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Volume 98 (1985)
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Volume 98, Issue 6
Displaying 1-38 of 38 articles from this issue
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Kunio TSURUGI, Kikuo OGATA1985 Volume 98 Issue 6 Pages 1427-1431
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe purified acidic ribosomal proteins (P1 and P2) in good yield from rat liver ribosomes by precipitation of ribosomes with MgCl2 prior to ethanol extraction and chromatography of the extract on a column of CM-cellulose at pH 4.8.
The newly-synthesized acidic ribosomal proteins in regenerating rat liver, labeled in vivo with [3H]leucine, were rapidly incorporated into cytoplasmic ribosomes without any detectable time lag and, after reaching a maximum at 30min, they gradually disappeared from the ribosomes, suggesting a short metabolic-life. However, it was found later that they were re-incorporated slowly when newly-labeled proteins were “chased” by an injection of a large amount of cold leucine intraperitoneally at 15 min after the injection of [3H]leucine. Furthermore, in a longterm experiment, acidic ribosomal proteins were found to disappear with a half-life of 100 h from the ribosomes. Thus, these results suggest that acidic ribosomal proteins have a long metabolic life and are exchangeable on cytoplasmic ribosomes in regenerating rat liver.View full abstractDownload PDF (1235K) -
Yukikazu SAEKI, Mitsuhiro NOZAKI, Kunio MATSUMOTO1985 Volume 98 Issue 6 Pages 1433-1440
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNADH oxidase, which catalyzes the oxidation of NADH, with the consumption of a stoichiometric amount of oxygen, to NAD+ and hydrogen peroxide was purified from Bacillus megaterium by 5'-AMP Sepharose affinity chromatography to homogeneity. The enzyme is a dimeric protein containing 1 mol of FAD per mol of subunit, Mr=52, 000. The absorption maxima of the native enzyme (oxidized form) were found at 270, 383, and 450 with a shoulder at 475 nm in 50 mm KP1, buffer, pH 7.0. The visible absorption bands at 383 and 450 nm disappeared on the addition of NADH under anaerobic conditions and reappeared upon the introduction of air. Thus, the non-covalently bound FAD functioned as a prosthetic group for the enzyme. We tentatively named this new enzyme NADH oxidase (NADH: oxygen oxidoreductase, hydrogen peroxide forming). This enzyme stereospecifically oxidizes the pro-S hydrogen at C-4 of the pyridine ring of NADH.View full abstractDownload PDF (1200K) -
Takao OHYASHIKI, Michiyo KODERA, Tetsuro MOHRI1985 Volume 98 Issue 6 Pages 1441-1446
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Cat2+-induced aggregation of porcine intestinal brush border membranes could be inhibited by addition of monovalent cations to the medium or by increasing the ionic strength of the medium, as measured by the change in optical density of the membrane suspension. The relative effectiveness of monovalent cations at 100mM in the inhibition was in the order, Na+ NH4+>K+ Rb+ Li+>choline+. The Ca2+ concentration dependence profile of the membrane aggregation showed that the Ca2+ threshold at which the aggregation began was distinctly shifted to a higher concentration by the addition of KCl. In addition, the results of fluorometric studies with 1-anilino-8-naphthalane sulfonate suggested that the inhibition of the membrane aggregation by extravesicular KCl is due to a decrease of the binding affinity of Ca2+ for the membranes as a result of neutralization of the surface charges. On the other hand, measurements of the incorporation of 1, 6-diphenyl-1, 3, 5-hex-atrience (DPH) into the membrane vesicles and of the anisotropy of DPH-labeled membranes suggested that the imposition of a salt gradient across the membrane vesicles (out>in) causes an increase of lipid fluidity of the membranes. Based on these results, a possible contribution of membrane surface charges and/or membrane fluidity to the Ca2+-induced aggregation of the membranes is discussed.View full abstractDownload PDF (465K) -
Mutsuo TAIJI, Shigeyuki YOKOYAMA, Tatsuo MIYAZAWA1985 Volume 98 Issue 6 Pages 1447-1453
