The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 85, Issue 2
Displaying 1-38 of 38 articles from this issue
  • Evidence Supporting Their Possible Functional Relationship
    Koji KIMURA, Hitoshi ENDOU, Jun-ichi SUDO, Fuminori SAKAI
    1979 Volume 85 Issue 2 Pages 319-326
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydro-genease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61×10-5M for NADP+, 4.93×10-5M for glucose 6-phosphate, and 2.14×10-4M for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH.
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  • Effects of Chemical Modification at Glutamic Acid-61 and Cystine-1 and of Organic Solvents on the Enzymatic Activity
    Sadamasa MINATO, Akira HIRAI
    1979 Volume 85 Issue 2 Pages 327-334
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    RNase U2 was purified and crystallized from the enriched culture medium (ammonium sulfate-urea-corn meal) of Ustilago sphaerogena and its characteristics were investigated. Chemical modification of RNase U2 was conducted with monoiodoacetic acid to carboxymethylate Glu-61 and with 2-methoxy-5-nitrotropone to nitrotroponylate the amino terminal residue. The amino terminal residue was modified reversibly by this reagent. Comparison of the 2'-AMP binding in the modified enzyme and the native one showed that Glu-61 is essential for the formation of the enzyme-substrate complex, while the amino terminal residue plays no important role in the enzymatic activity.
    The enzymatic activity and the structure of RNase U2 in aqueous organic solution were also investigated. The affinity of the enzyme for 2'-AMP, the inactivation by monoiodoacetic acid and the fluorescence intensity were examined. The profiles of the changes in the properties of the enzyme protein were consistent with those in the enzymatic activity. Fluorescence studies of the enzyme suggest that the tryptophan residue is closely related to the activity.
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  • Tomohiro MEGA, Yoshio MATSUSHIMA
    1979 Volume 85 Issue 2 Pages 335-341
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    β-Glucosidase [β-D-glucoside glucohydrolase EC 3. 2. 1. 21] and β-galactosidase [β-D-galactoside galactohydrolase, EC 3. 2. 1. 23] of Takadiastase were purified by acetone fractionation, DEAE-cellulose, and hydroxylapatite chromatography.
    Purity was confirmed by disc electrophoresis, ultracentrifugation and measurement of other glycosidase activities which coexisted in Takadiastase.
    Molecular weight of the β-glucosidase was 218, 000 by sedimentation equilibrium and 110, 000-116, 000 by SDS-disc electrophoresis. Molecular weight of the β-galactosidase was 112, 000 by sedimentation and 56, 000-59, 000 by SDS-disc electrophoresis. These values showed that both enzymes consisted of two subunits. Taka-β-N-acetylglucosaminidase also consisted of two subunits.
    Both enzymes were glycoproteins containing glucosamine and neutral sugar. Stability, pH optima, isoelectric points, and some specificities were observed.
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  • I. Biochemical Characterization and Electron Transport of Ascaris Microsomes
    Sadayuki MATUDA
    1979 Volume 85 Issue 2 Pages 343-350
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suurn. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components.
    The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424nm. However, the α-peak at 560nm was asymmetric with a shoulder at 555nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.
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  • II. Purification and Characterization of b-Type Cytochrome and NADH-Ferricyanide Reductase form Ascaris Muscle Microsomes
    Sadayuki MATUDA
    1979 Volume 85 Issue 2 Pages 351-358
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    A b-type cytochrome and NADH-ferricyanide (FC) reductase were solubilized from Ascaris muscle microsomes by detergents and purified by column chromatography.
    The purified b-type cytochrome displayed absorption bands at 560 (α-peak), 525 (β-peak), and 424nm (γ-peak), with a marked shoulder at 555nm in the reduced form, 415nm (γ-peak) in the oxidized form. This absorption spectrum was different from that of rat liver microsomal cytochrome b5.
    The molecular weight was estimated to be about 100, 000 by SDS-polyacrylamide gel electrophoresis, and the absorption spectrum of alkaline pyridine ferrohemochrome suggested that the prosthetic group of this cytochrome is protoheme.
    The molecular weight of the purified NADH-FC reductase was estimated to be about 55, 000 by SDS-polyacrylamide gel electrophoresis. The purified reductase required NADH as a specific electron donor. The reductase efficiently reduced some redox dyes with NADH, but the reduction of cytochrome c was much slower. The purified reductase, like the mem-brane-bound reductase, was not inhibited by thiol reagents.
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  • Two Kinds of Effects of Divalent Cations
    Koshin MIHASHI, Mikio NAKABAYASHI, Hideyuki YOSHIJURA, Hiroshi OHNUMA
    1979 Volume 85 Issue 2 Pages 359-366
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Two kinds of F-actin were prepared; one binds magnesium at the unique divalent cation binding site of actin protomer and the other binds calcium at this site. They were designated as F(Mg)-actin and F(Ca)-actin, respectively. The binding of fluorescein mercuric acetate (FMA) to F(Mg)-actin and F(Ca)-actin was studied spectroscopically. The absorption and fluorescence spectra of bound FMA differed slightly but distinctly between F(Mg)-actin and F(Ca)-actin. Moreover, FMA bound to F(Mg)-actin showed linear dichroism in the presence of 2mM MgCl2 (or 2mM CaCl2) in the solvent, while the dichroisrn was abolished by the removal of divalent cations from the solvent. In contrast, FMA bound to F(Ca)-actin did not show any appreciable linear dichroism irrespective of the presence (or absence) of divalent cations in the solvent. These results suggest that the structure of F-actin is characteristically regulated by divalent cations in a dual mode.
