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Volume 98 (1985)
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- Issue 4 Pages 863-
- Issue 3 Pages 585-
- Issue 2 Pages 269-
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Volume 98, Issue 2
Displaying 1-35 of 35 articles from this issue
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Akemichi UENO, Yeong-Man HONG, Naokatu ARAKAKI, Yoshiro TAKEDA1985 Volume 98 Issue 2 Pages 269-278
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA procedure was established for isolation of a low molecular weight polypeptide with insulin-stimulating activity in apparent homogeneity from a tryptic digest of bovine serum albumin on a semipreparative scale. Purification of this insulinstimulating peptide (ISP) was monitored by an adipose-explant assay in which stimulation of fatty acid synthesis from glucose by insulin was measured. The polypeptide was purified by a combination of DEAE-cellulose column chromatography, gel filtration on Bio-Gel P-10, hydrophobic chromatography on a semipreparative C18 reversed-phase HPLC column, and ion exchange chromatography on an SP-5PW HPLC column. The primary structure of ISP was deduced. ISP is a two-chain polypeptide consisting of 71 amino acid residues, and corresponds essentially to residues 115-143 and 144-184 (185) of bovine serum albumin connected to each other by a disulfide bridge. But comparison of the sequence of ISP with that of the relevant regions of bovine serum albumin determined by Brown indicated the presence of one tyrosine insertion between residues 155 and 156 of albumin. Therefore, the molecular weight of ISP was calculated to be 8, 496.View full abstractDownload PDF (1523K) -
Ribosomal RNA Synthesis in Mammalian Cells Permealized by Diethylaminoethyl (DEAE)-Dextran TreatmentYukio MISHIMA, Ryo KOMINAMI, Masami MURAMATSU1985 Volume 98 Issue 2 Pages 279-287
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEhrlich ascites tumor cells were made permeable to nucleoside triphosphates by treating them with diethylaminoethyl (DEAE)-dextran. Permeable cells incorporated labeled UTP into RNA at a constant rate for at least 30 min at 37°C and 100 min at 25°C, whereas no incorporation was detected without DEAE-dextran treatment. This RNA synthesis is dependent on added nucleoside triphosphates and is affected differentially by different concentrations of α-amanitin.
The results of both hybridization and Sl-nuclease protection mapping experiments with pulse-labeled RNA synthesized in this system indicate that reinitiation of rRNA transcription takes place from the physiological initiation site in cells made permeable by treatment with DEAE-dextran.
By using this system, we examined the kinetics of rRNA synthesis in mouse FM3A cells during inhibition of protein synthesis. The results reflected those previously obtained by analyses of nucleolar RNA synthesis in vivo during protein synthesis inhibition. This permeable cell system should provide a rapid and reproducible assay method for rRNA synthesis in mammalian cells.View full abstractDownload PDF (1494K) -
Nobuo TAMIYA, Tatsuhiko YAGI1985 Volume 98 Issue 2 Pages 289-303
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA “non-divergence theory” is proposed for the mechanism of evolution. The theory is based on the observation that comparison of the amino acid sequences of related proteins in various organisms gives inconsistent results from one type of protein to another, and on the occurrence of significant gene transfer among living organisms. Special attention is focused on the sequence comparisons of short- and long-chain neurotoxins and phospholipases A2 from the venoms of proteroglyphous snakes and those of microbial ferredoxins, rubredoxins, and flavodoxins.View full abstractDownload PDF (1294K) -
Subita SRIMAL, Toshiyuki MIYATA, Shun-ichiro KAWABATA, Takashi MIYATA, ...1985 Volume 98 Issue 2 Pages 305-318
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained:
1 10 20 30 _??_ D T N A P L _??_ L _??_ D E P G ILGRN _??_ L V T P E V K E K _??_
E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C
G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V
S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q
C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V
T Y N L E K D G F L C E S F R T C C G C P C R N Y
Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19, 675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an a-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The β-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.View full abstractDownload PDF (2055K) -
Keiji HASUMI, Reiko OTSUKI, Akira ENDO1985 Volume 98 Issue 2 Pages 319-325
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMouse mammary carcinoma FM3A cells, which are able to grow in a serum-free medium, have novel characteristics that could be valuable in biochemical and somatic cell genetic studies. In FM3A cells grown in the presence of serum, both sterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in the cholesterol biosynthetic pathway, were strongly suppressed by human low density lipoprotein (LDL). The addition of LDL (50 μg protein/ml) resulted in a 50%, decrease in the reductase activity within 3 h and a 95% reduction after 24 h. Similarly, over 90% suppression of the reductase activity was obtained by the addition of LDL or mevalonolactone when the cells were grown on a serum-free medium.
ML-236B (compactin), a specific inhibitor of HMG-CoA reductase, inhibited sterol synthesis from [14C]acetate by 80% at 1 μM. Reductase activity in FM3A cells was increased by 2.5- to 5-fold when the cells were treated with ML-236B (at 0.26-2.6 μM for 24 h).