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS2' and 3'-O-(N-acetyl-L-phenylalanyl) adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by highperformance liquid chromatography (HPLC). From the two isomers of [3H] Phe-tRNA in equilibrium, Ac-[3H] Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H] Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H] Phe-tRNA was found to be 0.20:0.80. Further, [3H] Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[β, γ-imido] triphosphate (GMP-P (NH) P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H] Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H] Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu•GTP and 0.04:0.96 in the complex with EF-Tu•GMP-P (NH) P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.View full abstractDownload PDF (602K) -
Tomio ONO, Yoshinobu KOIDE, Yuji ARAI, Kamejiro YAMASHITA1985 Volume 98 Issue 6 Pages 1455-1461
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA heat-stable 32K calmodulin-binding protein has been purified approximately 3, 670-fold from porcine testis to apparent homogeneity as judged by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and polyacrylamide gel electrophoresis under native conditions. The purification employed calmodulin-Sepharose 4B affinity chromatography; elution was performed with a free Ca2+ gradient. This provided a simple and efficient procedure, and approximately 1.62mg of pure heatstable calmodulin-binding protein was obtained from 390g of porcine testis with a yield of 47% in activity. The purified protein was asymmetric (ƒ/ƒ0=1.89) and consisted of a single polypeptide of Mr=32, 000. It is a highly acidic protein (pI=3.9) with a diffusion coefficient of 5.4×10-7 cm2/s, a sedimentation coefficient of 1.43S, and a Stokes radius of 39.5Å in its free form and 41.3Å in its complex form with calmodulin. The extent of inhibition of phosphodiesterase by the calmodulinbinding protein was affected by the order of addition of the agents to the reaction mixture. The extent of inhibition was maximal when phosphodiesterase was added last, while it was minimal when the calmodulin-binding protein was added last. This protein was indistringuishable from a heat-stable calmodulin-binding protein in rat testis (Ono, T., Koide, Y., Arai, Y., & Yamashita, K. (1984) J. Biol. Chem. 259, 9011-9016).View full abstractDownload PDF (1382K) -
Sang Jong LEE, Hideo AKUTSU, Yoshimasa KYOGOKU, Katsuhiko KITANO, Zenz ...1985 Volume 98 Issue 6 Pages 1463-1472
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe imino proton resonances of λ OR3 17 mer in water were observed at 500 MHz with the time-shared Redfield pulse train. All of the 17 imino proton resonances could be assigned specifically to individual base pairs by utilizing the trace of NOE connectivities between the imino and adenine C2H protons and between imino protons themselves. AT1 and 17 showed abnormally high chemical shifts in comparison with the other AT pairs. On raising the temperature, broadening of the signal occurred in a sequential manner from the terminals except for AT10 and AT11, which were broadened at a lower temperature than GC12. The relaxation rates of the imino protons were measured by the inversion recovery method. The rates at higher temperatures represent the exchange rates of the imino protons. From the temperature dependences, activation energies of about 15 kcal/mol for the AT imino protons and 23-26 kcal/mol for the GC imino protons were obtained.View full abstractDownload PDF (706K) -
Kikuo SHIMIZU, Hiroyuki ARAKI, Hideyuki OGAWA1985 Volume 98 Issue 6 Pages 1473-1485
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to construct an in vitro recombination system of T7 DNA, the reaction products of which resemble those in vivo in structure, T7 DNA-membrane complex which is free from concomitant DNase activity was purified from T7 phageinfected cells. T7-infected cells were lysed with T4 lysozyme/Brij58, and T7 DNA-membrane complex was purified through three successive density gradient centrifugations. The properties of the complex on exposure to defined nucleases and observation of the complex by electron microscopy revealed that in T7 DNA-membrane complex, both ends of a linear T7 DNA are bound with membrane components.