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  • Munenori NOGUCHI, Hiroshi INOUÉ, Kazumi KUBO
    1979 Volume 85 Issue 2 Pages 367-373
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Pellicles were isolated from Paramecium caudatum for a study of the properties of its insoluble ATPase [EC 3. 6. 1. 3] activity. Pellicular ATPase was solubilized by sonication and fractionated by sucrose density gradient centrifugation. The sedimentation coefficient of the ATPase was about 9S. The ATPase required Ca2+ for maximum activation. Addition of neutral salts to the assay medium inhibited the activity. Substrate specificity for ATP was low; other nucleoside triphosphates were hydrolyzed at about the same rate as ATP; AMP, pyrophosphate, and p-nitrophenyl phosphate were not hydrolyzed. The ATPase activity of the pellicle preparation had a pH optimum at pH 6.5, and a Michaelis constant of 9μM. On the other hand, the enzymatic properties of the ATPase were somewhat modified by the procedure of solubilization and fractionation. The pellicular ATPase does not resemble ciliary dynein ATPase or the soluble ATPase of Tetrahymena.
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  • Kumiko OHSUKA, Akio INOUE
    1979 Volume 85 Issue 2 Pages 375-378
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    A myosin-like protein was extracted and partially purified from a flowering plant, Egeria densa. It had no p-nitrophenyl phosphatase activity, but exhibited EDTA(K+)-ATPase [EC 3. 6. 1. 3] activity at high ionic strength. Its molecular weight as estimated by gel filtration was 4-5×105. The presence of a heavy chain (MW=about 1.8×105) was indicated by SDS-gel electrophoresis. Egeria myosin aggregated in an environment of low ionic strength and formed bipolar filaments. It bound with skeletal muscle F-actin with a periodicity of 40nm.
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  • Isolation and Properties of Novel Forms Lacking Tryptophan
    Hiroshi YOSHIDA, Tatsuyuki KUDO, Wataru SHINKAI, Nobuo TAMIYA
    1979 Volume 85 Issue 2 Pages 379-388
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phsopholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14, 000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue, whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, wheras III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A. T., Passey, R. B., & Toom, P. M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.
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  • X. Effcct of Hcmin and an Inhibitor on the Translation of Catalase Messenger RNA
    Terufumi SAKAMOTO, Tokuhiko HIGASHI
    1979 Volume 85 Issue 2 Pages 389-396
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Rat liver catalase mRNA was translated in a rabbit reticulocyte lysates and wheat germ cellfree system in the presence or absence of hemin and/or a translational inhibitor prepared from reticulocytes, liver cells, and wheat germs. Failure to add heroin to the lysates, or the addition of a hemin-regulated translational inhibitor (HRI) to the hemin-supplemented lysates caused a repressed translation. A preparation of inhibitor from rat liver showed activity similar to that of HRI for this translating system. The translation repression by rat liver inhibitor was reversed by eIF-2 (initiation factor) or GTP, but ATP enhanced the repression. The translation of catalase mRNA in the wheat germ system was not affected by the addition of hemin. An inhibitor prepared from wheat germ extracts, as well as the rat liver inhibitor, markedly decreased the rate of translation. eIF-2, GTP, and ATP behaved in the manner described above.
    Catalase synthesis in a cell-free system derived from rat liver (using endogenous mRNA) was not influenced by either hemin or the inhibitor.
    The possibilities are discussed that the synthesis of catalase in liver cells is controlled by a translational inhibitor at the level of chain initiation, and that the formation of the inhibitor from its inactive proinhibitor is regulated by the amount of heme.
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  • Michiko HAMATO, Tokuhiko HIGASHI
    1979 Volume 85 Issue 2 Pages 397-402
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Rats were injected with a single or repeated doses of heroin intraperitoneally, and the effect on liver catalase [EC 1. 11. 1. 6] was studied. A single administration of hemin caused a reduction in the concentration of liver catalase, both in enzymatic activity and in catalase protein determined immunochemically. The reduction occurred a few hours after the hemin injection, and is probably due to stimulated degradation. Disappearance of radioactivity from liver catalase prelabelled with [14C]leucine was enhanced following the administration of hemin. No evidence for a repression in vivo incorporation of [14C]leucine and [3H]δ-aminolevulinic acid into liver catalase was obtained with hemin-treated rats. When the hemin was given repeatedly at 12-h intervals, the level of liver catalase decreased considerably. However, the impairment in catalase-synthesizing activity of liver cells of rats thus treated was rather slight, when examined in a cell-free system. Some differences were noted between the results in the present study and those in previous investigations with Sedormid-treated rats.