Thus, in FM3A cells, HMG-CoA reductase activity responded well to LDL, as is observed in human skin fibroblasts. Along with other novel features of this cell line, the present observations indicate that FM3A cells should be useful in biochemical and somatic cell genetic analysis of cholesterol metabolism, especially as regards the regulation of HMG-CoA reductase activity.View full abstractDownload PDF (591K) -
Makoto KITO, Hitoshi TAKAMURA, Hiroshi NARITA, Reiko URADE1985 Volume 98 Issue 2 Pages 327-331
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives, which could be sensitively detected at 254 nm. The derivatives of 21 molecular species were resolved by high-performance liquid chromatography with an octadecylsilyl reversed-phase column. All the derivatives had the same peak area per mol, and peak areas were proportional to the amounts of the derivatives. Quantification was carried out at the picomole level.View full abstractDownload PDF (358K) -
Yoko SHIROYAI, Tatsuya SAMEJIMA1985 Volume 98 Issue 2 Pages 333-339
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhotooxidation of inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3. 6. 1. 1] from Bacillus stearothermophilus in the presence of rose Bengal resulted in rapid loss of enzymatic activity. The pH profile of the inactivation rate by the photooxidation showed an inflection point around pH 6.8, suggesting the involvement of histidyl residues in the inactivation. Amino acid analysis revealed that the loss of enzymatic activity was accompanied by the destruction of 3 histidyl residues per molecule. The presence of Mg2+ alone afforded partial protection against the inactivation, whereas inorganic pyrophosphate, the substrate, showed almost no protective effect against inactivation. The photooxidation of inorganic pyrophosphatase altered the circular dichroism spectrum and the difference UV spectrum induced by Mg2+ in the near ultraviolet region. These results suggested
that histidyl residues appear to be located at the binding site of Mg2+ and may contribute to the conformational change induced by Mg2+.View full abstractDownload PDF (646K) -
Masanori IWAMA, Tomoko TAKAHASHI, Norio INOKUCHI, Takashi KOYAMA, Masa ...1985 Volume 98 Issue 2 Pages 341-347
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of inhibition of the two glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives was investigated.
1. Hydrolysis of maltose by the glucoamylases was inhibited competitively by aminoalcohols at pH 5.0, and tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1, 3-propanediol and 2-aminocyclohexanol were relatively good inhibitors of the glucoamylases among the aminoalcohol derivatives tested.
2. One hydroxyl group and an amino group in these inhibitors were indispensable for the inhibitory action, and the addition of other hydroxyl, amino or ethyl groups was enhancing.
3. With an increase in pH from 4.0 to 6.0, the K1 values of the aminoalcohols decreased. This result suggested the participation of a carboxyl group, which was related to the glucoamylase activity and had a pKa of 5.7, in the binding of aminoalcohols.
4. The UV difference spectra induced on binding of the aminoalcohol analogues with the glucoamylases may indicate a change of the environment of tryptophan residues to a slightly higher pH on inhibitor binding.
5. The influence of aminoalcohols on the fluorescence intensity due to tryptophan residues and the CD-spectra of the glucoamylases was less than that of maltitol. Thus, the interaction of aminoalcohols with tryptophan residues in the glucoamylases might be less pronounced than that in the case of substrate analogues.
6. The modes of binding of the aminoalcohols with the two glucoamylases werevery similar. Therefore, the phenomenon might be a common feature of gluco amylases in general.View full abstractDownload PDF (537K) -
Bruno ROBERT, Sandra ANDRIANAMBININTSOA, Marc LUTZ1985 Volume 98 Issue 2 Pages 349-354
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResonance Raman spectroscopy provided evidence that high 800 nm-absorbing antennae from Rhodopseudomonas (Rps.) acidophila and Rps. palustris have similar structures around their dweller bacteriochiorophylls. These host-site structures are different from those of B 850-800 complexes from Chromatiaceae, which also exhibit a high absorbance at 800 nm. As also shown by previous biochemical data, these complexes might be stoichiometrically different from other antenna complexes, having one more BChI per minimal size unit of protein. A new classification of B 850-800 complexes is proposed, on the basis of resonance Raman and biochemical data: this classification distinguishes a class of B 850-800 S (involving the B 850-800 complexes from sulfur purple bacteria), two classes of B 850-800 NS (involving the B 850-800 complexes from non sulfur purple bacteria) and a class of H 800 complexes (involving the B 850-800 complexes from non sulfur purple bacteria exhibiting a high absorbance at 800 nm).View full abstractDownload PDF (469K) -
