude state, and whose union with GBM-proper can be easily broken by proteolytic digestion.View full abstractDownload PDF (1302K) -
I. Comparison of the Virion-Associated RNA Polymerase Activity of Various Strains of Influenza VirusKiyoshi KAWAKAMI, Akira ISHIHAMA, Michinari HAMAGUCHI1981 Volume 89 Issue 6 Pages 1751-1757
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA systematic and comparative study was performed on the polypeptide composition and the RNA polymerase activity associated with virions of various strains of influenza A virus, including four human and two avian viruses. Significant differences were found in the molecular weights of not only hemagglutinin (HA) but also both nucleoprotein (NP) and membrane protein (M), as determined by polyacrylamide gel electrophoresis under denaturing conditions. The results indicate that, among viruses sharing the same serotype determined by the surface proteins HA and NA (neuraminidase), considerable variations exist in the structure of viral proteins, including inner proteins. The relative contents of viral proteins also varied among these strains grown under similar conditions.
The total content of three P proteins, the putative RNA polymerase subunits, was within the range between 1.1 and 2.2% of total viral proteins and roughly paralleled the virion-associated RNA polymerase activity. The virion-associated RNA polymerase of all the strains tested were stimulated by the same dinucleotide primers, ApG or GpG, indicating that the specificity of transcription initiation is conserved among wide varieties of influenza virus.View full abstractDownload PDF (1223K) -
II. Influence of Oligonucleotide Chain Length on the Priming Activity of RNA Synthesis by Virion-Associated RNA PolymeraseKiyoshi KAWAKAMI, Toshiki TANAKA, Akira ISHIHAMA, Hideaki TAKASHIMA, E ...1981 Volume 89 Issue 6 Pages 1759-1768
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe virion-associated RNA-dependent RNA polymerase of influenza virus PR8 was activated by treatment with specific non-ionic detergents, and was remarkably stimulated by exogenously added oligonucleotide primers. Among synthetic oligo-nucleotides of various chain lengths, the dinucleotide ApG and the trinucleotide ApGpC were the best primers, exhibiting at least 15-fold stimulation; the Km values for these primers were within the range of 0.024-0.10mM. In spite of the potentially high affinity to the 3' terminal sequence of viral RNAs, two species of heptanucleotides, ApGpCpApApApA and ApGpCpGpApApA, complementary to the 3' termini of RNA segments no. 4, 5, and 8, and of segments no. 1, 2, 3, 6, and 7, respectively, stimulated the RNA polymerase activity by less than 3-fold, if at all, and thus were less efficient primers than di- and trinucleotides. It appears that the selective utilization of specific oligonucleotides as primers for transcription initiation is not a linear function of increased duplex stability between template RNA and complementary oligonucleotides but rather a reflection of the primer-binding properties of RNA polymerase at its product site.View full abstractDownload PDF (1247K) -
Tatsuhiko OHE, Yasuto WATANABE1981 Volume 89 Issue 6 Pages 1769-1776
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUrate oxidase [EC 1. 7. 3. 3] was purified to homogeneity from cell-free extracts of a strain of Streptomyces cyanogenus. The enzyme had a molecular weight of 100, 000 and consisted of three subunits each with a molecular weight of 32, 000. The isoelectric point was at pH 4.0. No evidence was found for the involvement of copper, iron or coenzymes in the urate oxidase reaction. The enzyme was most active at pH 8 and at 35°C, and was stable between pH 6 and 11 (35°C, 1 h) and below 50°C (pH 7.8, 10min). The enzyme was inhibited by cyanide and sulfhydryl reagents, but only slightly by heavy metal ions and chelating agents. The activity was inhibited by xanthine and 2-hydroxypurine. The enzyme was found not to be inhibited by high concentrations of uric acid when the activity was assayed in terms of hydrogen peroxide formation. Urea and racemic allantoin were formed from uric acid by the enzyme reaction in phosphate buffer, and urea and other ninhydrin-positive materials in borate buffer.View full abstractDownload PDF (1125K) -
Subcellular Localization and Properties of N-Acetylglutamate Synthase in Rat Small Intestinal MucosaChuzou UCHIYAMA, Masataka MORI, Masamiti TATIBANA1981 Volume 89 Issue 6 Pages 1777-1786
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN-Acetylglutamate synthase [EC 2. 3. 1. 1], which catalyzes the synthesis of N-acetylglutamate, a key effector of carbamoyl-phosphate synthase (ammonia) [EC 6. 3. 4. 16] in the liver of ureotelic animals, was demonstrated to be present in rat small intestinal mucosa. The activity of the enzyme was estimated to be 0.17 nmol N-acetylglutamate formed×(g mucosa)-1×min-1 at 25°C. Little activity was found in the muscle layer and serosa of the small intestine. The intestinal villous cells were separated from everted intestine, disrupted by nitrogen cavitation, and fractionated into nuclear, mitochondrial, microsomal, and soluble fractions. The mitochondria isolated by this method retained integrity of respiratory function. The mitochondrial fraction was further subjected to isopycnic centrifugation using Percoll (colloidal silica coated with polyvinylpyrrolidone). The activities of N-acetylglutamate synthase and the first two urea cycle enzymes, carbamoylphosphate synthase (ammonia) and ornithine carbamoyltransferase [EC 2. 1. 3. 3], were cofrac-tionated with mitochondrial marker enzymes during the cell fractionation and the isopycnic centrifugation.