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  • Tsunemi HASEGAWA, Shin-ichi ISHII
    1979 Volume 85 Issue 2 Pages 403-411
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    By a mild alkaline treatment of pyocin RI, the core particle was released from the contracted sheath. After sucrose density-gradient centrifugation, core-rich fractions were treated with anti-sheath serum and by a second density-gradient centrifugation, purified core particles were isolated. Homogeneity of the preparation was confirmed by observation under the electron microscope, immuno-precipitation reaction, and sucrose density-gradient centrifugation. The core particle exhibited a sedimentation coefficient of 37S. The quaternary structure of the core consists of a single kind of subunit protein with a molecular weight of 18, 000. No contamination by other proteins was detected by SDS-disc electrophoreses. Amino acid analysis revealed that the core is rich in glycine, alanine, valine, leucine, aspartic acid (or asparagine), glutamic acid (or glutamine), and serine. This amino acid composition bears some resemblance to that of T-even bacteriophage tail-core.
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  • Yoshimi OHNO, Ikuya YANO, Masamiki MASUI
    1979 Volume 85 Issue 2 Pages 413-421
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    A moderately halophilic bacterium, Pseudomonas halosaccharolytica ATCC 29423, can grow over a wide range of NaCl concentrations (0.5-4.25M), and high concentrations of NaCl enabled the bacterium to grow well at higher temperatures.
    The addition of glucose to a medium containing 2M NaCl resulted in an increase in the proportions of glucosylphosphatidyl glycerol and diphosphatidylglycerol with a concomitant decrease of phosphatidylglycerol, whereas there was no effect on the level of phosphatidylethanolamine.
    Increasing the cultivation temperature from a low level to the optimum for growth induced a marked increase in the relative amount of phosphatidylglycerol with a concomitant decrease of diphosphatidylglycerol, and further elevation of the temperature reversed the relation between the two phospholipids. Phosphatidylethanolamine gradually decreased as the temperature was increased, while the level of glucosylphosphatidylglycerol remained constant. The relative concentrations of C17 and C19 cyclopropanoic fatty acids increased with a concomitant decrease of corresponding unsaturated fatty acids as the growth temperature was increased.
    It was found that the total amount of negatively charged phospholipids in cells increased with NaCl concentration in the medium, from 0.7 times in 0.8M NaCl medium up to twice with 2M or more NaCl, compared with that of neutral phospholipid, phosphatidylethanolamine. Phosphatidylglycerol mainly contributed to the increased amount of negatively charged phospholipids. Furthermore, changes in the molecular species composition of phospholipids with different NaCl concentrations in the medium were observed; a high pro-portion of cyclopropanoic fatty acid-containing species, C16-17, C16-19 and C19-19, with a concomitant decrease of corresponding monounsaturated species was seen in cultures containing high NaCl concentrations at any growth phase, suggesting the induction of cyclopropane synthetase by high levels of NaCl.
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  • The Role of Lysyl Residues in the Catalytic and Regulatory Functions
    Aiko NAIDE, Katsura IZUI, Takeo YOSHINAGA, Hirohiko KATSUKI
    1979 Volume 85 Issue 2 Pages 423-432
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Phosphoenolpyruvate (PEP) carboxylase [EC 4. 1. 1. 31] of E. coli was inactivated by 2, 4, 6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, ε-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-α-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased.
    The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1, 6-bisphosphate (Fru-1, 6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1, 6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1, 6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1, 6-P2 and GTP.
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  • Fumito MATSUURA
    1979 Volume 85 Issue 2 Pages 433-441
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    From Monodonta labio a mixture of aminoalkylphosphonyl cerebrosides was isolated in a yield of 1.0% of the total complex lipids. The aminoalkylphosphonyl cerebrosides were found to be composed of a major portion of 1-O-[6'-O-(N-methylaminoethylphosphonyl)galactopyranosyl]ceramide and a minor portion of 1-O-[6'-O-(aminoethylphosphonyl)galactopyranosyl]ceramide.
    Palmitic (49.7%) and 2-hydroxy palmitic (12.2%) acids were the main fatty acid constituents and a small amount of oleic acid was also detected. The long chain bases had a charac-teristic pattern with 16-22 carbon chain lengths, and were classified into four groups: normal dihydroxy monounsaturated bases (40.6%), branched dihydroxy monounsaturated bases (34.0%), dihydroxy diunsaturated bases (22.0%), and trihydroxy bases (2.2%). Among them dihydroxy 18:1 and 18:2 and branched 19:1 bases were predominant.
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  • Seiki KURAMITSU, Kozo HAMAGUCHI
    1979 Volume 85 Issue 2 Pages 443-456
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300nm and had no isosbestic points. The ratio of the extinction difference at 245nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried tyrosyl residue on alkali denaturation.l The disulfide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of Tyr 23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25°C from the CD titration data. Comparison of the CD titration of turkey lysozyme with that of hen lysozyme suggested that Tyr 3 and Tyr 23 in turkey lysozyme have apparent pK values of 11.9 and 9.8, respectively.
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  • Hirofumi ONISHI, Shizuo WATANABE
    1979 Volume 85 Issue 2 Pages 457-472
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    A method was developed to obtain a preparation of chicken gizzard heavy meromyosin (HMM) that retains the two light-chain components of parent myosin: the 20, 000-dalton and 17, 000-dalton light-chains. The HMM preparation was also shown to retain two characteristics of the ATPase activity of the parent myosin: the characteristic effect of phosphorylation of the 20, 000-dalton light-chain component on the ATPase activity, and the characteristic dependence of the ATPase activity on the KCl concentration.