Tsukasa CHIBA, Toshio ASAKURA, Katsuko KAKINUMA1985 Volume 98 Issue 2 Pages 355-361
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCetiedil, α-cyclohexyl-3-thiopheneacetic acid 2-(hexahydro-1H-azepin-1-yl)-ethyl ester, was found to inhibit the generation of superoxide (O2-) by porcine neutrophils exposed to various stimulators. The concentration of cetiedil required for 50 inhibition was about 45 μM when neutrophils were stimulated by phorbol myristate acetate. Cetiedil not only decreased the rate of generation of O2-, but prolonged the lag time prior to the production of O2-. The inhibitory effect of cetiedil on the O2-generating activity of the NADPH oxidase in the membrane vesicles was less than that on whole cells; the concentration of cetiedil necessary for 50% inhibition was about 250 μM. To study the mechanism of cetiedil's effect on the mem brane, the transmembrane potential of neutrophils and the intracellular free Ca2+ concentration were monitored by using fluorescence probes, diS-C3-(5), and quin-2, respectively. Cetiedil caused depolarization of the membrane potential and increased the intracellular free Ca2+. These results indicate that integrity of ionic distribution is necessary to activate the O2--generating system of neutrophils.View full abstractDownload PDF (568K) -
Kouichi TAKIMOTO, Masato OKADA, Yoshihiro MATSUDA, Hachiro NAKAGAWA1985 Volume 98 Issue 2 Pages 363-370
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSmyo-Inositol-1-phosphatase [EC 3. 1. 3. 25] was purified from a cytosolic fraction of rat brain. The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 29, 000. The molecular weight of the native enzyme was 55, 000 as determined by molecular sieve chromatography. These values indicated that the native enzyme was composed of two identical subunits. The isoelectric point of the enzyme was 4.6.
The enzyme hydrolyzed inositol-1-phosphate, 2'-AMP, 2'-GMP, β-glycerophosphate, and α-glycerophosphate; the ratio of the reaction rates was 100:84:73:64:32. The Km values for inositol-1-phosphate, 2'-AMP, and β-glycerophosphate were 1.2×10-4M, 1.9×10-4M, and 7.7×10-4M, respectively. Mn2+ and Ca2+ were strong competitive inhibitors against Mg2+, with K1 values of 3 μM and 20 μM, respectively. This result suggests that myo-inositol-1-phosphatase might be regulated by intracellular Ca2+ and/or Mn2+. Li+, which is known to show a therapeutic effect on manic-depressive disease and also to prolong the intrinsic periods of circadian rhythms in various organisms, was a potent uncompetitive inhibitor and inhibited 50% of the activity at 1 mm. The possibility that myo-inositol-1-phosphatase and inositol phospholipid metabolism are involved in circadian rhythm oscillation is discussed in terms of Li actions.View full abstractDownload PDF (634K) -
Tomoko KATO, Masato KAKIUCHI, Shoji OKAMURAI1985 Volume 98 Issue 2 Pages 371-377
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSColchicine-binding protein (CBP) was purified from a cultured carrot cell extract by DEAE-Sephacel, phosphocellulose and Sephadex G 200 column chromatographies. The purified CBP separated into three bands on SDS-polyacrylamide gel electro phoresis. One of them reacted with a monoclonal antibody against chick brain α-tubulin and the other two with that against β-tubulin. Colchicine-binding activity of the purified protein was enhanced by tartrate and inhibited little by an excess of podophyllotoxin. It decayed following first order kinetics, but was more stable than the CBP in the crude extract. The binding constant of the purified CBP for colchicine was 0.57 μM-1 and the number of binding sites of colchicine permg protein was about 2 nmol. This binding constant is about ten times lower than that of porcine brain tubulin under identical conditions.View full abstractDownload PDF (3011K) -
Takeyuki KOHNO, Seiichi HASHIDA, Eiji ISHIKAWA1985 Volume 98 Issue 2 Pages 379-384
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG. Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde. The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20°C). Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound. The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37°C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to β-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al. (1978) J. Biochem. 84, 93-102).View full abstractDownload PDF (505K) -
Haruko KITAGAKI, Isamu MATSUMOTO, Hitomi SASAKI, Nobuko SENO1985 Volume 98 Issue 2 Pages 385-393
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA lectin highly reactive with dermatan sulfate (DS-lectin) was purified from adult chicken liver by gel filtration on Toyopearl HW-55 and subsequent affinity chromatography on new adsorbents which were prepared by immobilizing heparin or dermatan sulfate via the reducing ends on hydrazino-Toyopearl. The DS-lectin behaved as a single protein on polyacrylamide gel electrophoresis.