N-Acetylglutamate synthase, purified 8-fold from the acetone powder extract of small intestinal mucosa, had a high substrate specificity for L-glutamate and acetyl-CoA. The synthase reaction fitted normal Michaelis-Menten kinetics with respect to both L-glutamate (apparent Km, 2.5mM) and acetyl-CoA (apparent Km, 0.8mM). L-Arginine stimulated the enzyme activity by increasing the maximal velocity with no effect on apparent Km values for the substrates. These properties were similar to those of the rat liver enzyme (Shigesada & Tatibana (1978) Eur. J. Biochem. 84, 285-291). These results suggest that a function of the intestinal N-acetylglutamate is to activate carbamoyl-phosphate synthase (ammonia) and to allow citrulline synthesis in the tissue.View full abstractDownload PDF (748K) -
Hisahide HIURA, Tomisaburo KAKUNO, Jinpei YAMASHITA, Hiroshi MATSUBARA ...1981 Volume 89 Issue 6 Pages 1787-1792
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen the photoheterotroph, Rhodospirillum rubrum, was grown in the light, ferredoxin was excreted from the cells in a significant amount, as well as hydrogenase. The extracellular ferredoxin was purified to a homogeneous state. The molecular weight was approximately 9, 000, and the oxidation-reduction mid-potential was -0.29V (n=1) at pH 7.0 and 25°C. The amino acid composition was different from those of the intracellular ferredoxins, which were already known. The contents of non-heme iron and acid-labile sulfur were 10.6 and 7.9 mol/mol protein, respectively. The extracellular hydrogenase catalyzed the evolution of hydrogen gas from the ferredoxin in the reduced form. The Km for the ferredoxin was 4.1 μM, one-seven hundredth as low as that for methyl viologen. There is a possibility that hydrogenase here were functional for evolution of hydrogen gas outside the cells.View full abstractDownload PDF (345K) -
Kazuo HOSOI, Tadayoshi KIDA, Takao UEHA1981 Volume 89 Issue 6 Pages 1793-1798
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTrypsin-like and chymotrypsin-like esteroprotease isozymes of the mouse submandibular gland were separated by isoelectric focusing. In normal female mice the following pI-isozyme activities were found: pI-4.6, -5.6 (shoulder), -5.8, -7.1, and -9.9, hydrolytic activities for benzoylarginine ethylester (BAEE) (trypsin-like enzymes), and pI-4.7 and -10.3 hydrolytic activities for acetyltyrosine ethylester (ATEE) (chymotrypsin-like enzymes). In mice with testicular feminization (Tfm mice), only pI-4.6 hydrolytic activity for BAEE was found; no ATEE hydrolytic activity was detected.
In normal female mice, both 5α-dihydrotestosterone (5α-DHT) and tri-iodo-L-thyronine (T3) significantly increased all these isozymes except the pI-4.6 hydrolytic activity for BAEE. In Tfm mice, T3 also increased all these isozymes except the pI-4.6 hydrolytic activity for BAEE, but 5α-DHT had no effect on any enzymes.
