The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 95 , Issue 3
Showing 1-42 articles out of 42 articles from the selected issue
  • Kihachiro HORIIKE, Hiromasa TOJO, Toshio YAMANO, Mitsuhiro NOZAKI
    1984 Volume 95 Issue 3 Pages 605-609
    Published: 1984
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    In the study of chemical modification of proteins, it has been a common practice to plot the fractional remaining activity against the number of residues modified per protein molecule. Extrapolation of the initial, nearly linear portion of the curve to the axis giving numbers of residues has often been presumed to specify the actual number of critical groups modified, i.e., the stoichiometry of the modification and the inactivation reactions. However, this extrapolation method is not generally applicable (Horiike, K. & McCormick, D. B. (1979) J. Theor. Biol. 79, 403-414). This paper describes further examination of the underlying theoretical framework of the extrapolation method. The properties and features of the extrapolated values are considered and presented with numerical and graphical examples. And the theoretical conditions with which the extrapolated value gives the number of essential residues are derived.
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  • Noriaki ISHIOKA, Toshiaki ISOBE, Toshihiko KADOYA, Tsuneo OKUYAMA, Tak ...
    1984 Volume 95 Issue 3 Pages 611-617
    Published: 1984
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    A simple method has been developed for the large scale purification of neuronspecific enolase [EC 4. 2. 1. 11]. The method consists of ammonium sulfate fractionation of brain extract, and two subsequent column chromatography steps on DEAE Sephadex A-50. The chromatography was performed on a short (25cm height) and thick (8.5cm inside diameter) column unit that was specially devised for the large scale preparation. The purified enolase was crystallized in 0.05M imidazole-HCl buffer containing 1.6M ammonium sulfate (pH 6.39), with a yield of 0.9g/kg of bovine brain tissue.
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  • Kinzo NAGASAWA, Hideki UCHIYAMA
    1984 Volume 95 Issue 3 Pages 619-626
    Published: 1984
    Released: November 18, 2008
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    Three kinds of heparins from different animal sources - hog-intestinal heparin (167 USP units/mg), bovine-lung heparin (149 USP units/mg) and whale-intestinal heparin (Balaenoptera borealis L., 174 USP units/mg)-were fractionated through an antithrombin III-Sepharose column and high-affinity (HA) subfractions (1.2-1.8M NaCl eluted fractions) were separated. These HA heparin specimens, which were confirmed to be equivalent in strength of binding to antithrombin III, were compared with respect to the anticoagulant properties by means of [1] whole blood clotting assay and [2] thrombin or [3] factor Xa inhibition assay. The activities of whale HA heparin were [1] 82%, [2] 38%, and [3] 45%, respectively, of those of the hog and bovine HA heparins (which showed approximately identical activities).
    Since it was confirmed that the affinity for antithrombin III of whale heparin is of the same nature as those of hog and bovine heparins by (a) fluorometric titration and (b) affinity chromatography on an antithrombin III-Sepharose column saturated with hog HA heparin, the above difference in anticoagulant properties between the whale HA heparin and hog and bovine HA heparins may be considered to be due to a difference in undescribed saccharide-sequence structures at sites other than the well-characterized antithrombin III-binding sites, suggesting strongly that some heparin saccharide structures other than antithrombin III-binding oligosaccharide participate in the anticoagulant activity dependent on antithrombin III-heparin binding-at least in the manifestation of anti-thrombin activity and the enhancement of anti-Xa activity.
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  • Koichi NISHIGAKI, Yuzuru HUSIMI, Masaaki MASUDA, Kiyomitu KANEKO, Toyo ...
    1984 Volume 95 Issue 3 Pages 627-635
    Published: 1984
    Released: November 18, 2008
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    Precise analysis using the denaturant gradient gel electrophoresis which was devised by Fischer and Lerman (Cell 16, 191-200, 1979), was found to present specific patterns of fine structures for double stranded DNA fragments. These seem to be a mobility change of DNA fragments caused by specific denaturation processes. The effect of denaturants on DNA melting was ascertained to be similar to the effect of temperature. The observed patterns, in comparison with the melting processes of DNAs theoretically obtained, were closely related to DNA meltings and strand dissociations. A number of electrophoretic mobility transitions showed the retardation correspondent to each of the cooperative meltings. Strand dissociations occurred under the conditions theoretically predicted. The degree of retardation in electrophoresis for DNA fragments seemed to correspond to the size of melted regions. Methods presented here were proved to have advantages over the conventional ones for the study of DNA stability maps.
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  • Tsutomu KODAKI, Nobuyuki FUJITA, Isamu KAMESHITA, Katsura IZUI, Hirohi ...
    1984 Volume 95 Issue 3 Pages 637-642
    Published: 1984
    Released: November 18, 2008
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    An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli. (i) Fructose 1, 6-bisphosphate (Fru-1, 6-P2) and GTP-the allosteric activators-were competitive with each other in the activation. (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator. (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1, 6-P2 or GTP but had no effect on the sensitivity to other effectors.
    It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1, 6-P2 by reaction with 2, 4, 6-trinitrobenzene sulfonate. The activation by the analogs was not observed in the presence of 200mM ammonium sulfate. In the presence of lower concentrations (0.1mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700mM but had an inhibitory effect on the desensitized enzyme. These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1, 6-P2 site of the enzyme or the active site depending on the reaction conditions.