    1. Two distinct stages were observed in the Mg-ATPase reaction catalyzed by gizzard HMM and rabbit skeletal actin in the presence of gizzard “native” tropomyosin (NTM) and Ca2+ ions: an early lag phase, in which the reaction rate gradually increased, and a subsequent steady state, in which the reaction proceeded at a high, constant rate. Urea-gel electrophoresis revealed that the 20, 000-dalton light-chain component was gradually phosphorylated in the lag phase, and was fully phosphorylated in the steady state. It was also observed that addition of EGTA (to remove Ca2+ ions) at various times in the lag phase caused neither a further increase nor a decrease in the reaction rate, and that addition of EGTA in the steady state caused no change in the reaction rate. These observations imply that the ATPase activity increased as the amount of phosphorylated 20, 000-dalton light-chain component increased, and also that Mg-ATPase of acto-phosphorylated HMM was no longer calcium-sensitive.
    2. The Mg-ATPase activity of HMM in the presence of gizzard NTM and Ca2+ ions or EGTA was studied as a function of the concentration of rabbit skeletal actin. The maximal activity (Vmax) and the apparent affinity constant of acto-HMM (KA) were thus estimated from the double-reciprocal plot of Eisenberg-Moos: the Vmax and KA values for phospho-rylated HMM (in the presence of Ca2+ ions) were 5s-1 and 5.5mg/ml actin, respectively, and the Vmax value for unphosphorylated HMM (in the presence of EGTA) was 0.3 s-1, assuming that the KA value with unphosphorylated HMM is equal to that with phosphorylated HMM.
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  • Kiyoshi ABE, Nobuo MAKINO, F. Koichi ANAN
    1979 Volume 85 Issue 2 Pages 473-479
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The pH-dependence of the kinetic parameters in H2O2 decomposition by beef liver catalase was investigated. At pH 7.0, the ternary complex (ESS) decomposition rate was about 100 times faster than ESS formation (42μm H2O2), and the value of the Michaelis constant was 0.025M.
    From ethanol competition experiments, two different proton dissociation constants of the enzyme (pKe1, =5.0, pKes2=5.9) were obtained for the binding of first and second H2O2 molecules. Another pKa value (pKes1) of 4.2 was obtained from the pH dependence of overall rate constant (k0). The reaction mechanism of catalase was discussed in relation to these ionizable groups.
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  • Yukihiro SANADA, Nobumasa YASOGAWA, Nobuhiko KATUNUMA
    1979 Volume 85 Issue 2 Pages 481-483
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Morphological changes occurred in myofibrils prepared from the glycerinated psoas muscle of rabbit during incubation with a serine protease crystallized from rat skeletal muscle. Two notable phenomena were observed: (1) loss of the Z band in the early stage of incubation and (2) complete disappearance of the A band after swelling of the myofibrils. The results in-dicate that the serine protease has an action on myofibrils different from that of Ca2+-depend-ent neutral protease.
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  • pH Dependence of Delayed Fluorescence, Electron Transfer and Degree of Coupling
    Hiroyuki ARATA, Mitsuo MISHIMURA
    1979 Volume 85 Issue 2 Pages 485-494
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The effects of pH on the thermodynamic properties of the proton-translocating cyclic electron transfer system in a purple photosynthetic bacterium Chromatium vinosum were studied. Two thermodynamic parameters, the flux (Je) and force (Δ_??_e) of the electron transfer process, were analyzed. The rate of electron transfer in the re-reduction of photooxidized reactioncenter bacteriochlorophyll was used as Je. Δ_??_e was determined from the intensity of the delayed fluorescence from bacteriochlorophyll. Δ_??_e is composed of the redox potential difference and the electrical potential difference between two electron transfer components. In the steady state under illumination, the flux-to-force ratio is determined by the following relationship:
    Je=(1-q2)Lee Δ_??_e
    where q is the “degree of coupling” of electron transfer to proton translocation and Lee is the value of Je/Δ_??_e when there is no back pressure by formation of Δ_??_H+ (electrochemical potential difference of H+). The value of (1-q2)Lee increased with increasing pH in the neutral pH range. Uncouplers and ionophores that dissipate Δ_??_H+ increased Je and decreased Δ_??_e. The effects were more prominent in the lower pH range. Therefore, q must be smaller at higher pH. The coupling is probably tight when redox components are saturated with protons. The experimental results agreed with the theoretical predictions for a system where a hydrogen-translocating component functions as an electron-proton symport carrier.
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  • Hiromichi KUMAGAI, Eisuke NISHIDA, Hikoichi SAKAI
    1979 Volume 85 Issue 2 Pages 495-502
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Microtubule (MT) assembly was investigated in the presence of ATP and Ca ions using both crude extract (CE) and purified microtubular proteins (PMP) prepared from porcine brains. ATP inhibited MT assembly from CE prepared with the reassembly buffer containing 1mM GTP. Half-maximal inhibition occurred at an ATP concentration of 0.4-0.5mM. Calcium ions, on the contrary, cancelled the ATP-induced inhibition, 1-2 μm calcium ions supporting maximal MT assembly. The ATP-induced inhibition in PMP was not so prominent as in CE, but occurred significantly in the presence of RNA. In PMP dissolved in the reassembly buffer containing ATP and yeast tRNA, the content of the ring fraction decreased significantly as compared with PMP containing only RNA. Furthermore, microtubule-associated proteins were found to be capable of binding ATP. The significance of the ATP-induced inhibition of MT assembly and the release of the inhibition by Ca2+ was discussed.