These results suggest that the pI-4.6 hydrolytic activity for BAEE is non-inducible by the two hormones. Androgen does not seem to be involved in the inductions of these esteroproteases by T3.View full abstractDownload PDF (341K) -
Kohei HOSAKA, Toshiro KIKUCHI, Noboru MITSUHIDA, Akihiko KAWAGUCHI1981 Volume 89 Issue 6 Pages 1799-1803
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rapid and sensitive spectrophotometric assay for free fatty acids using acyl-CoA synthetase and acyl-CoA oxidase is described. It is sensitive to as low as 5 nmol of free fatty acids, and the standard curve is linear up to 100 nmol. The assay consists of the measurement of H2O2 produced from free fatty acids by acyl-CoA synthetase and acyl-CoA oxidase. The quantity of H2O2 is determined by the absorbance at 550 nm in the presence of catalase and 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole (AHMT). This method shows a broad specificity to long-chain fatty acids and the recoveries of added fatty acids (C12-C18) are more than 90%. The presence or absence of serum components or Escherichia coli cell-free extracts has no significant effect on the recovery of added palmitic acid.View full abstractDownload PDF (359K) -
Tetsuya ASAKAWA, Yoichi YAZAWA, Naomi AZUMA1981 Volume 89 Issue 6 Pages 1805-1814
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Abalone myosin consisted of one kind of heavy chain and two kinds of light chain according to SDS gel electrophoresis. The molecular weights of the light chains were estimated as 16, 600 daltons (Light Chain-1:LC-1) and 14, 500 daltons (Light Chain-2:LC-2).
2. The amino acid composition of LC-2 was not appreciably different from that of LC-1 except that the valine residue was 1 mol per mol of LC-2.
3. Both LC-1 and LC-2 showed a calcium-induced UV absorption difference spectrum though the apparent binding constants for Ca2+ were low (2.5×10-3M for LC-1 and 3.2×10-4M for LC-2).
4. The modification of carboxyl groups of LC-2 by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide caused the disappearance of the calcium-induced UV absorption difference spectrum.
5. Manganese ion could also induce a UV absorption difference spectrum with these light chains although the concentration of Mn2+ required to produce the difference spectrum was very high.
6. Abalone LC-2 bound to desensitized scallop myosin and restored the calcium sensitivity of the myosin but LC-1 did not.View full abstractDownload PDF (1690K) -
Mamoru ISEMURA, Hiroshi MUNAKATA, Zensaku YOSIZAWA1981 Volume 89 Issue 6 Pages 1815-1819
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA particulate fraction was separated from endometrial scrapings of the uterus of hormone- or sham-treated ovariectomized rabbit. The effects of estrogen and progesterone on the incorporation of [35S]sulfate from 3'-[35S]phosphoadenosine 5'-phosphosulfate (PAPS) into endogenous acceptors in the particulate fraction were investigated. Estrogen increased the incorporation of [35S]sulfate, but progesterone suppressed this effect. The results of DEAE-Sephadex A-25 (Cl- form) column chromatography of the pronase digest of the 35S-labeled substances indicated that the fraction eluted with 0.9M NaCl (0.9 Fr) was most sensitive to the hormones. The major component in 0.9 Fr was resistant to crude heparinase, whereas the minor component was susceptible to this enzyme. The present observation, together with previous findings, suggested that the former was sulfated glycopeptide and the latter heparan sulfate. The results of the present study indicated that PAPS can serve as the direct “activated” sulfate donor in the enzymatic sulfation of sulfated glycoprotein in the particulate fraction, that the sulfation is greatly stimulated by pre-treatment of the rabbit with estrogen, and that the estrogen effect is suppressed by progesterone.View full abstractDownload PDF (331K) -
Kosuke ICHIHARA, Kiyomi ISHIHARA, Emi KUSUNOSE, Masamichi KUSUNOSE1981 Volume 89 Issue 6 Pages 1821-1827
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmong many different tissues of rabbit, hexadecane hydroxylation activity was found in only small intestine and liver microsomes. Both activities for hexadecane and decane hydroxylation in intestinal microsomes were significantly higher than those in liver microsomes. In contrast, the hydroxylation activities of p-xylene, benzo(a)pyrene, and decanol, and the demethylation activity of aminopyrine in the former were much lower than those in the latter. The intestinal microsomes converted [1-14C]hexadecane to cetyl alcohol, but not further to palmitic acid. Hexadecane hydroxylation activity was found to be markedly stimulated by non-ionic detergents such as Triton X-100, Nonidet P-40, and Emulgen 913. Phosphatidylcholine and phosphatidylethanolamine stimulated the activity to a lesser extent. The hydroxylation was inhibited by various aliphatic hydrocarbons with carbon numbers larger than 10, but not by aromatic and polycyclic hydrocarbons. Hexadecane hydroxylation activity was solubilized from the intestinal microsomes and reconstituted with a partially purified cytochrome P-450 fraction, and intestinal NADPH-cytochrome c reductase, or spinach ferredoxin and ferredoxin-NADP reductase. The chromatography of the crude cytochrome P-450 preparation on hydroxylapatite separated at least two cytochrome P-450 fractions; one preferentially hydroxylating hexadecane, and the other preferentially hydoxylating myristic acid. The results suggest that rabbit intestinal mucosa microsomes had a cytochrome P-450 species specialized for hexadecane hydroxylation.View full abstractDownload PDF (496K) -