A mixture of 32P-labeled T7 DNA-membrane complex and BU-labeled T7 DNA-membrane complex was incubated with T7 exonuclease and T7 DNA-binding protein, and the reaction products with intermediate density were purified. Most of the products were found to have structures similar to that of the recombination intermediate found in T7-infected cells upon electron microscopic examination.View full abstractDownload PDF (4247K) -
Tetsuya UEDA, Yukio MORIMOTO, Mamoru SATO, Tomisaburo KAKUNO, Jinpei Y ...1985 Volume 98 Issue 6 Pages 1487-1498
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtein complexes (photochemical reaction complex; PR complex) bound to both light-harvesting bacteriochlorophyll-1 (LH-Bchl-1) and reaction center Bchl (RC-Bchl) were purified from Rhodospirillum rubrum (wild and carotenoid-less), Rhodopseudomonas sphaeroides (wild), and Chromatium vinosum (wild). Another protein complex (LH-2 complex) bound to LH-Bchl-2 was also purified from Rps. sphaeroides. The bacteria were grown in the presence of a [14C] amino acid mixture. The purification procedure included molecular-sieve chromatography in the presence of cholate-deoxycholate, and non-equilibrated isoelectric electrophoresis with 3- [(3-cholamidopropyl) dimethylamino] -1-propanesulfonate. The purified complexes were separated into their constituent proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The molar ratios of the proteins were determined by comparing their radioactivities divided by their molecular weights after consideration of the molecular masses of the complexes. The PR complexes all contained per mol: 1 mol each of RC H-, M-, and L-subunits, 10-13 (probably 12) mol each of two other proteins with molecular weights of 11-12K and 8-11K, 28-32 mol Bchl, 13-15 mol carotenoids (except in the carotenoid-less mutant), 2.6-3.9 mol ubiquinone (or menaquinone in Chr. vinosum), and 53-79 mol phosphate without phospholipid. The LH-2 complex contained per mol:1 mol 52K protein, about 13 (probably 12) mol each of 9K and 8K proteins, 30 mol Bchl, 10 mol carotenoids, and 38 mol phosphate without phospholipid. The PR complexes and LH-2 complex showed similar X-ray diffraction patterns, implying that they had similar, highly organized molecular structures.View full abstractDownload PDF (922K) -
Takashi KURASAWA, Shinji YOKOYAMA, Yasuko MIYAKE, Taku YAMAMURA, Akira ...1985 Volume 98 Issue 6 Pages 1499-1508
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rate of cholesteryl ester transfer between intrinsic high and low density lipoproteins (HDL and LDL) was measured in human serum in the absence of lecithin: cholesterol acyltransferase (LCAT) and very low density lipoproteins. The rate was calculated according to the equilibrium transfer model between the two lipoprotein cholesteryl ester pools. The average rate of the transfer was 213±95 nmol/h/ml (mean±S. E.) for all 20 normal and 13 hyperlipoproteinemic subjects. The transfer rate in individual serum was higher by several times than the cholesterol esterification rate, showing that cholesteryl ester generated by LCAT on HDL is promptly distributed among various lipoprotein subclasses. The rate of cholesteryl ester transfer in the serum was significantly proportional to the product of intrinsic HDL and LDL concentrations, a possible indicator of the frequency of collision between these lipoproteins, for the range of physiological lipoprotein concentrations. Among the subjects analyzed was a patient with remarkable hyperalphalipoproteinemia accompanied by hypertriglyceridemia, who showed a very low rate of cholesteryl ester transfer in relation to his LDL and HDL concentrations. Compositional analysis of lipoprotein lipids of the patients also supported the possibility of impaired neutral lipids transfer activity among lipoproteins in the blood.View full abstractDownload PDF (873K) -
Keizo TESHIMA, Yuji SAMEJIMA, Saju KAWAUCHI, Kiyoshi IKEDA, Kyozo HAYA ...1985 Volume 98 Issue 6 Pages 1509-1517
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hydrolysis of 1, 2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by a cobra (Naja naja atra) venom phospholipase A2, was studied at 25°C and ionic strength 0.1 in the presence of 3-10mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The Km value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n-alkylphosphorylcholines (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n-dodecylphosphorylcholine (n-C12PC). These results indicated that the substrate and n-C12PC compete for the same site on the enzyme molecule.