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  • Masaru TANOKURA, Kazuhiro YAMADA
    1984 Volume 95 Issue 3 Pages 643-649
    Published: 1984
    Released: November 18, 2008
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    Microcalorimetric titrations were carried out in order to analyze the structural changes of calmodulin caused by Ca2+-binding. Measurements were made both in the absence and in the presence of Mg2+, at 5, 15, and 25°C. Titrations of calmodulin with Ca2+ in the absence of Mg2+ showed that the Ca2+-binding reaction is endothermic and thus is driven solely by the large entropy change. Following the method of Sturtevant (1977), the magnitudes of the hydrophobic and intramolecular vibrational contributions to the heat capacity and entropy changes of calmodulin on Ca2+-binding were estimated. In both the absence and the presence of Mg2+, Ca2+-binding to every site of calmodulin gives rise to an increase in hydrophobic entropy and thus to an “assembling” of nonpolar groups, which are scattered on the surface of the molecule in Ca2+-free calmodulin. Ca2+-binding to calmodulin also gives rise to an increase in vibrational entropy, indicating a “softening” of the overall structure.
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  • Noriko MURAKAMI, Sueo MATSUMURA, Akira KUMON
    1984 Volume 95 Issue 3 Pages 651-660
    Published: 1984
    Released: November 18, 2008
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    A high salt extract of bovine brain was found to contain a protein kinase which catalyzed the phosphorylation of heavy chain of brain myosin. The protein kinase, designated as myosin heavy chain kinase, has been purified by column chromatography on phosphocellulose, Sephacryl S-300, and hydroxylapatite. During the purification, the myosin heavy chain kinase was found to co-purify with casein kinase II. Furthermore, upon polyacrylamide gel electrophoresis of the purified enzyme under non-denaturing conditions, both the heavy chain kinase and casein kinase activities were found to comigrate. The purified enzyme phosphorylated casein, phosvitin, troponin T, and isolated 20, 000-dalton light chain of gizzard myosin, but not histone or protamine. The kinase did not require Ca2+-calmodulin, or cyclic AMP for activity. Heparin, which is known to be a specific inhibitor of casein kinase II, inhibited the heavy chain kinase activity. These results indicate that the myosin heavy chain kinase is identical to casein kinase II.
    The myosin heavy chain kinase catalyzed the phosphorylation of the heavy chains in intact brain myosin. The heavy chains in intact gizzard myosin were also phosphorylated, but to a much lesser extent. The heavy chains of skeletal muscle and cardiac muscle myosins were no_??_phosphorylated to an appreciable extent. Although the light chains isolated from brain and gizzard myosins were efficiently phosphorylated by the same enzyme, the rates of phosphorylation of these light chains in the intact myosins were very small. From these results it is suggested that casein kinase II plays a role as a myosin heavy chain kinase for brain myosin rather than as a myosin light chain kinase.
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  • Naoto ITOH, Masao KAWAKITA
    1984 Volume 95 Issue 3 Pages 661-669
    Published: 1984
    Released: November 18, 2008
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    Interaction between Gd3+ and Tb3+ ions and Ca2+, Mg2+-ATPase of sarcoplasmic reticulum was studied. Three classes of lanthanide-ion binding sites with different affinities were distinguished. Binding of Gd3+ to the site with the highest affinity seemed to occur at less than 10-6M free Gd3+ and resulted in severe inhibition of ATPase activity. The reaction rates of both E-P formation and decomposition in the forward direction were inhibited in parallel with this binding, whereas ADP-dependent decay of E-P in the backward direction was not. At these Gd3+ concentrations, Ca2+-binding to the transport site was not inhibited. Binding of Gd3+ and Tb3+ to the Ca2+-transport site did occur, but more than 10-5M free Gd3+ or Tb3+ was required for effective competition with Ca2+ for that site. Gd3+ bound to the transport site in place of Ca2+ did not activate the E-P intermediate formation. Addition of 10-4M Tb3+ to a suspension of sarcoplasmic reticulum membranes resulted in marked enhancement of Tb3+ fluorescence, which is due to an energy transfer from aromatic amino acid residues of ATPase to Tb3+ ions bound to the low affinity site of the enzyme. Gd3+ and Mn2+ competed with Tb3+ for that site, but Ca2+, Zn2+, and Cd2+ did not.
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  • Koji FURUNO, Kaname MIYAMOTO, Toyoko ISHIKAWA, Keitaro KATO
    1984 Volume 95 Issue 3 Pages 671-678
    Published: 1984
    Released: November 18, 2008
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    Autolysosomes were isolated from leupeptin-treated rat liver (Furuno, K., Ishikawa, T., & Kato, K. (1982) J. Biochem. 91, 1943-1950). They were disrupted by hypotonic treatment and subfractionated by centrifugation in a discontinuous sucrose gradient into three distinct parts: the membranes, the soluble contents, and the insoluble remnants. The content fraction contained the bulk of the activities of lysosomal enzymes with a protein yield of about 36%. The membrane fraction, representing about 5% of protein of the autolysosomes, had a high specific activity of acid phosphatase. In SDS-polyacrylamide gel electrophoretic analysis, the autolysosomal membranes showed protein profiles similar to those of normal lysosomal membranes. The aggregates of partially digested cellular components, including organelles, in the autolysosomes were recovered as granular materials with a very high density (designated as the remnant fraction). This fraction accounted for more than half of protein of the autolysosomes but contained little of the activities of lysosomal enzymes. Lipid analyses revealed that the autolysosomes were poor in lipids because the lipid content of the insoluble remnants was very low. Measurements of the rate of protein degradation in vitro in the crude lysosomal fraction and the isolated autolysosomes from leupeptin-treated rat liver showed that proteolysis was suppressed within the autolysosomes. It was suggested that lipids of sequestered cellular components were preferentially digested within the autolysosomes due to the inhibition of proteolytic activity by leupeptin, and the resulting massive accumulation of proteins was responsible for the enhanced autolysosomal density.