Fumio FUKAI, Akira MURAYAMA1981 Volume 89 Issue 6 Pages 1829-1837
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnalysis of the cytoplasmic estrogen receptor (ER) system of gilt uterus by protecting ER from proteolysis showed that the ER system of gilt uterus is similar to that of cow uterus described previously. There was only one native unit molecule [native “4 S” ER (sedimentation coefficient, 4.5 S; Stokes radius, 45 A; molecular weight, 82, 000)] with specific affinity towards estradiol. This molecule was further designated as vero-ER. “8S” ER-forming factor [(“8S”ER)-FF] (Stokes radius, 51 A) was separated from the cytosol under hypotonic (low salt) conditions, and this was further dissociated into component A [“5 S” ER-forming factor, (“5 S”ER)-FF] (Stokes radius, 37 A) and component B (Stokes radius, 18.5 A) in the presence of sodium thiocyanate. Vero-ER was proteolyzed in the absence of Ca2+ ion by a cytoplasmic protease into modified “4 S” ER (sedimentation coefficient, 4.5 S; Stokes radius, 35 A; molecular weight, 65, 000) which was further designated as secto-ER. The constituents of the cytoplasmic ER system of cow and gilt uteri cross-reacted with each other to undergo similar molecular assembly as in their own systems. When analyzed for various uterine specimens, fluctuation of (“8 S” ER)-FF-level (1-50 units/g tissue) was more remarkable than that of ER-level (3-6×10-12 mol/g tissue). Component B labeled with [14C] iodoacetic acid was purified over 1, 500-fold. Mixture of vero-ER and component B labeled with [14C] iodoacetic acid formed “6 S” ER with 14C-activities under hypotonic conditions. This clearly excluded the possibility that component B is a catalyzer of self-association (dimerization) of vero-ER.View full abstractDownload PDF (611K) -
I-Yih HUANG, Masami KIMURA, Akiko HATA, Hajime TSUNOO, Akira YOSHIDA1981 Volume 89 Issue 6 Pages 1839-1845
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of thionein-II, one of the two major mouse liver thionein components, was determined. The main fragmentation of thionein-II, which consists of 61 amino acid residues, was accomplished by digesting the S-[14C]-carboxymethylated protein and the cyanogen bromide-treated oxidized protein with trypsin. The peptides obtained by papain digestion of S-[14C] carboxymethylated thionein-II were used to align the major tryptic peptides. The sequence was determined by a combination of automated and manual Edman degradation techniques. Remarkable structural homology is observed in mouse thionein-I, mouse thionein-II, and thioneins from man and horse.View full abstractDownload PDF (517K) -
Hidenori HAYASHI, Tsunenori NOZAWA, Masahiro HATANO, Sigehiro MORITA1981 Volume 89 Issue 6 Pages 1847-1852
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacteriochlorophyll (Bchl) protein complexes containing light harvesting (LH) Bchls were isolated from Chromatium vinosum, and their CD spectra were measured in the near-infrared region. These isolated Bchl protein complexes retained the CD signals of LH Bchls that were observed in situ in chromatophores, The CD spectrum of fraction A (containing B890) consisted of paired positive and negative bands (a double CD) having a zero-crossing at 800 nm and a single negative band at around 900 nm attributed to the B890. For low 850 fraction B (B800 and B850), a double CD having a zero-crossing at 795 nm and a single negative band at around 850 nm attributed to the B850 were found. High 850 fraction B exhibited a double CD having a zero-crossing at 795 nm, a negative band at around 860 nm and a positive band at around 840 nm. For fraction C (B800 and B820), a double CD having a zero-crossing at 795 nm and a single negative band at around 830 run, which was attributed to the B820, were found. The double CD was attributed to the B800 in fractions A, B, and C. There was no additional CD besides the CD of the isolated Bchl protein complexes in the CD spectra of chromatophores.View full abstractDownload PDF (423K) -
Hidenori HAYASHI, Tsunenori NOZAWA, Masahiro HATANO, Sigehiro MORITA1981 Volume 89 Issue 6 Pages 1853-1861