The pH dependence curve of the kinetic parameter, kcat/Km, exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20). The second group was assigned as the a-amino group on the basis of its pK value, which had been determined from the pH dependence of the rate constant for the reaction of p-bromophenacyl bromide (BPB) with His 48 (Teshima et al. (1984) J. Biochem. 96, 1903-1913). The third group was tentatively assigned as the invariant Tyr 52, located in close proximity to the imidazole ring of His 48.View full abstractDownload PDF (743K) -
Atsuo HIWATASHI, Isao HAMAMOTO, Yoshiyuki ICHIKAWA1985 Volume 98 Issue 6 Pages 1519-1526
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS3β-Hydroxysteroid dehydrogenase was purified from bovine adrenocortical microsornes and its properties were studied. The purified dehydrogenase gave a single homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed no steroid Δ5-Δ-4isomerase activity. The molecular weight of the dehydrogenase was estimated to be 41, 000 for the monomer and the isoelectric point was determined to be at pH 6.3.
The Km values of the dehydrogenase were 6.2 μM for NAD+, 4.9mM: for NADP+, 2.0 μM for pregnenolone, and 5.3 μM for 17α-hydroxypregnenolone. The mechanism of inhibition by trilostane of the dehydrogenase was also examined kinetically. The inhibition was found to be competitive, with K1 values of 0.14 μM for 17α-hydroxy-pregnenolone and 0.38 μM for pregnenolone.View full abstractDownload PDF (1169K) -
Minoru TANAKA, Kiyoko SUGISAKI, Kunio NAKASHIMA1985 Volume 98 Issue 6 Pages 1527-1534
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChicken brain enolase was found to show multiple forms (I, II and III) separable by DEAE-cellulose column chromatography, whereas enolase from chicken skeletal muscle showed a single form. Brain enolase I, enolase III and muscle enolase were purified to electrophoretic homogeneity. These three isozymes were dimeric enzymes, each being composed of two identical subunits, α, γ and β, having molecular weights of 51, 000±600, 52, 000±550 and 51, 500±650, respectively, as determined by SDS-polyacrylamide gel electrophoresis analysis. Brain enolases I. II and III and muscle enolase had similar catalytic parameters, including almost the same Km values and pH optima.
Specific antibodies against brain enolase I, enolase III and muscle enolase, raised in rabbit, showed no cross-reactivity with each other. Antibodies for brain enolases I and III also reacted with brain enolase II, indicating that brain enolase II was the hybrid form (αγ) of brain enolases I (αα) and III (γγ). Enolases from chicken liver, kidney and heart reacted with the antisera for brain enolase I, but not with those for brain enolase III or muscle enolase.