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  • Jun NAGAI, Minoru TANAKA, Kunio NAKASHIMA
    1984 Volume 95 Issue 3 Pages 679-684
    Published: 1984
    Released: November 18, 2008
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    In vitro activation of human reovirus transcriptase by α-chymotrypsin digestion of viral outer shell proteins was completely dependent on the ionic size of the monovalent cation in the medium. Cations with nonhydrated ionic radii larger than 1.3 Å showed full potency of activation of chymotrypsin digestion, and produced transcriptionally active virus cores. Smaller cations having ionic radii of 0.6 Å or 0.95 Å, on the other hand, promoted the chymotrypsin digestion to lesser extents, and yielded subviral particles showing latent or very low transcriptase activities. Differential conformational changes would be induced in viral outer shell proteins by these monovalent cations, resulting in the varied accessibility to chymotrypsin. Electron microscopic analyses under denaturing conditions of the cross-linked reovirus core genome RNAs with the AMT photoreaction revealed that they were almost evenly cross-linked by the psoralen adducts forming no reproducible size of “bubbles.” This result suggests that the double helical reovirus genome may not be bound tightly by the inner viral proteins forming such nucleoprotein structures as nucleosomes in eukaryotic chromatin.
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  • Kazuo ASAOKA
    1984 Volume 95 Issue 3 Pages 685-696
    Published: 1984
    Released: November 18, 2008
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    Conditions have been investigated for the use of triazine dye agarose as an affinity matrix for the purification of glutathione S-transferases from bovine liver. Orange A agarose was most suitable for this purpose among various dye agaroses tested. The enzymes were adsorbed on the dye agarose column and then completely eluted with the buffer containing 1mM reduced glutathione. Thus, a simple and rapid method for purification of bovine liver transferases was developed, which uses column chromatographies on orange A agarose followed by DEAE-Sephacel. One step of the affinity chromatography provided 40-fold purification. Upon chromatography on DEAE-Sephacel, the enzyme activity separated into two major forms (I and II), which were purified to apparent homogeneity as examined by polyacrylamide gel electrophoreses. The pI values of the two forms, I and II, were 6.7 and 6.1, respectively. The overall extents of purification of I and II were about 40-fold and 50-fold, respectively. The activities of the two major enzymes toward various substrates were roughly similar. The optimum pH values of these enzymes were 7.5 as measured with o-dinitrobenzene as a substrate. The activities were significantly inhibited by Cu2+, Hg2+, Cd2+, and Zn2+. In sodium dodecyl sulfatepolyacrylamide gel electrophoresis, both enzymes showed one band with a molecular weight of about 27, 000. Both enzymes, however, were eluted as a single peak from a Sephadex G-150 column at a position corresponding to a molecular weight of about 49, 000. These results show that each enzyme consists of two subunits bound to each other non-covalently. The amino acid compositions showed characteristically high contents of leucine and aspartic acid residues. Double immunodiffusion showed complete identity of the two forms reacting with both rabbit anti-enzyme sera. The two enzymes had an identical amino-terminal amino acid sequence as follows:
    H-Pro-Met- Ile -Leu-Gly-Tyr-Trp-Asp- Ile -Arg-
    Gly-Leu-Ala-His-Ala- Ile -Ser-Leu-Leu-Leu-.
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  • Yoshiki MATSUURA, Masami KUSUNOKI, Wakako HARADA, Masao KAKUDO
    1984 Volume 95 Issue 3 Pages 697-702
    Published: 1984
    Released: November 18, 2008
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    A complete molecular model of Taka-amylase A consisting of 478 amino acid residues was built with the aid of amino acid sequence data. Some typical structural features of the molecule are described. A model fitting of an amylose chain in the catalytic site of the enzyme showed a possible productive binding mode between substrate and enzyme. On the basis of the difference Fourier analysis and the model fitting study, glutamic acid (G1u230) and aspartic acid (Asp297), which are located at the bottom of the cleft, were concluded to be the catalytic residues, serving as the general acid and base, respectively.
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  • Shin-ichi KUWAHARA, Nobuhiro HARADA, Hidefumi YOSHIOKA, Toshiyuki MIYA ...
    1984 Volume 95 Issue 3 Pages 703-714
    Published: 1984
    Released: November 18, 2008
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    Four forms of cytochrome P-450, tentatively designated PB-1, PB-2, MC-1, and MC-2, were purified from liver microsomes of rats treated with phenobarbital (PB-1 and PB-2) or 3-methylcholanthrene (MC-1 and MC-2). Each purified form showed a single protein-staining band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56, 000 (MC-1), 53, 000 (PB-1), 53, 000 (MC-2), or 49, 000 (PB-2). PB-1 and MC-1 were the major cytochrome P-450 components inducible by phenobarbital (PB) and 3-methylcholanthrene (MC), respectively. Antibodies prepared against each form of purified cytochrome P-450 did not crossreact with heterologous antigens in Ouchterlony double diffusion tests, confirming the immunological distinctness of the four forms. The CO-compounds of reduced PB-1 and PB-2 had an absorption maximum at 450 nm, whereas those of MC-1 and MC-2 had a maximum at 447 nm. Judging from the oxidized absolute spectra, MC-2 was of high spin type and the others were of low spin type. Amino acid analysis revealed considerable differences among the purified four forms of cytochrome P-450, and the amino acid sequences of their NH2-terminal portions confirmed that the four forms were different proteins.
    In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, PB-1 and PB-2 oxidized benzphetamine at high rates, but their oxidation of benzo (a) pyrene was much slower than that by MC-1, which catalyzed rapid hydroxylation of benzo (a) pyrene but had low activity with benzphetamine. The quantity of each form of cytochrome P-450 in microsomes was determined by quantitative immunoprecipitation, and selective induction of PB-1 and MG-1 by PB and MC, respecively, was confirmed. Some induction of PB-2 and MC-2 by the corresponding inducers was also noticed. PB group P-450's were not increased by MC treatment, nor were MC group P-450's by PB.