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacteriochlorophyll (Bchl) protein complexes containing light harvesting (LH) Bchls were isolated from Chromatium vinosum, and their CD spectra were measured in the near-infrared region. These isolated Bchl protein complexes retained the CD signals of LH Bchls that were observed in situ in chromatophores, The CD spectrum of fraction A (containing B890) consisted of paired positive and negative bands (a double CD) having a zero-crossing at 800nm and a single negative band at around 900nm attributed to the B890. For low 850 fraction B (B800 and B850), a double CD having a zero-crossing at 795nm and a single negative band at around 850nm attributed to the B850 were found. High 850 fraction B exhibited a double CD having a zero-crossing at 795nm, a negative band at around 860nm and a positive band at around 840nm. For fraction C (B800 and B820), a double CD having a zero-crossing at 795nm and a single negative band at around 830 run, which was attributed to the B820, were found. The double CD was attributed to the B800 in fractions A, B, and C. There was no additional CD besides the CD of the isolated Bchl protein complexes in the CD spectra of chromatophores.View full abstractDownload PDF (630K) -
Masaharu SAKAI, Yoshiaki FUJII-KURIYAMA, Tsunehiro SAITO, Masami MURAM ...1981 Volume 89 Issue 6 Pages 1863-1868
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant plasmids containing DNA complementary to protamine messenger RNA of rainbow trout (Salmo gairdnerii) have been isolated and sequenced. One of the clones contained the entire coding sequence of a protamine gene together with the complete 3' non-coding region. Another clone had an identical nucleotide sequence in the coding region but four base substitutions in the 3' non-coding region. On the basis of a comparison of the nucleotide sequences available at present, the structure and divergence of the protamine gene family are discussed.View full abstractDownload PDF (1095K) -
Yoshiaki FUJII-KURIYAMA, Tadatsugu TANIGUCHI, Yuzuru MIZUKAMI, Masahar ...1981 Volume 89 Issue 6 Pages 1869-1879
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome P-450 mRNA has been partially purified from membrane-bound polysomes of the livers of phenobarbital-treated rats by SDS-phenol-chloroform extraction, followed by poly(U)-Sepharose chromatography and by centrifugation through a sucrose density gradient. Cytochrome P-450 mRNA activity was detected near 18 S in the sucrose density gradient, accounting for approximately 5 % of total mRNA activity on the basis of [3H] leucine incorporation in an in vitro translation system of wheat germ. Complementary DNA (cDNA) which had been synthesized on the partially purified mRNA by AMV reverse transcriptase was inserted into the Pst I site of pBR 322. After bacterial transformation, and in situ colony hybridization using [32P] cDNA as a probe, a colony carrying cytochrome P-450 cDNA sequence was identified by a hybridization-arrested translation assay. Sequence complementarity of the inserted DNA sequence to cytochrome P-450 mRNA was further confirmed by a positive hybridization-translation assay. The mRNA isolated from the partially purified mRNA preparation by hybridizing it with the recombinant DNA (III-8-10) showed enriched synthesis of a protein product whose apparent molecular weight was consistent with that of cytochrome P-450, and which was immunoprecipitable with anti-cytochrome P-450 antibody.View full abstractDownload PDF (3795K) -
Yukio ISHIMI, Yoshiki OHBA, Hideyo YASUDA, Masa-atsu YAMADA1981 Volume 89 Issue 6 Pages 1881-1888
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structural changes caused by H1 histone depletion of calf thymus chromatin or nucleosome monomer were studied by using the criterion of accessibility to micro-coccal nuclease. When H1 histone was depleted from monomer, the DNA structure unfolded and became sensitive to the nuclease at a particular site on the nucleosome core, 105 base pairs from a chain terminus of core DNA.
In reconstitution experiments with chromatin, this sensitive site on the core could be protected from the attack of the nuclease only when H1 histone was present.
These results indicate that Hl histone interacts not only with the nucleosome linker region, but also with the core particle.View full abstractDownload PDF (2412K) -
Kazuko KAWASHIMA, Mitsuo IZAWA1981 Volume 89 Issue 6 Pages 1889-1901
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly(ADP-ribose) synthetic activity in isolated nucleoli from rapidly growing mouse ascites tumor cells and ADP-ribosylation of the nucleolar proteins in vitro were studied. The specific activity of the synthesis in the nucleoli was significantly higher than that in the chromatin. The optimum magnesium and NAD+ concentrations, and the effect of RNase treatment on the reaction in the nucleoli were also distinctly different from those in the chromatin. Hydrolysis of the reaction product of the nucleoli with snake venom phosphodiesterase and with calf thymus poly(ADP-ribose) glycohydrolase yielded 5'-AMP and 2'-(5''-phosphoribosyl)5'-AMP, and ADP-ribose, respectively. The average chain length of the polymer formed in the nucleoli was found to be about 4 as a whole, but the distribution was heterogenous, from 1.2 to over 12. Analysis of ADP-ribosylated proteins in the nucleoli by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that several non-histone proteins with molecular weights of over 100, 000 were highly ADP-ribosylated compared with other proteins including histones. This pattern was also different from that of the chromatin.