Developmental changes in enolase isozyme distribution were observed in chicken brain and skeletal muscle. In brain, the ay and γγ forms were not detected in the early embryonic stage and increased gradually during the development of the brain, whereas the αα form existed at an almost constant level during development. In skeletal muscle, complete switching from as enolase to ββ was observed during the period around hatching.View full abstractDownload PDF (1100K) -
Yasuhide SEKI, Yuko NAGAI, Makoto ISHIMOTO1985 Volume 98 Issue 6 Pages 1535-1543
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA desulfoviridin-type sulfite reductase having the α band at 638 nm was purified from Desulfovibrio africanus Benghazi (NCIB 8401) by chromatography on DEAF-cellulose, Sephadex G-200, and DEAE-Sepharose columns and by disc gel electrophoresis. The content of desulfoviridin in the soluble protein was estimated to be about 6% from the purification indexes. Like the typical desulfoviridin from D. vulgaris Miyazaki K, it formed mainly trithionate besides thiosulfate and sulfide in sulfite reduction coupled to hydrogenase and methyl viologen. No significant differences in the amino acid compositions, CD patterns in the UV (205-250 nm) region, and subunit structures were found, except for a pl value about 1 unit larger (pI 5.3). The split Soret (410±2 nm, less intense peak at 391±2 nm with a shoulder around 380 nm) and β (584±2 nm) band maxima of the enzyme as isolated, and the visible absorption and fluorescence spectra of the acidic acetone-extracted chromophore were almost identical to those ascribed to sirohydrochlorin in spite of the reported difference in the native enzyme (α band maxima at 638 nm as against 628±2 nm in a typical desulfoviridin). Iron was the only significant chelatable metal contained in the chromophore. Some differences between africanus and vulgaris desulfoviridins were observed in the CD patterns in the UV to near UV region (250-340 nm) and also in the visible absorption spectra in the presence of dithionite.View full abstractDownload PDF (730K) -
Takahisa KANDA, Yoshihiko AMANO, Kazutosi NISIZAWA1985 Volume 98 Issue 6 Pages 1545-1554
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo different endo-1, 4-β-xylanases [1, 4-β-D-xylan xylanohydrolases, EC 3.2.1.8], named Xylanases I and III, were purified to homogeneity by gel filtration and ion exchange column chromatography from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The purified enzymes were found to be homogeneous on polyacrylamide disc electrophoresis and their specific activities toward xylan were increased approximately 28.7 and 19.8 times, respectively. The activities of each enzyme were considerably inhibited by Hg2+, Ag+, and Mn2+. Their molecular weights were estimated to be approximately 38, 000 and 62, 000 by gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, respectively. Their carbohydrate contents were 2.5% and 8.0% as glucose, and their amino acid composition patterns resembled each other, showing high contents of acidic amino acids, serine, threonine, alanine, and glycine. Both enzymes were most active at pH 6.0 but Xylanase I was more stable as to pH. Their optimum temperatures were 60°C and 70°C, respectively.
Xylanase I split up to 34.5% of larchwood xylan whereas Xylanase III split only 18.9% of it. The products with the former were mainly xylose (X1), xylobiose (X2), and xylotriose (X3), whereas X2 and X3 were the main products with the latter. Both enzymes did not hydrolyze X2. Xylanase I produced almost equal quantities of X, and X2 from X3, while Xylanase III did not attack this substrate. Both enzymes showed no activity toward glycans, other than xylan, such as starch, pachyman and Avicel (microcrystalline cellulose), except the almost one twentieth activity of Xylanase III toward sodium carboxymethyl cellulose (CMC).View full abstractDownload PDF (1374K) -
Nobuyuki YAMASAKI, Tomomitsu HATAKEYAMA, Gunki FUNATSU1985 Volume 98 Issue 6 Pages 1555-1560
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of ricin D with specific saccharides was investigated by ultraviolet difference spectroscopy. Upon binding to saccharides, ricin D displayed ultraviolet difference spectra with maxima at 280 nm and 288 nm. Such difference spectra suggest that the environment of a tyrosine residue (s) located at or near the saccharidebinding site is changed by the binding of saccharide. In addition to the two positive peaks, a small trough was observed around 300 nm in the complexes with galactose-containing saccharides but not in the complex with N-acetylgalactosamine or galactosamine, suggesting the participation of tryptophan in the binding with galactose-containing saccharides. The magnitude of the difference maxima increased with increasing concentration of saccharides until the binding site was saturated. From the variation of the maximum at 288 nm as a function of saccharide concentration, the association constants were obtained for the binding of saccharides to ricin D at various temperatures and pH's. The saccharide binding of ricin D decreased with increasing temperature and with decreasing pH below pH 6.0. It was suggested that difference maximum at 288 nm observed in the ricin D-saccharide interaction reflects the binding of saccharides to the high-affinity saccharide-binding site of ricin D.View full abstractDownload PDF (458K) -