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  • Kazumori MASAMOTO
    1984 Volume 95 Issue 3 Pages 715-719
    Published: 1984
    Released: November 18, 2008
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    Dependence on the salt concentration of the activity of microsome-bound arylsulfatase C [EC 3.1.6.1] from rat liver was examined. The activity increased with increasing salt concentration in the reaction medium in the whole pH range tested. This effect can be explained by the dependence of the reaction rate on the surface pH and the surface concentration of the ionic substrate. The dependence on salt concentration of the activity of the microsome-bound arylsulfatase C and the pH-dependences of Vmax and Km of the enzyme were used for the estimation of pH at the microsomal surface. The two values of the surface pH (surface potential) and the salt concentration were applied to the Gouy-Chapman equation. The value of -0.39±0.08×10-3 elementary charge/Å2 was obtained as the surface charge density in the vicinity of the microsome-bound arylsulfatase C. This was smaller than the over-all value for microsomes (-1.08±0.04×10-3 elementary charge/Å2; Masamoto, K. (1982) J. Biochem. 92, 365-371). This suggests that the anion concentration in the vicinity of the enzyme on microsomes is lower than that in the bulk aqueous phase and is higher than the average value at the microsomal surface when the salt concentration is low.
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  • Eisaku KATAYAMA, Takeyuki WAKABAYASHI, Fernando REINACH, Tomoh MASAKI, ...
    1984 Volume 95 Issue 3 Pages 721-727
    Published: 1984
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    MF-18, one of the monoclonal antibodies generated to chicken myosin, cross-reacted with rabbit skeletal myosin subfragment-1 (S1). Utilizing an improved procedure of immuno-blotting, a decrease in reactivity of MF-18 to S1 by trinitrophenylation was observed. This indicates that the reactive lysyl residue is very close to the hapten site. This is consistent with the evidence that the hapten site resides in the 26, 000 dalton tryptic fragment of S1. Use of such antibodies as labels may open the way to determining the location of specific hapten sites in the three-dimensional image of actin-S1 complex reconstructed from the electron micrographs.
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  • Tadashi MABUCHI, Satoshi NISHIKAWA, Kazuhiko WAKABAYASHI
    1984 Volume 95 Issue 3 Pages 729-736
    Published: 1984
    Released: November 18, 2008
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    We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-lll, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322. These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 μ DNA. They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E. coll. No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome. These sequences were located in intergenic regions.
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  • Mutsumi SUGITA, Tatsuya INOUE, Osamu ITASAKA, Taro HORI
    1984 Volume 95 Issue 3 Pages 737-742
    Published: 1984
    Released: November 18, 2008
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    1. Specific and high titer antisera against 4-O-methylglucuronic acid-containing glycosphingolipid (Lipid IV) from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii, were raised in rabbits.
    2. The antisera were found to agglutinate spermatozoa of three fresh-water bivalves, H. schlegelii, Anodonta woodiana, and Cristaria plicata (Palaeoheterodonta), but they did not agglutinate those of Corbicula sandai (Heterodonta).
    3. The specificity of the agglutination was examined by an inhibition test using various carbohydrates, from which it was concluded that an antigenic determinant is GicA4Me-Fuc.
    4. Immunohistochemical studies indicated that Lipid IV exists on the cell surface of the spermatozoa.
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  • Akihito MORITA, Yasuaki ARATANI, Etsuro SUGIMOTO, Yasuo KITAGAWA
    1984 Volume 95 Issue 3 Pages 743-750
    Published: 1984
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The endogenous protein phosphorylation stimulated by catecholamines was compared in 3T3-L1 preadipocytes and adipocytes. Phosphorylation of a protein with an approximate molecular weight of 57, 000 was stimulated both in preadipocytes and adipocytes of 3T3-L1. Stimulated phosphorylation of four other proteins with approximate molecular weights of 90, 000, 62, 000, 48, 000, and 32, 000 was observed only in 3T3-L1 adipocytes. All of these proteins appeared to be localized in the microsomal fraction. Phosphorylation of these proteins was stimulated by norepinephrine, epinephrine, isoproterenol, dibutyryl cyclic AMP, theophylline, or 1-methyl-3-isobutylxanthine, but not by A23187. Among the phosphorylated proteins in 3T3-L1 adipocytes, the 62, 000 dalton protein was most evident. Using this protein as a marker, it appeared that epinephrine and norepinephrine were effective in stimulating the phosphorylation at the same concentration range. This result was in clear contrast to the different affinities of these catecholamines for β-receptors of 3T3-L1 adipocytes reported by Lai, Rosen, and Rubin (J. Biol. Chem. (1982) 257, 6691-6696). The phosphorylation of the 62, 000 dalton protein in 3T3-L1 adipocytes was observed 1min after the addition of norepinephrine, and dephosphorylation was observed within 10min aster the addition of propranolol.
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  • Masachika IRIE, Hideaki WATANABE, Kazuko OHGI, Mayumi TOBE, Go MATSUMU ...
    1984 Volume 95 Issue 3 Pages 751-759
    Published: 1984
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    In order to clarify the subsite structure of ribonuclease A (RNase A), the interactions of pdTp, pAp, dTpdAp, and pdTpdAp with RNase A were investigated by means of kinetic studies and 31P NMR spectroscopy. The pH profile of the 31P NMR spectrum of RNase A-pdTp complex indicated the interaction of the 3'- and 5'-phosphates with RNase A. The signal of 3'-phosphate of pdTp in the presence of RNase A gave a characteristic titration curve indicating the participation of more than 2 ionic groups in the P1 subsite. A similar 31P NMR titration was observed in the case of 5'-phosphate of pAp in the presence of RNase A. The results indicated that pAp interacted with RNase A at the P1, B2, and P2 sites.
    dTpdAp and pdTpdAp inhibited RNase A action more markedly than dTpdA, indicating the contribution of 3'- and 5'-terminal phosphate groups attached to dTpdA to the affinity of RNase A. The 31P NMR spectra of RNase A-dTpdAp and pdTpdAp complexes excluded the possible interaction of the monoester type phosphate of the inhibitors with the P1 site of RNase A, thus indicating the binding of the 3'-side phosphates with the P2 subsite of RNase A.