These experimental results demonstrate that the nucleoli are independent from the chromatin as regards poly(ADP-ribose) synthesis in vitro.View full abstractDownload PDF (2666K) -
Shuichi TSUJI, Kazutomo IMAHORI, Yoshiaki NONOMURA1981 Volume 89 Issue 6 Pages 1903-1912
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystals of RNA polymerase of T. thermophilus were examined by electron microscopic observation of the negatively stained and sectioned materials. Three types of crystals were observed: ordered aggregates (Type I), cylindrical duplei (Type II), and plane (Type III) forms. It was deduced mainly from sectioned images that Type I crystal is a precursor or a premature form of Type II crystal. Type III corresponds to the flattened layer of Type II. In Type III crystal the enzyme molecules are arranged in an orderly two-dimensional lattice and thus we could analyze the molecular structure by optical filterling of the negatively stained images. On the basis of these results, a quaternary structure is proposed for RNA polymerase.View full abstractDownload PDF (3572K) -
Tomoko YAMANISHI, Syozo TUBOI1981 Volume 89 Issue 6 Pages 1913-1921
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was demonstrated that γ-cystathionase [EC 4. 4. 1. 1] incorporates the S-atom of L-cystine as a labile sulfane S-atom in the course of the cystine cleavage reaction.
The 35S-labeled native γ-cystathionase gradually released 35S. However, when the enzyme protein was denatured with 5 % trichloroacetic acid, the release of 35S from the enzyme protein was considerably suppressed. The release of 35S from the native enzyme protein was markedly enhanced by incubation with L-cystine. 35S was eliminated from the native enzyme with L-cystine as the sulfide ion, which was identified by trapping with lead acetate. The apparent Km of L-cystine for the 35S release (2.7×10-5 M) was comparable to that of [35S] cystine for 35S incorporation into the enzyme protein (2.5×10-5 M).
SH compounds such as dithiothreitol could also release the S-atom from the enzyme protein. The elimination reaction by these SH compounds seems to be nonenzymatic, since these SH compounds could eliminate 35S from the denatured 35S-labeled enzyme.
Iodoacetamide did not inhibit the homoserine deaminase, cystathionine lyase or cystine cleavage activities of γ-cystathionase. However, the incorporation of the S-atom from L-cystine into the enzyme protein and the release of the S-atom from the enzyme protein with L-cystine were markedly inhibited in the presence of 5mM iodoacetamide, indicating that iodoacetamide inhibits the S-atom incorporation by trapping the thiol formed from L-cystine by cystine cleavage. The thiol trapped with iodoacetamide was analyzed by using [14C] iodoacetamide, [14C] cystine or [35S] cystine. The compounds formed from the thiol and iodoacetamide were identified as cystine-thioglycolamide mixed disulfide and thioglycolamide disulfide, which was enzymatically derived from the mixed disulfide.
From these results, the mechanism of the cystine cleavage reaction catalyzed by γ-cystathionase is proposed to be as follows: (1) L-cystine is cleaved to pyruvate, NH3 and thiocysteine; (2) the thiocysteine interacts with a disulfide bond in the enzyme protein followed by incorporation of the S-atom of the thiocysteine to form the cystine trisulfide structure; (3) the cystine trisulfide structure is cleaved by consuming 2 mol of cysteine to eliminate the S-atom incorporated from the thiocysteine as a sulfide ion, accompanied by the reformation of a disulfide bond in the enzyme and the oxidation of cysteines to cystine.View full abstractDownload PDF (1444K) -
Keiko KOBAYASHI, Tomoko YAMANISHI, Syozo TUBOI1981 Volume 89 Issue 6 Pages 1923-1931
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFumarases located in the cytosolic and mitochondrial fractions were separately purified and crystallized from rat liver.
Polyacrylamide gel electrophoresis of these two crystallized fumarases in the presence of dodecyl sulfate gave a single protein band in a position corresponding to a molecular weight of about 49, 000 in each case. A single protein band was also obtained by acidic polyacrylamide gel electrophoresis of the cytosolic or mitochondrial fumarase in the presence of 8M urea. In both experiments, the subunits of these two fumarases migrated to the same position, indicating that both native enzymes are composed of subunits which could not be differentiated from each other by these procedures.
The molecular weight of both fumarases was estimated to be 200, 000 by sucrose density gradient centrifugation and molecular sieve chromatography with Sephadex G-200. A subunit cross-linking experiment using glutaraldehyde revealed that both native enzymes were composed of four identical subunits.