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  • Nobuhito SONE, Masasuke YOSHIDA, Hajime HIRATA, Yasuo KAGAWA
    1979 Volume 85 Issue 2 Pages 503-509
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    H+-Translocating ATPase, which catalyzes ATP synthesis in biomembranes, is composed of a head piece (F1) and a membrane moiety (F0). Using highly-purified Fo from a thermophilic bacterium PS3 (TF0), the following results were obtained.
    1. Inhibition by N, N'-dicyclohexylcarbodiimide (DCCD) of H+ conduction through TF0 followed pseudo-first-order kinetics. The second-order rate constant for inhibitor-enzyme interaction was 5×103M-1•min-1
    2. H+ conductivity blocked by DCCD was proportional to the amount of DCCD incorporated in the band 8 protein of TF0. When only one-third of the band 8 protein was labeled with DCCD, TF0 hardly transported any H+.
    3. By extracting TFo with chloroform-methanol, the band 8 protein was obtained as a proteolipid. Polyacrylamide gel electrophoresis with dodecyl sulfate and urea showed that the molecular weight was about 6, 000.
    4. The amino acid composition of band 8 protein indicated that this protein contained an extremely high percentage of hydrophobic amino acids (0.29 in polarity) and was devoid of histidine, tryptophan, cysteine, and lysine. Its minimum molecular weight was 6, 500.
    5. The role of band 8 protein (DCCD-binding protein) in H+ conduction through TF0 is discussed on the basis of these results.
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  • Effect of the Sulfate Group on the Hydrolysis
    Gen'ichiro NONAKA, Yasuo KISHIMOTO, Yousuke SEYAMA, Tamio YAMAKAWA
    1979 Volume 85 Issue 2 Pages 511-518
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Saponification of cerebroside sulfate (sulfatide) by refluxing with 1 N KOH in 90% n-butanol for 1h yielded ceramide, sphingosine, lysosulfatide (psychosine-3'-sulfate ester) and a hitherto unknown compound. The latter compound was identified as 3, 6-anhydrogalactosyl sphingosine (3', 6'-anhydropsychosine) from its mass spectrum. The structure of lysosulfatide was confirmed by reacylating it to sulfatide by condensing it with lignoceroyl chloride. The resulting sulfatide, which was chromatographically identical to control sulfatides, was not oxidized by periodate. The sulfatide was also permethylated and methanolyzed. The sugar moiety obtained was identified as methyl 2, 4, 6-tri-O-methylgalactoside by gas-liquid chromatography and thin-layer chromatography. The presence of the sulfate group in lysosulfatide was further confirmed by IR spectroscopy and the presence of radioactivity when it was prepared from [35S]sulfatide. The effect of the sulfate group on cleavage of the galactoside linkage and on the formation of the 3, 6-anhydro derivative is discussed.
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  • A Decrease in Sulfation of Chondroitin Sulfate with Aging
    Atsushi HONDA, Midori ABE, Sei-itsu MUROTA, Yo MORI
    1979 Volume 85 Issue 2 Pages 519-528
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The amount of glycosaminoglycan (GAG) in dry costal cartilage tissue of rats decreased with aging, while the GAG content in mg DNA (unit cartilage cell) remained the same with aging. These results can be explained by the finding that the total number of cartilage cells decreased with aging.
    Electrophoretic analysis showed that chondroitin 4-sulfate was the major GAG in rat costal cartilage of various ages.
    Rat costal cartilage of different ages was incubated with radioactive precursors, and newly synthesized GAG was prepared and the radioactivity analyzed to determine the biosynthetic activity. As to changes in the radioactivity uptake with aging per mg dry cartilage tissue, aging influenced [35S]sulfate incorporation into GAG more significantly than [3H]glucosamine incorporation into GAG. There was a significant decrease in the specific radioactivity of [35S]-sulfate per mg DNA (unit cartilage cell), whereas the specific radioactivity of [3H]glucosamine per mg DNA did not change significantly with aging.
    Both the total sulfotransferase activity and the specific activity per mg DNA decreased significantly with aging.
    Analysis of disaccharide units formed after chondroitinase ABC digestion of labeled GAG isolated from young and old cartilage showed that the percentage of incorporation of [3H]-glucosamine into ΔDi-OS increased significantly with aging. These results suggested that the appearance of nonsulfated positions in the structure of the chondroitin sulfate chain increased with aging.
    On the basis of gel chromatography on Bio-Gel A-1.5m no significant difference in the approximate molecular size of chondroitin sulfate was observed between the young and old GAG samples.
    The present study indicated that the sulfation of chondroitin sulfate chains from rat costal cartilage decreased with the process of aging.