On excitation at 280 nm, the DS-lectin emitted fluorescence centered at 336 nm, which was attributable to tryptophan residues and could be quenched by the addition of specific saccharides. The affinity constants of the DS-lectin with specific saccharides were calculated from the changes in intensities of fluorescence-difference spectra induced by the saccharides. Dermatan sulfate and protuberic acid, which is composed of L-iduronic acid and D-glucuronic acid (1:2), had the highest affinity constants among the polysaccharides tested. Partially N-desulfated heparin had a higher affinity constant than that of native heparin while dextran sulfate showed no affinity. D-Glucuronic acid and N-acetylneuraminic acid induced weak but significant quenching, but not N-acetylgalactosamine or cellobiose. These results were essentially in good agreement with those of hemagglutination inhibition tests and indicated that DS-lectin has a strong affinity for L-iduronic acid residues and probably carboxyl groups in the saccharides, while sulfate groups on the saccharides interfere with the specific interaction.View full abstractDownload PDF (1337K) -
J. Yuzuru HOMMA, Motohiro MATSUURA, Shiro KANEGASAKI, Yasuaki KAWAKUBO ...1985 Volume 98 Issue 2 Pages 395-406
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-{2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-0-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl}-3-O-[(R)-3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranose 1, 4'-bis(phos-phate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the I or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.View full abstractDownload PDF (3341K) -
Mitsushi INOMATA, Masami HAYASHI, Megumi NAKAMURA, Kazutomo IMAHORI, S ...1985 Volume 98 Issue 2 Pages 407-416
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSome endogenous substrates were incubated with two forms of calcium-activated neutral protease (CANP) with high (μCANP) and low (mCANP) sensitivities to calcium ions. In addition to analyses of the processes of their degradation, changes in the molecular properties of these CANPs were also examined. Among the tested substrate proteins, the myosin heavy chain of rabbit skeletal muscle myofibrils and spectrin or band 3 protein of human erythrocyte membranes were degraded relatively rapidly. So far as these proteins were concerned, a higher degradation velocity was observed for μCANP than for mCANP. Vimentin from ascites tumor cells was degraded most rapidly and no difference was observed in degradation velocity between μCANP and mCANP. In all cases, μCANP and mCANP produced different proteolytic peptide fragments, suggesting the different substrate-specificities of these CANPs.
The degradation of substrates always accompanied the autodigestion of CANPs, and the small subunits of both CANPs were degraded in the early stage of the autodigestion. The large subunit of μCANP (79K) was converted to a 76K polypeptide via a 77K polypeptide as an intermediate. The autodigested μCANP with 76K polypeptide retained sufficient protease activity and, moreover, its calcium-sensitivity was higher than that of intact μCANP. The possibility is thus proposed that restricted autodigestion is a necessary activation step for the appearance of activity of μCANP. No such transition was observed for mCANP.View full abstractDownload PDF (4135K) -
Chikao NAKAYAMA, Mineo SANEYOSHI1985 Volume 98 Issue 2 Pages 417-425
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVarious 5-substituted 1-β-D-xylofuranosyluracil 5'-triphosphates (hydrogen, methyl-, ethyl-, n-propyl, n-butyl, fluoro-, chloro-, bromo-, and iodo derivatives) and some of the 3'-deoxyribofuranosyl nucleotides (3'-deoxy UTP and 3'-deoxy TTP) were synthesized chemically and their inhibitory effects on DNA-dependent RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied systematically. These 3'-modified UTP analogues could not be utilized as substrates in place of UTP, but they did inhibit the incorporation of UMP into RNA in vitro. In contrast, 2'-modified UTP analogues, such as 2'-dTTP and Ara TTP, were neither substrates nor inhibitors. Kinetic analysis showed that the inhibition by these compounds was essentially competitive with substrate UTP. The K1 values of RNA polymerase I for the analogues were smaller (2-6, μM) than the Km value for UTP (8 μM), but those for xylo-EtUTP, xylo-PrUTP, and xylo-BuUTP were larger (about 20 μM) than the Km for UTP. In contrast to these alkyl groups with steric and electron-donating effects, halogen groups have electron-withdrawing effects on the uracil nucleus. Therefore, it was concluded that the inhibitory activity of these analogues on RNA polymerase I was not affected by the inductive effects of substituent groups at the 5-position of uracil nucleus but by their steric effects. On the other hand, all of the K1 values of RNA polymerase II for UTP analogues were smaller (0.4-3 μM) than the Km value for UTP (4 μM). In this case, neither steric effect nor an inductive effect of substituents on UTP analogues influenced the inhibitory activity towards RNA polymerase II.View full abstractDownload PDF (639K) -