    The effects of pA, Ap, and adenosine on the RNase A-s4Up complex were studied by difference spectroscopy. The results indicated the binding of Ap at the B2 and P2 sites of RNase A without affecting the RNase A-s4Up complex. The rates of enzymatic cleavage of UpApA and UpApG were 2.47 and 5.17 times larger than that of UpA, respectively and were different from each other. This supports the existence of the B3 site besides the P2 site in the active site of RNase A. These results clearly indicated the presence of P2 and B3 sites in RNase A in addition to the P0, B1, P1, and B2 sites previously reported.
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  • Masao IWAMORI, Junko SHIMOMURA, Setsuko TSUYUHARA, Yoshitaka NAGAI
    1984 Volume 95 Issue 3 Pages 761-770
    Published: 1984
    Released: November 18, 2008
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    Gangliosides were extracted from various tissues of rat (Wistar strain, male, 3 months old) and their structures were elucidated by enzymatic and chemical procedures including the analysis by negative ion fast atom bombardment mass spectrometry. All tissues analyzed contained gangliosides in various but characteristic concentrations. GDla was detected in the various extraneural tissues (erythrocytes, buffy coat, bone marrow, testis, spleen, and liver) in amounts corresponding to more than 30% of total lipid-bound sialic acid, and surprisingly, it was the sole ganglioside found in buffy coat. The extraneural tissues were classified into several categories according to the nature of the asialo-oligosaccharides of gangliosides as follows: (1) gangliosides with ganglio-N-tetraose were exclusively present (buffy coat and erythrocytes), (2) the concentration of ganglio-N-tetraose-containing gangliosides was higher than that of lactose-containing gangliosides (testis and bone marrow), (3) ganglio-N-tetraose-containing gangliosides amounted to 25-30% of lactosecontaining gangliosides (liver and spleen), (4) ganglio-N-tetraose-containing gangliosides amounted to 7-11% of lactose-containing gangliosides (lung and stomach), (5) more than 90% of gangliosides were lactose-containing gangliosides (heart, intestine, and kidney). In addition, the following gangliosides were characteristically detected in high concentration in the following tissues: GM4 in kidney. GM2 in bone marrow, fucosyl GM1 and GM1 in erythrocytes and GM3 with 2-hydroxy fatty acid, phytosphingosine and N-glycolylneuraminic acid in intestine.
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  • Toshiaki IMAGAWA, Takahide WATANABE, Takao NAKAMURA
    1984 Volume 95 Issue 3 Pages 771-778
    Published: 1984
    Released: November 18, 2008
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    Linoleate monohydroperoxide (L-HPO), methyl linoleate monohydroperoxide (ML-HPO), and methyl hydroperoxy-epoxy-octadecenoate (ML-X) inhibited state 3 respiration of mitochondria when palmitate, palmitoyl CoA, or L-palmitoylcarnitine was used as a substrate. L-HPO was the most effective, and 50% inhibition of palmitate-supported respiration was observed with 2, 3.3, and 6.5 nmol/mg protein of L-HPO, ML-X, and ML-HPO, respectively. Almost the same values were obtained when palmitoyl CoA or L-palmitoylcarnitine was used in place of palmitate. L-HPO inhibited the reaction of β-oxidation in mitochondria in a similar concentration range (4 nmol/mg protein for 50% inhibition) when L-palmitoylcarnitine was used as a substrate. L-HPO also inhibited the formation of 3-hydroxypalmitoylcarnitine from the same substrate. Carnitine palmitoyltransferase activity of mitochondria was inhibited by L-HPO, 50% inhibition occurring at 12 nmol/mg protein. These inhibitory effects of L-HPO were weaker when ATP was removed by hexokinase and glucose. ATP-dependent formation of carnitine ester of L-HPO was also suggested. It was deduced that L-HPO (and ML-X and ML-HPO after hydrolysis) was converted to carnitine ester and inhibited the palmitate metabolism at the site(s) of intramitochondrial carnitine palmitoyltransferase (and possibly acyl CoA dehydrogenase).
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  • Yoshiro MIURA, Harumi HISAKI, Sen-ichi ODA
    1984 Volume 95 Issue 3 Pages 779-784
    Published: 1984
    Released: November 18, 2008
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    The substrate specificity and other properties of a fatty acid hydroxylase system in liver microsomes of the Japanese house musk shrew, Suncus murinus, were examined. Shrew liver microsomes catalyzed the hydroxylation of various saturated fatty acids (C8-C18) to the corresponding ω- and (ω-1)-hydroxy derivatives. The relative activities of the hydroxylase with these substrates were as follows: C12 (100, actual conversion: 7.05 nmol/mg of microsomal protein/min), C10 (90), C14 (87), C16 (23), C8 (19), and C18 (11). The specific activity of the fatty acid hydroxylase in shrew liver was much higher than that in other species. The ω/ω-1-hydroxylation ratio decreased with increasing chain length of fatty acid substrates (C10-C18), but it was 0 for the C8 fatty acid. Both NADPH and O2 were required for hydroxylase activity, and NADH had little effect. The apparent Km value for laurate was 1.6×10-5M. The hydroxylase activity was 92% inhibited by CO at a CO-O2 ratio of 9. p-Chloromercuribenzoate (0.1mM) inhibited hydroxylation by 94% whereas iodoacetate (0.1mM) inhibited it by only 8%. SKF 525-A (1mM) and menadione (0.01mM), respectively, caused 41% and 29% inhibition of the activity. It is concluded that the hydroxylase catalyzing fatty acid hydroxylation in shrew liver microsomes is a typical cytochrome P-450-linked monooxygenase.