The amino acid composition of the cytosolic fumarase was shown to be very similar to that of the mitochondrial one by amino acid analysis. From the data obtained by amino acid analysis, the molecular weights of both native enzymes were calculated to be about 195, 000.
Kinetic constants such as the apparent Km, the optimal pH and the specific activity of the cytosolic fumarase were quite similar to those of the mitochondrial enzyme.
Furthermore, these two fumarases could not be differentiated from each other by immunochemical techniques, since a single precipitin band was formed between the rabbit antiserum against the cytosolic fumarase and the cytosolic or mitochondrial fumarase. In addition, these precipitin bands fused completely with each other on the Ouchterlony double immunodiffusion test.
These results may suggest that the fumarases located in the cytosolic and mitochondrial fractions of the rat liver cell may be products of the same nuclear gene, and also that the structures of these two mature fumarases are very similar, if not identical.View full abstractDownload PDF (2589K) -
Masashi KOSAKAI, Zensaku YOSIZAWA1981 Volume 89 Issue 6 Pages 1933-1944
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPorcine heparin, whale heparin, and a solvolyzed porcine heparin were deaminated, and sulfated oligosaccharides, compounds 3f, 4f, 3s, 4s, 5, 6, 7s, 10, 11f, 11s, and 13 were isolated from the deamination products by Dowex 1×2 (Cl- form) column chromatography and high voltage paper electrophoresis and/or gel filtration on Sephadex G-25. Based on the results of chemical, 1H and 13C NMR spectral analyses, and of Smith degradation, together with previous observations, the structures of these sulfated oligosaccharides are proposed to be as follows: compound 3f, IdUA(2S)α1→4GlcNAcα1→4GlcUA; compound 4f, IdUAα1→4GlcNAc(6S)α1→4GlcUA; compound 3s, IdUA(2S)α1→4GlcNAcα1→4GlcUAβ1→4aMan; compound 4s IdUAα1→4GlcNAc(6S)α1→4GlcUAβ1→4aMan; compound 5, IdUA(2S)α1→4aMan; compound 6, GlcUAβ1→4aMan(6S); compound 7s, IdUAα1→4aMan(6S); compound 10, IdUA(2S)α1→4GlcNAc(6S)α1→4G1cUAβ1→4aMan; compound 11f, IdUA(2S)α1→4GlcNAcα1→4GlcUAβ1→4aMan(6S); compound 11s, IdUAα1→4GlCNAc(6S)α1→4GIcUAβ1→4aMan(6S); compound 13, IdUA(2S)α1→4aMan(6S).
For the sulfated disaccharides, the same results as those reported in our previous papers were obtained. On the other hand, the proportion of total sulfated tri- and tetrasaccharides from whale heparin was 1.9 times higher than that from porcine heparine, reflecting a higher content of GlcNAc in the former. Also, the yields of compound 11s from these two heparins were comparable to their anticoagulant activities. In addition, certain 2-O-sulfates on IdUA flanked with GlcNS(6X) (X=H or S) in the heparin molecule are suggested to be important for the activity.View full abstractDownload PDF (744K) -
Kazuyuki AKASAKA, Shigeru FUJII, Robert KAPTEIN1981 Volume 89 Issue 6 Pages 1945-1949
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExposure of aromatic residues, Tyr 7, Tyr 75, Tyr 93, His 43, His 106, and Trp 86, was studied by laser-induced photo-CIDNP in the 1H NMR spectrum of Streptomyces subtilisin inhibitor at 360 MHz. Only Tyr 7 and Tyr 75 gave strong CIDNP signals, whereas the rest of the aromatic residues gave no detectable signals in the temperature range 25-55°C. From the temperature dependence data, it is concluded that Tyr 7 is well exposed at all temperatures, whereas the exposure of Tyr 75 increases with temperature, in agreement with the conclusion obtained by other methods. Agreements and discrepancies between the conclusions derived from the CIDNP data and the results so far obtained by other methods are compared for all the aforementioned aromatic residues.View full abstractDownload PDF (338K) -
Takashi MIKAWA, Shinichi TAKEDA, Teruo SHIMIZU, Takashi KITAURA1981 Volume 89 Issue 6 Pages 1951-1962
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a micro two dimensional gel electrophoretic system and silver stain method, we examined the isoform patterns of myofibrillar proteins in single muscle fibers of adult chicken. Several isoforms of myosin light chains, tropomyosin, troponin T, troponin I, and troponin C were identified. Analysis of pectoral, anterior latissimus dorsi, sartorius and soleus muscle revealed that single fibers contained either fast or slow form of each of the troponin subunits, indicating that troponin exists as a ‘homocomplex.’ The form of troponin in a given cell was associated with that of myosin light chains. The form of tropomyosin, however, was not associated with those of myosin light chains and troponin, but was tissue specific. Overall, we found four combinations of isoforms of myosin light chains, troponin subunits and tropomyosin subunits.