Shonen YOSHIDA, Rika SUZUKI, Shigeo MASAKI, Osamu KOIWAI1985 Volume 98 Issue 2 Pages 427-433
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has been shown that DNA primase activity is tightly associated with 10 S DNA polymerase α from calf thymus and that the ribonucleotide-dependent DNA synthesis is more sensitive to araCTP than DNA-primed DNA synthesis (Yoshida, S., et al. (1983) Biochim. Biophys. Acta 741, 348-357). Here we measured DNA primase activity using poly(dT) template or M 13 bacteriophage single-stranded DNA template and primer RNA synthesis was coupled to the reaction by Escherichia coli DNA polymerase I Klenow fragment. By this method, the primer RNA synthesis can be measured independently of the associating DNA polymerase α. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparent K1 for araATP was 21 μM and the ratio of K1/Km (for rATP) was as low as 0.015. With poly(dI, dT) or M 13 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using [α-32P]rATP showed that araATP inhibited the elongation of primer RNA. However, it is not likely that arabinosyl nucleotides act as chain-terminators, since incubation of primer RNA with araATP did not abolish its priming activity. From these results, it is suggested that arabinosyl nucleotide inhibits the initiation as well as elongation of Okazaki fragments in mammalian cells.View full abstractDownload PDF (1192K) -
Seung-Ho KIM, Saburo HARA, Sumihiro HASE, Tokuji IKENAKA, Hiroko TODA, ...1985 Volume 98 Issue 2 Pages 435-448
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequences of three variants of the Kunitz-type trypsin inhibitors, Tia, Tib, and Tic, obtained from some cultivars of soybean were determined by conventional methods. All three inhibitors consisted of 181 amino acid residues. The differences in the amino acid sequences are as follows:
Tia E12 G55 Y62 H71 S74 M114 L120 P137 L176
Tib S F N R V I T V
Tic E
The amino acid sequences of Pro (60) -Ser (61) and Asp (154) -Asp (155) -Gly (156) His (157) of Tia reported previously (Koide & Ikenaka (1973) Eur. J. Biochem. 32, 417-431) were amended to Ser (60) -Pro (61) and His (154) -Asp-Asp-Gly (157), respectively.View full abstractDownload PDF (1576K) -
I. Partial Purification of Isopentenyl Pyrophosphate Isomerase, Farnesyl Pyrophosphate Synthetase, and Geranylgeranyl Pyrophosphate SynthetaseTanetoshi KOYAMA, Minoru MATSUBARA, Kyozo OGURA1985 Volume 98 Issue 2 Pages 449-456
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIsopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, and gera nylgeranyl pyrophosphate synthetase were detected in cell-free extracts of Bombyx mori and were partially purified by hydroxyapatite and Sephadex G-100 chromatography. Two forms of farnesyl pyrophosphate synthetase were chromatographically separated. They were designated as farnesyl pyrophosphate synthetases I and II in the order of their elution from hydroxyapatite. Both enzymes catalyzed the exclusive formation of (E, E)-farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl pyrophosphate or geranyl pyrophosphate. However, they were not interconvertible, unlike the enzyme from pig liver. These two enzymes resembled each other in pH optima and molecular weights but differed in susceptibility to metal ions. Farnesyl pyrophosphate synthetase II was stimulated by Triton X-100 while synthetase I was inhibited by the same reagent.View full abstractDownload PDF (607K) -
II. Formation of the Juvenile Hormone Skeletons by Farnesyl Pyrophosphate Synthetase IITanetoshi KOYAMA, Minoru MATSUBARA, Kyozo OGURA1985 Volume 98 Issue 2 Pages 457-463
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComparative substrate specificities of farnesyl pyrophosphate synthetases I and II purified from larvae of silkworm, Bombyx mori, were studied by use of the possible biosynthetic intermediates of juvenile hormones in the insect. In the presence of Mn2+ ions farnesyl pyrophosphate synthetase II showed higher activity than synthetase I and the corresponding enzyme from pig liver with the following substrate homologues: (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, (2E, 6Z)-3, 7-dimethyl-2, 6-nonadienyl-, and (2E, 6Z)-3-ethyl-7-methyl-2, 6-nonadienyl pyrophosphate. When (Z)-3-methyl-2-pentenyl-, 3-ethyl-3-butenyl-, and isopentenyl pyrophosphate were mixed and incubated with farnesyl pyrophosphate synthetase II, (2E, 6E, 10Z)-3, 11-dimethyl-7-ethyl-2, 6, 10-tridecatrienyl-, (2E, 6 E, 10Z)-3, 7, 11-trimethyl-2, 6, 10-tridecatrienyl, and a trace amount of (2E, 6E, 1OZ)-3, 7-diethyl-ll-methyl-2, 6, 10-tridecatrienyl pyrophosphate, whose carbon skeletons were the same as those of juvenile hormone I, II, and O, respectively, were formed.