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  • Choemon KANNO, Kunio YAMAUCHI
    1979 Volume 85 Issue 2 Pages 529-534
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Large amounts (66-97%) of marker enzymes such as alkaline phosphatase, 5'-nucleotidase, phosphodiesterase I, and γ-glutamyl transpeptidase of bovine milk fat globule membrane (MFGM) were selectively solubilized by nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK, and Emulgen 109-P. On the other hand, the extractability of MFGM protein with these detergents was less than 50%. Judging from the recovery of total activity, it is likely that alkaline phosphatase, phosphodiesterase I, and γ-glutamyl transpeptidase are activated by nonionic detergents, whereas 5'-nucleotidase is somewhat inhibited by the detergents, except for Tween 20, and acid phosphatase is strongly inhibited by all detergents. In addition, solubilization of the protein with the nonionic detergents was found to be somewhat selective by SDS-polyacrylamide gel electrophoresis. There was no appreciable difference between the five brands of nonionic detergents used as regards the extractability of protein and the enzymatic activity of the extracted marker enzymes of MFGM, except that the solubilizing ability of Tween 20 was relatively low.
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  • Miyako TANAKA, Hidehiro TANAKA
    1979 Volume 85 Issue 2 Pages 535-540
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    A method for the almost complete extraction of myosin from smooth muscle fibers of the anterior byssal retractor muscle (ABRM) of Mytilus edulis was developed, and functional reformation of thick filaments in the fibers was achieved. Complete removal of myosin from the glycerol-extracted ABRM fibers with a solution containing 600mM KCl, 5mM MgCl2, and 5mM ATP was difficult. However, successive treatments of the ABRM fibers with glycerol and saponin made the plasma membrane permeable to Mg•ATP and myosin. The extraction of myosin completely eliminated the tension induced by the addition of Mg•ATP. Partial recovery of tension development was observed by irrigation of myosin into fibers from which myosin had been extracted. Similar results were obtained using rabbit myosin instead of ABRM myosin. Addition of heavy meromyosin, on the other hand, had a suppressive effect on the tension development, as is the case in glycerinated rabbit psoas muscle fibers.
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  • Junki KO, Shiro HORIUCHI, Masahiro YAMAGUCHI
    1979 Volume 85 Issue 2 Pages 541-548
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    1. Crayfish (Procambarus clarki) myosin was obtained from abdominal flexor muscle. The Ca2+-ATPase activity of crayfish myosin was much lower than that of rabbit skeletal myosin. However, F-actin-activated Mg2+-ATPase of crayfish and its superprecipitation closely resembled those of rabbit skeletal myosin. This fact suggests that the ability of crayfish myosin to combine with F-actin is essentially the same as that of skeletal myosin, although the chemical structures of both the myosin molecules when involved in their Ca2+-ATPase activity must be different from each other.
    2. Crayfish and rabbit skeletal myosins were subjected to SDS-polyacrylamide gel electrophoresis. Crayfish myosin was found to have one heavy chain and two distinct light chain components (CF-g1 and CF-g2), which have molecular weights of 18, 000 and 16, 000, respectively. These light chains correspond in molecular weight to the light chains (SK-g2 and SK-g3) in rabbit skeletal myosin.
    3. CF-gl could be liberated from the crayfish myosin molecule reacting with 5, 5'-dithio-bis (2-nitrobenzoic acid), (Nbs2), without recovery of ATPase activity by the addition of DTT. These properties are equivalent to those of SK-g2 in rabbit skeletal myosin, although Nbs2-treated crayfish myosin did not recover its ATPase activity at all.
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  • Yoshihiro FUKUOKA, Yoshio HOJIMA, Shuichi MIYAURA, Chiaki MORIWAKI
    1979 Volume 85 Issue 2 Pages 549-557
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The cat submaxillary gland contains 1, 000-2, 400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6-7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930-1, 260 KU in the dog vasodilator assay, and hydrolyzed 15-25 and 9-12 μmol of N-α-benzoyl-L-arginine ethyl ester (BAEE) and N-α-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in I min at 25°C and pH 8.0. The Km's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34mM, respectively. Hydrolysis of N-α-benzoyl-L-tyrosine ethyl ester (BTEE), N-α-benzoyl-arginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5×104 by Sephadex G-100 gel filtration and 4.7×104 by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.
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  • Masayuki MITSUKA, Takenori YAMADA, Hiroshi SHIMIZU
    1979 Volume 85 Issue 2 Pages 559-565
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The shortening of myosin-extracted skinned single fibers (MESS fibers) was studied in detail under irrigations with myosin, heavy meromyosin (HMM), and myosin subfragment-1 (S-1). The MESS fibers were carefully prepared according to Tawada et al. (J. Biochem. (1976) 80, 121-127) with a modification as regards the extraction temperature. In addition, the fibers were kept at 40°C for more than 10min in order to inactivate any trace amount of myosin, if present. The MESS fibers were confirmed to recover their shortening ability when placed in myosin, HMM, and S-1 solutions, as reported by Oplatka et al. (Biochim. Biophys. Acta (1976) 440, 241-258). The velocity of the shortening was very slow but was proportional to the original length. A quasi-biphasic behavior was observed in the extent of the shortening of the MESS fibers, at least in heavy meromyosin solutions, with respect to the temperature as well as the concentration of Mg-ATP. The molecular mechanism of the shortening can be discussed in terms of the metachronal rotation model of myosin heads. It is pointed out that close parallels exist between the shortening of MESS fibers with heavy meromyosin and the active streaming of heavy meromyosin solutions in a stream cell (Yano, M. et al. (1978) J. Biochem. 84, 277-283; Yano, M. & Shimizu, H. (1978) J. Biochem. 84, 1087-1092; Shimizu, H. & Yano, M. (1978) J. Biochem. 84, 1093-1102). Therefore, the dynamic cooperativity among elementary cycles of the chemo-mechanical conversion seems to be an important factor in the shortening of MESS fibers.