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  • Hideyoshi HIGASHI, Kazuyoshi IKUTA, Shigeharu UEDA, Shiro KATO, Yoshio ...
    1984 Volume 95 Issue 3 Pages 785-794
    Published: 1984
    Released: November 18, 2008
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    Heterophile, Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acidcontaining glycosphingolipids (GSLs) were detected as tumor-associated foreign antigens of a Marek's disease lymphoma-derived cell line, MSB 1, by enzyme-immunoassay with chicken antibody against N-glycolylneuraminyl-lactosylceramide (anti-NeuGc-LacCer). At least three species of HD antigen-active GSLs were detected by two-dimensional thin-layer chromatography (TLC) combined with enzyme-immunoassay. The reactivities of the GSLs with anti-NeuGc-LacCer, their behaviors on two-dimensional TLC and the results of an endo-β-galactosidase digestion study indicated that these three GSLs were NeuGc-LacCer (NeuGcα2-3Galβ1-4Glc-Cer), NeuGc-nLcOse4Cer (NeuGcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer) and NeuGc-nLcOse6Cer (NeuGcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer).
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  • Kazuyuki MORIHARA, Hiroshige TSUZUKI, Minoru HARADA, Tatsuo IWATA
    1984 Volume 95 Issue 3 Pages 795-804
    Published: 1984
    Released: November 18, 2008
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    Human α1-proteinase inhibitor was purified according to a modification of the method of Kurecki et al. (Anal. Biochem. 99, 415 (1979)), with Affi-Gel Blue treatment before Zn-affinity column chromatography. The inhibitor was inactivated in the presence of Pseudomonas aeruginosa elastase (1/2, 000 molar ratio) for 2h at pH 7.5 and 25°C. The inactivated inhibitor was purified by column chromatography on Sephadex G-75 and DE-52. Little or no difference was observed between the native and inactive inhibitors in immunological response, amino acid composition or far-ultraviolet CD spectrum. On the other hand, a considerable difference was observed in the near-ultraviolet CD spectrum. Two amino-terminal sequences were found in the inactive inhibitor in almost the same ratio; one was the same as that of the intact inhibitor and the other was Met-Ser-Ile-Pro-. The two components were separated by high-performance liquid chromatography using 0.1% trifluoroacetic acid containing 30-70% CH3CN (gradient) as the eluent. Amino acid analysis and N- and C-terminal amino acid sequence analyses indicated that one fraction corresponded to the sequence of 1-357 of the α1-proteinase inhibitor and the other to 358-394. We concluded that P. aeruginosa elastase can inactivate human α1-proteinase inhibitor by splitting the peptide bond of Pro357-Met358, leading to local change near the active site but little change in the structure as a whole. The split carboxy-terminal fragment binds tightly to the rest of the inhibitor.
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  • Tetsuo MAITA, Masaki HAYASHIDA, Genji MATSUDA
    1984 Volume 95 Issue 3 Pages 805-813
    Published: 1984
    Released: November 18, 2008
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    Tryptic peptides of the α and β chains from silvery marmoset (Callithrix argentatus) and cotton-headed tamarin (Saguinus oedipus) hemoglobins were isolated and sequenced, respectively, by conventional methods. The ordering of the peptides in each chain was deduced from the homology of their sequences with those of human adult hemoglobin. The primary structures thus deduced are compared with those of other primate hemoglobins, and the rate of evolution in New World monkey hemoglobins is discussed.
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  • Toshiaki ODA, Arata ICHIYAMA, Satoshi MIURA, Masataka MORI
    1984 Volume 95 Issue 3 Pages 815-824
    Published: 1984
    Released: November 18, 2008
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    Processing and uptake of the precursor of serine: pyruvate aminotransferase [EC 2. 6. 1. 51] by mitochondria were studied in vitro and in vivo. Serine: pyruvate aminotransferase was synthesized mainly on free ribosomes as judged by immunoprecipitation of puromycin-labeled nascent peptides prepared from free and bound ribosomes. The precursor of rat liver serine: pyruvate aminotransferase (pSPT) synthesized in vitro was post-translationally processed to an apparently mature form by isolated rat liver mitochondria. Available evidence indicated that the processed product was localized in the matrix of mitochondria.
    Mature serine: pyruvate aminotransferase did not inhibit the in vitro processing, suggesting that the extra peptide was necessary for the mitochondrial uptake of the precursor. In the livers of rats fed a vitamin B6-deficient high-protein diet, the induction by glucagon of serine: pyruvate aminotransferase occurred and most of the induced enzyme existed in mitochondria as the apo-form, suggesting that pSPT was taken up by mitochondria and processed in the apo-form under the conditions employed. In the in vitro system, on the other hand, the processing of pSPT proceeded both in the absence and presence of pyridoxal 5'-phosphate. Should the precursor also bind the prosthetic molecule, therefore, it would be transported into mitochondria in both the apo- and holo-forms.
    When isolated rat hepatocytes were labeled with [35S] methionine, labeled pSPT appeared in the cytosolic fraction and was transported rapidly into mitochondria in association with the processing. This uptake and processing were inhibited by a fluorescent laser dye, rhodamine 123, and the precursor accumulated in the cytosol in the presence of the dye.
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  • Makoto KANEDA, Hiroshi OHMINE, Hiroo YONEZAWA, Naotomo TOMINAGA
    1984 Volume 95 Issue 3 Pages 825-829
    Published: 1984
    Released: November 18, 2008
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    Cucumisin is a diisopropyl fluorophosphate-sensitive enzyme. The inactivation by DFP is accompanied by the formation of I mol of labeled serine residue per mol of enzyme. From the soluble portion of the chymotryptic digest of the diisopropyl phosphoryl derivative of cucumisin, two peptides containing phosphorus were isolated; their amino acid sequences were determined to be Gly-Thr-Ser (P) -Met and Asn-Ile-Ile-Ser-Gly-Thr-Ser (P) -Met, respectively. The four residues Gly-Thr-Ser-Met in the above amino acid sequence are identical with those of subtilisin.