(Z)-3-Methyl-2-pentenyl pyrophosphate was produced from 3-ethyl-3-butenyl pyrophosphate as a single product by the action of silkworm isopentenyl pyrophosphate isomerase, though the enzyme activity was much lower with this substrate than with the usual substrate, isopentenyl pyrophosphate.View full abstractDownload PDF (574K) -
Keizo SHIMADA1985 Volume 98 Issue 2 Pages 465-473
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe composition of the light-harvesting system of Rhodopseudornonas sphaeroides forma sp. denitrificans was investigated. When chromatophores were solubilized by sodium dodecyl sulfate (SDS) at 0°C and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), at least two B800-B850 pigment-protein complexes, three B 870 pigment-protein complexes, a reaction center (RC) complex and two pigmented bands which contained B 800, B 850, and B 870 were resolved. In the re-electrophoresis, the B 870 pigment-protein complexes gave rise to a series of multiple pigmented bands. All of these multiple pigment-protein complexes showed almost the same polypeptide composition and absorption spectrum characteristic of the B 870 complex. The apparent molecular weights of these B 870 complexes showed a regular interval of about 7, 000 indicating that these complexes were oligomers of a subunit. It was also found that a predominant B 800-B 850 pigmentprotein complex could be degraded into a small complex via some intermediates. These results indicate that essentially two kinds of pigment-protein complexes construct the light-harvesting system of this bacteria and, upon treatment with SDS, these complexes are degraded into many classes of subunit aggregates showing a complicated profile of pigmented bands on the gel. Pigmented bands which contained both of B 800-B 850 and B 870 complexes were considered to arise from occasional co-migration of distinct B 800-B 850 and B 870 pigment-protein complexes.View full abstractDownload PDF (3588K) -
Toshihiko IKEDA, Kimie AIBA, Kuniaki FUKUDA, Minoru TANAKA1985 Volume 98 Issue 2 Pages 475-482
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe induction of peroxisome proliferation in rat liver was examined after administration of perfluoro-n-decanoic acid (PFDA, C10), perfluoro-n-octanoic acid (PFOA, C8), perfluoro-n-butyric acid (PFBA, C4), 1-H, 1-H-pentadecafluoro-n-octanol (PFOL, C8), perfluorododecane (PFD, C12), and perfluorooctane (PFO, C8). The peroxisome proliferation in the liver was detected by the following methods; 1) measurement of liver weight, 2) assay of hepatic catalase activity, 3) analysis of 600×g supernatant of liver homogenates by SDS-polyacrylamide gel electrophoresis to observe the induction of the bifunctional enoyl-CoA hydratase in peroxisomes (80 K-protein) and 4) observation by electron microscopy. The oral administration of powdered chow containing 0.02%-PFOA and PFBA to male rats of the SpragueDawley strain for 2 weeks and the single intraperitoneal injection of corn oil mixed with PFDA, PFOA, and PFOL at the dose of 100mg/kg induced peroxisome proliferation markedly. PFOL, which has two hydrogen atoms around the hydroxylated carbon, should be metabolized to PFOA, which is an active inducer. Perfluorinated paraffins, PFD and PFO, did not show any induction, indicating the importance of the carboxylic group in the molecule for the peroxisome proliferation. Although the participation of thyroid hormone cannot be excluded, PFOA annears to act directly on the liver.View full abstractDownload PDF (2541K) -
Yoshikazu ICHIHARA, Kazuhiro SOGAWA, Kenji TAKAHASHI1985 Volume 98 Issue 2 Pages 483-492
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe total RNAs were extracted from human, swine, rat, and calf gastric mucosae, and translated in vitro in the presence of radiolabeled amino acids using a wheat germ cell-free system. Upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the translation products, a protein band with a molecular weight of about 43, 000 was obtained in each case as one of the major products. These products could be specifically immunoprecipitated with a corresponding anti-pepsinogen or anti-chymosin antiserum. Radiosequence analysis of these translation products purified by SDS-polyacrylamide gel electrophoresis showed that each of them is a precursor form, i.e., prepepsinogen or preprochymosin, having an amino-terminal extension peptide (signal sequence) comprising 15 (human and swine) or 16 (rat and calf) amino acid residues. The primary structures of these signal sequences were determined to be as follows:
_??_
These signal sequences share common characteristics with those of other pre-secretory proteins, i.e., the presence of positive charges in the NH2-terminal region, hydrophobic amino acid clusters in the interior part, and amino acids with short side chains at the site of cleavage by the signal peptidase.View full abstractDownload PDF (1593K) -
Yoshihiro FUKUMORI, Koji NAKAYAMA, Tateo YAMANAKA1985 Volume 98 Issue 2 Pages 493-499
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c oxidase (cytochrome aa3-type) [EC 1. 9. 3. 1] was purified from Pseudo-monas AM 1 to an electrophoretically homogeneous state and some of its properties were studied. The oxidase showed absorption peaks at 428 and 598nm in the oxidized form, and at 442 and 604 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 602 nm. The enzyme molecule was composed of two kinds of subunits with molecular weights of 50, 000 and 30, 000 and it contained equimolar amounts of heme α and copper atom.