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  • Attribution of Most of the Enzymic Activity to an Actomyosin-Like Protein
    Toshihiro TSUDZUKI
    1979 Volume 85 Issue 2 Pages 567-574
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Synaptic vesicle fractions, which were isolated from bovine cerebral cortex, caudatolenticular nuclei, and chick brain according to Kadota and Kadota (1), contained an Mg2+-dependent ATPase showing maximum activity at around pH 6.5-6.8 in common. The ATPase was similar to that in cow-adrenal chromaffin granules as regards the optimum pH (16) and susceptibility to inhibition by NEM (18). SDS-polyacrylamide gel electrophoresis revealed that the vesicle fractions contained protein components which were tentatively identified as a myosin-like protein (220, 000 daltons), tubulin (54, 000), and actin (45, 000), as reported previously (6, 17, 19). Further purification of the vesicles by sucrose density gradient centrifugation brought about a decrease in the content of the myosin-like protein, accompanied by a decrease in the Mg2+-ATPase activity. These results suggest that most of the Mg2+-ATPase in the vesicle fractions is due to the (acto)myosin-like protein, whose Mg2+-ATPase shows maximum activity at around pH 6.8 (21), and that the protein could be separated from the synaptic vesicles.
    The synaptic vesicle fraction of caudatolenticular nuclei bound tritiated dopamine in a saturable manner at pH 6.7 and 25°C, but the binding was not accelerated by ATP. The pH-dependency of the binding of dopamine did not resemble that of the Mg2+-ATPase activity. Hence the Mg2+-ATPase appeared not to be involved in the uptake of dopamine by the synaptic vesicles.
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  • Takao OHYASHIKI, Takamitsu SEKINE
    1979 Volume 85 Issue 2 Pages 575-580
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Spectrofluorometric studies on the conformational changes in tropomyosin associated with depolymerization of the molecules were carried out using 1-anilino-8-naphthalene sulfonate (ANS). When ANS-probed tropomyosin was depolymerized to its monomer, the fluorescence intensity markedly increased, with a decrease in fluorescence polarization. On the other hand, the emission maxima of the ANS-tropomyosin complexes of both forms were the same. The temperature dependence of the polarization of the complexes at various KCl concentrations suggested that the segmental motion of a moiety containing the fluorophore was considerably activated by depolymerization of tropomyosin. In the polymerized and oligomeric forms, a thermal transition in the polarization was observed with a transition temperature of 30°C. Titration curves of tropomyosin with ANS showed simple saturation kinetics with both monomer and polymer, and the apparent dissociation constants were estimated to be 9.93×10-5M (monomer) and 7.43×10-5M (polymer). On the other hand, the number of the ANS-binding sites increased from 0.5 to 2.0 per tropomyosin monomer on depolymerization of the molecules.
    Based on these results, the conformational state of tropomyosin in the polymerized form is discussed.
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  • Takashi KUMAZAKI, Shin-ichi ISHII
    1979 Volume 85 Issue 2 Pages 581-590
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    A commercial preparation of bovine trypsin was treated with methyl acetimidate-HCl, and most of the lysine residues were converted to trypsin-resistant residues retaining their cationic charges. The modified preparation was then fractionated by ion-exchange chromatography on SE-Sephadex C-50 into two active components, amidinated α- and amidinated β-trypsins. The latter component (Am-β-trypsin), which consisted of a single polypeptide chain, was al-lowed to autolyze at pH 7.8, 25°C for 3.5h and a new active component named Am-δ-trypsin was isolated from the autolysate. Several lines of experimental evidence indicated that Am-δ-trypsin was derived by primary cleavage of the bond Argl05-Val106. Cleavage at Arg55 Leu56, on the other hand, appeared to lead to inactivation of Am-β-trypsin.
    The kinetic properties of the catalytic hydrolyses of synthetic substrates and the affinity to Gly-Gly-Arg immobilized on Sepharose were compared among Am-δ-, Am-β-, and Am-α-trypsins. Am-δ-trypsin resembled Am-β-trypsin in these properties, but did not resemble Am-α-trypsin which had a cleavage at Lys131-Ser132.
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  • III. Multiplicity of β-Glucosidase, and Purification and Properties of a Second Component
    Toshiya HIRAYAMA, Hideo NAGAYAMA, Kazuo MATSUDA
    1979 Volume 85 Issue 2 Pages 591-599
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    To determine the relationship between the induction patterns of three components of β-glucosidase of Pyricularia oryzae and carbon sources in the growth medium, various culture conditions were examined. Avicel, hydroxyethylcellulose and methyl-β-D-glucoside as the carbon source induced both β-glucosidase components, GB-1 and GB-2, whereas cellobiose and gentiobiose induced only one component, GB-1. Thus, these two components were induced independently and hence thought to be isozymes.