In adult dystrophic chicken the isoform patterns of tropomyosin and troponin T in single fibers of pectoral muscle markedly deviated from the normal patterns.View full abstractDownload PDF (4487K) -
Tomoe BEPPU, Yousuke SEYAMA, Takeshi KASAMA, Tamio YAMAKAWA1981 Volume 89 Issue 6 Pages 1963-1973
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIndividual non-sulfated bile acids and sulfated lithocholic acid in serum were determined by mass fragmentography. A hexafluoroisopropyl ester-trifluoroacetyl derivative of bile acid was prepared by the method of Imai et al. (J. Chromatogr. 120, 181, 1976). Deuterium labeled deoxycholic acid was used as an internal standard monitoring at m/z 623. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were determined by monitoring the intensities of m/z 622, m/z 620, m/z 620, m/z 620, and m/z 618, respectively. A serum sample of 200 μl including 500 ng of internal standard was hydrolyzed with strong alkali, then acidified to pH 1 with 2 N HCl under cooling on ice, and extracted with diethyl ether immediately. Ether extracts were derivatized without further purification. Besides this assay of non-sulfated bile acids, total serum lithocholic acid including the sulfated form was determined as follows: extraction was performed after mixing the acidified (pH 1 with 2 N HCl) hydrolysate with ether and incubating at 40°C for 2 h. Bile acid peaks in the mass fragmentogram were not affected by other materials in these serum extracts. The average values of individual non-sulfated bile acids in sera from healthy fasting subjects (n=15) were as follows: lithocholic acid, 0.049 μg/ml; deoxycholic acid, 0.462 μg/ml; chenodeoxycholic acid, 0.671 μg/ml; ursodeoxycholic acid, 0.070 μg/ml; and cholic acid, 0.217 μg/ml. Total lithocholic acid (non-sulfated and sulfated) in sera was 0.166 μg/ml.View full abstractDownload PDF (1246K) -
Yasuo KIKUCHI, Nobuo TAMIYA1981 Volume 89 Issue 6 Pages 1975-1976
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA high content of methionine sulfoxide was observed in the resiliums (internal hingeligaments) of surf clams. As no isolation procedure which might cause the oxidation of methionine to methionine sulfoxide was involved and the hydrolysis was carried out in vacuo, it is the first solid evidece for the presence of methionine sulfoxide as a constituent of natural protein.View full abstractDownload PDF (152K) -
Satoshi SAIGO1981 Volume 89 Issue 6 Pages 1977-1980
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKinetic difference spectra during the alkaline isomerization of ferricytochrome c were obtained by the pH-jump method in the range of 540 to 655 nm. The spectrum of the transient intermediate, which appears during the course of the isomerization, was reproduced from the spectra. The intermediate showed an intense absorption band at 600 nm, indicating that it is a high spin or mixed spin species. This is in contrast to the stable neutral and alkaline forms which are low spin species. The transient spin-state change during the isomerization was also observed upon rapid oxidation of ferrocytochrome c at alkaline pH.View full abstractDownload PDF (240K) -
Michio MUGURUMA, Kiyotaka KOBAYASHI, Toshiyuki FUKAZAWA, Kazuyo OHASHI ...1981 Volume 89 Issue 6 Pages 1981-1984
Published: June 01, 1981
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new protein with a chain weight of approximately 220, 000 was isolated from 0.6M KI extracts of I-Z-I brushes of rabbit and chicken skeletal muscles, using (NH4)2SO4 precipitation and three column chromatographic procedures in succession. It was only possible to separate the high molecular weight protein from actin and α-actinin in the presence of 6M urea or 0.1% sodium dodecyl sulfate (SDS). The purified protein migrated as a single band on SDS gel electrophoresis. The amino acid composition of the 220, 000 dalton protein was distinct from any known proteins found in myofibrils, e. g., α-actinin and actin binding protein (ABP; filamin). An indirect immunofluorescence technique revealed that the new protein was exclusively located in the Z lines of myofibrils of chicken breast muscle. There is, however, a possibility that the 220, 000 dalton protein is identical with synemin recently isolated from chicken gizzard (Granger, B. L. and Lazarides, E. (1980) Cell 22, 727).View full abstractDownload PDF (1239K)
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