The enzyme rapidly oxidized Candida krusei and horse ferrocytochromes c as well as Pseudomonas AM 1 ferrocytochrome c. The reactions catalyzed by the enzyme were strongly inhibited by KCN.View full abstractDownload PDF (1903K) -
Akio SAITO, Hyogo SINOHARA1985 Volume 98 Issue 2 Pages 501-516
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMurinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr=210, 000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat α-1-macro-globulin but not towards rat or human α-2-macroglobulin. The chemical compo-sitions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat α-1- or α-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two α-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murino-globulin, but not the α-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two α-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two α-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two α-macroglobulins.View full abstractDownload PDF (5522K) -
Mitsuhiro FUKUDA, Hiroshi SHIMA, Shigeru KUNUGI1985 Volume 98 Issue 2 Pages 517-525
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDetailed kinetic analyses of carboxypeptidase P-catalyzed reactions were carried out spectrophotometrically using 3-(2-furyl) acryloyl-acylated peptide substrates. The maximum kcat/Km was observed at around pH 3.5 for the synthetic peptide substrates. The kcat/Km value decreased with increasing pH, with an apparent pKa value of 4.43. However, the maximum kcat was observed at neutral pH (pH_??_6) and the pKa was 4.49. These apparently different pH profiles for kcat/Km and kcat of this enzyme were due to the decreasing Km value in the acid pH region. The pressure and temperature dependences of these kinetic parameters were also measured. N-Benzoylglycyl-L-phenyllactate (Bz-Gly-OPhLac) gave dependences similar to those of the peptide substrate, suggesting that there is no distinct difference in the catalytic mechanism between the peptide and the ester hydrolyses.View full abstractDownload PDF (679K) -
Jun HITOMI, Yoko MURAKAMI, Fumio SAITOH, Naoyuki SHIGEMITSU, Haruki YA ...1985 Volume 98 Issue 2 Pages 527-533
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn endo-β-N-acetylglucosaminidase which hydrolyzes the N, N'-diacetylchitobiosyl linkage in asparagine-linked oligosaccharides was purified from the enzyme product of Aspergillus oryzae. Its substrate specificity was similar to that of endo-β-N-acetylglucosaminidase H from Streptomyces griseus with respect to the relative activities toward the glycopeptides obtained from ovalbumin and bovine IgG. The present endoglycosidase exhibited a broad optimum pH range and was relatively stable. Metal ions, chelating agents and D-mannose did not have a significant effect on the enzyme activity.View full abstractDownload PDF (657K) -
Takashi FUNATSU, Shin'ichi ISHIWATA1985 Volume 98 Issue 2 Pages 535-544
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the physico-chemical properties and the functions of β-actinin by using a β-actinin preparation having the same properties as those reported by Maruyama et al. (J. Biochem. 81, 215-232, 1977). β-Actinin was composed of two components with molecular weights (estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis) of 35, 000 and 31, 000 daltons. Their isoelectric points in 8M urea were, respectively, 5.9 and 5.4, clearly distinguishable from those of tropomyosin, troponin T and some enzymes having similar molecular weights. β-Actinin suppressed the polymerization of actin onto the free end, i.e., the pointed end, of thin filaments in an I-Z-I brush prepared by dissolving thick filaments of a myofibril at high ionic strength. Further, β-actinin suppressed the association of actin to the whole region of an I-Z-I brush. The present study indicates that β-actinin is composed of two components and functions as a suppressor of elongation at the pointed end of thin filaments, supporting the conclusions of Maruyama et al. (J. Biochem. 81, 215-232, 1977).View full abstractDownload PDF (5138K) -
Ken-ichi NAGAI, Keiko TADANO-ARITOMI, Kichitaro KAWAGUCHI, Ineo ISHIZU ...1985 Volume 98 Issue 2 Pages 545-559
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe composition and contents of acidic glycolipids in the kidney of a striped dolphin (Stenella coeruleoalba, the order Cetacea, whales) were determined. The following eight acidic glycolipids were isolated and characterized: SM4s (124.2 nmol/g tissue), SM3 (8.7), GM3 (NeuAc) (12.3), GM3 (NeuGc) (31.6), GD3 (NeuAc-NeuAc) (14.7), GD3 (NeuAc-NeuGc) (II3α(NeuAcα2-8NeuGc)-LacCer) (9.8), GD3 (NeuGc-NeuAc) (IIh3α(NeuGcα2-8NeuAc)-LacCer) (5.3), and GD3 (NeuGc-NeuGc) (15.8). The assignment of the four types of GD3 was further confirmed as described below. Evidence indicating 2-8 linkages of the disialosyl residues of GD3 was obtained on methylation analysis of sialic acid. GD3 (NeuAc-NeuAc) and GD3 (NeuGc-NeuAc) were degraded to GM3 (NeuAc), and GD3 (NeuAc-NeuGc) and GD3 (NeuGc-NeuGc) yielded GM3 (NeuGc) on mild acid hydrolysis. Fragment ions characteristic of the carbohydrate and lipophilic moieties of the permethylated GD3 were observed in direct inlet-electron impact-mass