    The GB-2 was purified to homogeneity by ion exchange and gel filtration chromatog-raphies from two different cultures on methyl-β-D-glucoside and Avicel. The specific activity of GB-2 when salicin was used as substrate was approximately 5.9mg glucose/min/mg protein. GB-2 was found to be an oligomeric glycoprotein, which consisted of two subunits with molecular weight of approximately 120, 000, comprising a relatively large number of acidic amino acids and mannose, as is the case with GB-1.
    These two isozymes were clearly different in thermostability, GB-2 being more thermolabile than GB-1. However, the same carboxyl group (pKa 4.2-4.8) was found to be strongly implicated in the formation and dissociation of the enzyme-substrate complex for both of the enzymes, from the analysis of kinetic parameters as a function of pH.
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  • The Formation of Ternary Complex of Streptomyces Subtilisin Inhibitor, α-Chymotrypsin, and Proflavine
    Kuniyo INOUYE, Ben'ichiro TONOMURA, Keitaro HIROMI
    1979 Volume 85 Issue 2 Pages 601-607
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    The present paper deals with the inhibition and the binding of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), against α-chymotrypsin. Both α-chymotrypsin and trypsin have been reported not to be inhibited by this inhibitor when casein is used as a substrate (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067-1074). However, in this study, SSI was shown to inhibit α-chymotrypsin competitively with an inhibitor constant, K1=3.8μM, by using p-nitrophenyl acetate as a substrate. The dissociation constant of α-chymotrypsin-SSI complex determined by static titration utilizing the small ultraviolet absorption difference spectra observed on the binding of the enzyme and SSI (Kd) is 2.2μM, and that obtained utilizing the visible absorption difference spectra of proflavine bound to the active site of the enzyme (Kd') is 2.9μM. These values are almost 104 times larger than the inhibitor constant estimated for subtilisin BPN', which was of the order of 10-10M (Inouye, K. et al. (1977) J. Biochem. 82, 961-967).
    It was demonstrated by spectrophotometric titration and equilibrium dialysis that proflavine bound at the active site of α-chymotrypsin is not displaced by SSI binding, but forms a ternary complex with the enzyme and the inhibitor. The dissociation constant of the α-chymotrypsin-proflavine complex into the enzyme and proflavine (Kp) was determined to be 51±1μM by spectrophotometric titration and 64±5μM by equilibrium dialysis; the dissociation constant of proflavine from α-chymotrypsin-SSI-proflavine complex (Kp') was determined to be 132±8μM by spectrophotometric titration and 160±8μM by equilibrium dialysis.
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  • Shigeru SAKIYAMA
    1979 Volume 85 Issue 2 Pages 609-613
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
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    Polysomal RNA of rat ascites hepatoma AH 7974 cells was fractionated into poly(A)+ and poly(A)- mRNAs and these RNAs were translated into polypeptides in a protein-synthesizing system derived from wheat germ. The analysis of polypeptides synthesized in vitro by twodimensional gel electrophoresis revealed that there are three classes of polypeptides. The first group can be synthesized equally by both poly(A)+ and poly(A)- mRNAs; the second and third groups are synthesized predominantly by poly(A)+ and poly(A)- mRNAs, respectively. These results suggest that the three classes of polypeptides can be characterized by the presence or absence of poly (A) tails of the corresponding mRNAs.
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  • Isolation and Amino Acid Sequence
    Yoshihide OHE, Hiroaki HAYASHI, Koichi IWAI
    1979 Volume 85 Issue 2 Pages 615-624
    Published: February 01, 1979
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    The amino acid sequences of human histones have been investigated for studies of histone evolution. The whole histone was prepared from human spleen and was separated into 3 fractions, H4+H3+H2A, H2B, and HI, by our technique of CM-cellulose chromatography. The H2B fraction was further purified by Bio-Gel P-60 chromatography. For sequence determination, the H2B molecule was first split into 4 major fragments, I to IV, by limited chymotryptic digestion at pH 5.0 and 15°C, followed by Sephadex G-50 chromatography. Fragments I and III were then digested with trypsin, yielding 18 and 16 peptides, respectively, on column and paper chromatographies. Sequence analyses of these tryptic peptides, as well as chymotryptic fragments II and IV, showed no differences from the corresponding parts of calf thymus H2B sequence, making it possible to locate fragments I to IV at residues 1-40, 41-42, 43-121, and 122-125 of the total sequence. The only new findings were microhetero-geneities at residues 39 (75% valine and 25% isoleucine) and 124 (70% serine and 30 alanine). The sequence of the most basic cluster at residues 27-34, -Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-, was confirmed with a peptide obtained from fragment I by staphylococcal protease digestion. Thus, it is concluded that the H2B sequence of lower mammals was conserved during the evolutionary process leading to man.
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  • 1979 Volume 85 Issue 2 Pages 625a
    Published: 1979
    Released on J-STAGE: November 18, 2008
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  • 1979 Volume 85 Issue 2 Pages 625b
    Published: 1979
    Released on J-STAGE: November 18, 2008
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