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  • Edwin O. NGAHA, Isaac O. OGUNLEYE, Michael A. MADUSOLUMUO
    1984 Volume 95 Issue 3 Pages 831-837
    Published: 1984
    Released: November 18, 2008
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    Gentamicin has been shown to induce renal tubular damage in man and laboratory animals and to result in elevated urinary excretion of some enzymes associated with specific cell regions in the kidney. In the present investigation, the possible protective effect of selenium against gentamicin-induced renal damage was tested by measuring the urinary excretion of some enzymes in the presence and absence of selenium. Our results show that a prior subcutaneous injection of selenium to rats for two days followed by a simultaneous S. C. injection of gentamicin and selenium resulted in a marked reduction in the excretion of such biochemical systems as the urine volume, urinary proteins, alkaline and acid phosphatases, β-glucuronidase, muramidase, and glutamate dehydrogenase. Renal functional studies revealed that selenium-treated rats suffered less adverse effects compared to rats treated with gentamicin alone. Urinary acid phosphatase, β-glucuronidase and muramidase, the three lysosomal enzymes tested, appeared to respond most readily to protection by selenium.
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  • Shigeru CHAEN, Hiroshi SHIMIZU
    1984 Volume 95 Issue 3 Pages 839-845
    Published: 1984
    Released: November 18, 2008
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    The contraction characteristics of the dorsal longitudinal muscle of Lethocerus derollei were investigated by applying small sinusoidal length changes (±1% of resting length) to glycerinated muscle bundles and studying the effect of varying the frequency from 0.1 to 10 Hz and the concentration of MgATP from 35 μM to 2.3mM. The maximum work done by the muscle per cycle increased as the MgATP concentration was decreased from 2.3mM to 52 μM. Between 52 and 35 μM, the maximum work suddenly changed from a positive to a negative value. The optimal frequency for maximal work shifted from low to high values with increase in the MgATP concentration. As the temperature was increased, the optimal work frequency in 2.3mM MgATP solution shifted to a higher value. As the MgATP concentration was increased, the optimal frequency for maximal power increased. The maximal value of the power was an increasing function of the MgATP concentration, reaching a plateau above 52 μM MgATP. The muscle stiffness was a decreasing function of the MgATP concentration, and above 52 μM MgATP it reached a minimum of about 22% of that in the rigor solution. These results are discussed in relation to the crossbridge kinetics.
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  • Toshio TANAKA, Susumu OI
    1984 Volume 95 Issue 3 Pages 847-854
    Published: 1984
    Released: November 18, 2008
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    A β-transglycosylase was purified to a homogeneous state from the extract of a wheat bran Koji culture of Trichoderma longibrachiatum by column chromatography. The purified enzyme showed a typical disproportionation reaction with cellopentaose as the substrate, producing a high molecular component (a waterinsoluble glucan). The enzyme showed neither cellulase nor β-glucosidase activity. The reaction was optimal at pH 6.0 and 37°C. The molecular weight of the enzyme was estimated to be 11, 000 by gel filtration using a TOYOPEARL HW-55F column. The amount of the glucan synthesized by the enzyme increased with prolonged incubation in a reaction with cellopentaose, and soluble cellooligosaccharides, such as cellobiose, cellotriose, cellotetraose, and cellohexaose, were also produced. No glucose was produced in the reaction even when it was carried out for a long time. The total number of molecules (cellooligosaccharides) in the reaction mixture remained at the initial substrate level during the entire reaction. The β-transglycosylase proved to be a specific transferase showing transfer activity of glucosyl, cellobiosyl, and cellotriosyl moieties from one cellopentaose to an acceptor molecule from cellopentaose upwards with almost 100% efficiency.
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  • Tetsuo HIYAMA, Takashi OHTSUKA, Hidehiro SAKURAI
    1984 Volume 95 Issue 3 Pages 855-860
    Published: 1984
    Released: November 18, 2008
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    A new low temperature electron paramagnetic resonance (EPR) signal with a g-value of 1.97 was found in Photosystem-1 particles from a blue-green alga, Anacystis nidulans, illuminated at room temperature. A similar signal was also found in spinach Photosystem-1 particles treated with thiophenol to decrease interference from a signal due to Center A. In the dark, the signal appeared only when the Anacystis particles were at redox potentials lower than -0.5 volts where Centers A and B were also reduced. The signal is most likely due to another iron-sulfur cluster, tentatively designated as Center C. Center C could be photoreduced at low temperatures like Center A when Centers A and B were partially reduced prior to illumination, indicating possible close association of these centers in Photosystem 1 of green plant and algal photosynthesis.
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  • Masanori KATO, Shigeo AIBARA, Yuhei MORITA, Hiroshi NAKATANI, Keitaro ...
    1984 Volume 95 Issue 3 Pages 861-870
    Published: 1984
    Released: November 18, 2008
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    Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme.
    The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the_??_-cation radical.
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  • Tetsuji HIRAO, Kaoru HARA, Kenji TAKAHASHI
    1984 Volume 95 Issue 3 Pages 871-879
    Published: 1984
    Released: November 18, 2008
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    Cathepsin B was purified about 11, 000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24, 000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, β-neoendorphin, α-neoendorphin, dynorphin (1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.