spectra. The presence of these four types of GD3 in a tissue has not been reported previously. GD3 containednon-hydroxy (69-84%) and hydroxy fatty acids (16-31%) with 16-24 carbons. The long chain base of all GD3, except GD3 (NeuGc-NeuAc) (not analyzed), consisted of 4-sphingenine (d 18:1) and 4-hydroxysphinganine (t18:0) in almost equal amounts. The total amount of renal lipid-bound acidic groups (sulfate and sialic acid) of the dolphin (190 μmol/animal) is considerably higher than that of a terrestrial mammal (88 μmol/animal) with a body weight comparable to that of the dolphin. This deviation suggests that the amount of renal acidic amphiphiles required to maintain the osmotic balance of body fluids in marine mammals might be higher than that in terrestrial ones.View full abstractDownload PDF (3055K) -
Akihito MORITA, Yasuo KITAGAWA, Etsuro SUGIMOTO1985 Volume 98 Issue 2 Pages 561-568
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied the effects of monensin on post-translational modification and intracellular transport of precursors of laminin subunits in parietal endoderm-like F9 cells. At concentrations higher than 0.1 μM, monensin inhibited the processing of high-mannose type precursors for all three subunits and caused their cytoplasmic accumulation. Furthermore, the secretion of mature subunits of laminin was inhibited. Instead, polypeptides with similar molecular weights to those of intra-cellular precursors were secreted. These polypeptides were immunologically related to laminin subunits and were sensitive to digestion with β-N-acetylglucosaminidase H (Endo H). This indicated that Golgi complexes of the cells can transport the precursors of laminin subunits even with their terminal glycosylation inactivated by monensin. Tunicamycin induced the accumulation of unglycosylated precursors and strongly reduced their secretion into the medium.View full abstractDownload PDF (4413K) -
Hitoshi SOHMA, Michio YAZAWA, Fumi MORITA1985 Volume 98 Issue 2 Pages 569-572
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOne of the two regulatory light chains, RLC-a, of scallop smooth muscle myosin was fully phosphorylated by myosin light chain kinase of chicken gizzard muscle. The residue phosphorylated was Ser. It may be the Ser at number 11 from the N-terminal. The sequence of 9 residues around the Ser-11, QRATSNVFA, is identical with that around the phosphorylatable Ser of LC20 of chicken gizzard myosin. RLC-a was also phosphorylated slowly by cAMP-dependent protein kinase. The phosphorylation of RLC-a may be involved in the regulatory system for the catch contraction of scallop muscle.View full abstractDownload PDF (1486K) -
Ashok Kumar, Devendra Kumar AGRAWAL, Sultan AHMAD, Edwin Embrey DANIEL ...1985 Volume 98 Issue 2 Pages 573-575
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTarget size of the 5'-nucleotidase in six different smooth muscles was determined by radiation inactivation. The enzyme in the soluble fraction of rat myometrium and vas deferens gave a target size of approximately 80, 000 daltons. The plasma membrane bound 5'-nucleotidase however, gave target size of 80, 000 to 110, 000 daltons in rat gastric fundus and vas deferens and dog stomach and ileum, 135, 000 daltons in rat mesenteric artery and 210, 000 daltons in rat myometrium.View full abstractDownload PDF (242K) -
Takafumi IWASA, Nobuhiro INOUE, Eishichi MIYAMOTO1985 Volume 98 Issue 2 Pages 577-580
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA multifunctional calmodulin-dependent protein kinase in the canine cardiac cytosol was purified to near homogeneity. The purified enzyme inactivated glycogen synthase by means of phosphorylation. The enzyme also phosphorylated phospho-lamban and several other proteins. In view of its physicochemical properties and substrate specificity, the enzyme differed from myosin light chain kinase and phosphorylase kinase, and was considered to belong to a class of similar calmodulin-dependent protein kinases from brain, liver, and skeletal muscle. The results suggest that the enzyme mediates multiple Ca2+-dependent functions in the heart.View full abstractDownload PDF (2251K) -
Masashi SUZUKI, Takemi ENOMOTO, Fumio HANAOKA, Masa-atsu YAMADA1985 Volume 98 Issue 2 Pages 581-584
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conditions for dissociation of the DNA polymerase α-primase complex (DNA polymerase α1) have been examined. It was revealed that 50% ethylene glycol effectively dissociated the complex. The dissociated DNA polymerase and primase were purified to eliminate cross-contaminating activities by column chromatography using buffers containing 50% ethylene glycol. The sedimentation coefficients of the purified DNA polymerase and primase were 7.1 S and 5.7 S, respectively. These two enzymes were mixed in the presence of 20%. ethylene glycol and the mixture was sedimented through a glycerol gradient containing no ethylene glycol. The DNA polymerase and primase activities co-sedimented at 9.1 S which corresponds to the S value of intact α1, indicating the reconstitution of the DNA polymerase α-primase complex.View full abstractDownload PDF (372K)
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