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  • Takao OHYASHIKI, Masayoshi TAKEUCHI, Tetsuro MOHRI
    1984 Volume 95 Issue 3 Pages 881-886
    Published: 1984
    Released: November 18, 2008
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    Cation specificity and effects of pH and temperature on membrane aggregation of porcine intestinal brush borders were studied in terms of the turbidity change of the membrane suspensions due to the addition of polyvalent cations. Monovalent cations (K+ and Na+) did not induce the membrane aggregation at 5mM or below. Divalent and trivalent cations induced membrane aggregation even below 0.5mM in the following orders of effectiveness: Hg2+_??_Cd2+>Zn2+>Mn2+>Ca2+_??_Ba2+>Sr2+_??_Mg2+ and Lu3+>Yb3+>Nd3+=Er3+=Tb3+>Ce3+>La3+, respectively. The aggregation of membranes induced by these polyvalent cations was reversible. Membrane aggregation induced by either divalent or trivalent cations followed saturation kinetics with increasing cation concentration and the apparent dissociation constants of these cations for the membranes were estimated to be at 4-6mM for divalent cations (Ca2+, Mn2+, and Sr2+) and 1-2mM for trivalent cations (Lu3+, Tb3+, and La3+). Cd2+ and Hg2+ had peculiarly much lower dissociation constants than any other divalent cations tested, being rather comparable to trivalent ones. The membrane aggregation induced by Ca2+, Mn2+, or Sr2+ was independent of pH in the range of 6.6 to 8.0, whereas that induced by Tb3+ or Cd2+ was linearly enhanced with increasing pH, very steeply above pH 7.0. The membrane aggregation induced by Cd2+, Hg2+, or Tb3+ was enhanced steadily with increasing temperature between 15 and 45°C. On the other hand, Ca3+-induced aggregation showed a peak at around 30°C in the temperature dependence profile, still being enhanced above 40°C.
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  • Noriyoshi SAKABE, Nobuo KAMIYA, Kiwako SAKABE, Hiroshi KONDO
    1984 Volume 95 Issue 3 Pages 887-890
    Published: 1984
    Released: November 18, 2008
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    X-ray diffraction photographs of a chicken gizzard G-actin•DNase I complex crystal have been recorded using the synchrotron radiation beam emitted by the Synchrotron Radiation Source at Daresbury and the Photon Factory at Tsukuba. The resolution limit was extended to 2.4 Å and the exposure time was reduced approximately by a factor of 10, when data recorded at the Photon Factory, were compared with those recorded with a conventional rotating-anode source. Using a newly designed Weissenberg camera equipped with a multi-layer line screen, the diffraction data in a 36 oscillation range were recorded on a single film up to 3.5 Å resolution.
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  • Masato HIRATA, Tetsuaki INAMITSU, Toshihiko HASHIMOTO, Toshitaka KOGA
    1984 Volume 95 Issue 3 Pages 891-894
    Published: 1984
    Released: November 18, 2008
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    An inhibitor of lipoxygenase of arachidonic acid, nordihydroguaiaretic acid, shortens actin filaments, as judged by electron microscopic observation of negatively stained actin filaments, actin sedimentability and measurement of specific viscosity, at concentrations that inhibit the migration of phagocytic cells. At higher concentrations (more than 10 μM), nordihydroguaiaretic acid strikingly inhibits myosin ATPase activity. Thus nordihydroguaiaretic acid has a severe side effect on the cytoskeleton.
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  • Kenji IKEHARA, Hiroko OKADA, Kaeko MAEDA, Akemi OGURA, Kin-ichi SUGAE
    1984 Volume 95 Issue 3 Pages 895-897
    Published: 1984
    Released: November 18, 2008
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    Both Bacillus subtilis BR 16 S (rel+) and BR 16 R (relA-) cells accumulated ppGpp after a temperature shift-down from 37 to 0°C. This indicates that a ppGpp accumulation system is present in B. subtilis vegetative cells, which is induced upon cold-shock treatment and is mediated by a relA gene-independent product.
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  • Hirofumi ONISHI, Shizuo WATANABE
    1984 Volume 95 Issue 3 Pages 899-902
    Published: 1984
    Released: November 18, 2008
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    n our previous reports, ATP was shown to induce a drastic change in the conformation of gizzard myosin molecules. For example, the sedimentation constant of unphosphorylated myosin (UM) increased from 6 S to 10 S although an ATP-induced change in the sedimentation constant did not occur with phosphorylated myosin (Suzuki et al. (1978) J. Biochem. 84, 1529). We now report the finding that the ATP-induced formation of 10 S-myosin is associated with a drastic change in the papain digestibility of gizzard UM. With 10 S-myosin, the cleavage by papain was strongly inhibited at two regions on heavy chains and at one region on light chains; that is, the junction between the 72 K dalton and 22 K dalton fragments (i.e., a cleavable site in myosin head), the one between the 22 K dalton and 130 K dalton fragments (i.e., a head-tail junction), and the one between the 3 K dalton and 17 K dalton fragments of 20 K dalton light chains. An even more intimate correlation between the myosin conformation and the papain digestibility of myosin was demonstrated by using thiophosphorylated myosin (thioPM); the cleavages by papain at the 72 K-22 K dalton junction and the 22 K-130 K dalton junction were not inhibited when thioPM was digested.
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  • Hirofumi ONISHI, Takeyuki WAKABAYASHI
    1984 Volume 95 Issue 3 Pages 903-905
    Published: 1984
    Released: November 18, 2008
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    Previously, we (Onishi, H. & Wakabayashi, T. (1982) J. Biochem. 92, 871) reported that the ATP-induced disassembly of chicken gizzard “thick filaments” resulted in myosin monomers with “looped” tails. In the present study, we found that these monomers assembled themselves into antiparallel dimers when they were placed in a medium of low ionic strength (_??_2mM).
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  • 1984 Volume 95 Issue 3 Pages 907b
    Published: 1984
    Released: November 18, 2008
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  • 1984 Volume 95 Issue 3 Pages 907c
    Published: 1984
    Released: November 18, 2008
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  • 1984 Volume 95 Issue 3 Pages 907a
    Published: 1984
    Released: November 18, 2008
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