Masao IWAMORI, Machiko IWAMOTO, Kaori HAYASHI, Kaoru KIGUCHI, Yoshitak ...1985 Volume 98 Issue 6 Pages 1561-1569
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGangliosides of thymuses from rabbit, mouse, rat, calf, and man were analyzed. The ganglioside compositions of the thymuses showed species specificities, and the compositions of the species other than the rabbit were found to be markedly different from that of the rabbit, which contained characteristically substantial amounts of IV3NeuGc-nLc4Cer and VI3NeuGc-nLc6Cer (Iwamori, M. & Nagai, Y. (1981) Biochim. Biophys. Acta 665, 214-220). The inter-species differences in the thymus ganglioside compositions were not remarkable in the 8 mouse and 2 rabbit strains examined. Rabbit thymocytes, but not those of mouse and rat, were lysed with human Hanganutziu-Deicher serum in the presence of guinea pig complement, reflecting the high content of gangliosides containing N-glycolylneuraminic acid in rabbit thymus. As to age-dependent changes of gangliosides in rabbit thymus and spleen, the concentrations gradually decreased with age, while the molar ratio of total gangliosides to total phospholipids was constant in the spleen throughout life and in the thymus at 3, 4, and 6 weeks of age. It was noted that old (180 weeks of age) rabbit thymus, which is occupied largely by fat tissue, still contained a significant amount of neolactoseries gangliosides.View full abstractDownload PDF (2845K) -
Akio ITO, Tadashi OGISHIMA, Weija OU, Tsuneo OMURA, Haruhiko AOYAGI, S ...1985 Volume 98 Issue 6 Pages 1571-1582
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOne common and characteristic feature of the extension peptides of mitochondrial enzyme precursors is the presence of repeating short stretches of uncharged amino acids linked by basic amino acids. We synthesized several model peptides having this particular feature of the extension peptides. The peptides contained arginine or lysine as a basic amino acid residue linking sequences of two to four residues of leucine and alanine. We examined the effects of the peptides on the import of the precursors of two mitochondrial enzymes, cytochrome P-450 (SCC) and adrenodoxin, and found that the peptides were generally inhibitory to the import of the precursors into mitochondria. The effective concentrations of some of the inhibitory peptides were as low as a few μM. The peptides containing lysine instead of arginine had an essentially similar inhibitory effect on the import. The peptides did not inhibit the binding of pre-P450 (SCC) to the surface of mitochondria. The synthetic model peptides uncoupled oxidative phosphorylation of mitochondria prepared from either rat liver or bovine adrenal cortex, and induced leakage of enzymes from the inner compartments of mitochondria. However, the synthetic model peptides did not solubilize membrane-bound enzymes from mitochondria, suggesting that their effect on the membranes is different from that of detergents. The synthetic model peptides seem to bind to the membranes causing significant perturbation in the membrane structure, which is possibly related to the functions of the particular common sequence found in the extension peptides of mitochondrial enzyme precursors.View full abstractDownload PDF (4504K) -
Yasuhide HIBINO, Nobuhiko SUGANO1985 Volume 98 Issue 6 Pages 1583-1590
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleosomes released by the incubation (autodigestion) of rat-liver nuclei were fractionated by sucrose-density gradient centrifugation, and subjected to nuclease assay with heat-denatured 3H-DNA from Escherichia coli as an exogenous substrate. With increasing incubation time, the nuclease activity was enhanced and localized in the mono/tetra-, hexa/hepta-, and long-chain oligonucleosome fractions. In contrast, independent of the nucleosome size, the activities of 0.35M NaCl-soluble fractions from them were found to be almost equal in terms of specific activity (dpm/nucleosomal DNA). Such nuclease activity was not detected in the sucrose gradient (top region) lacking nucleosomes and/or chromatin. When the chromatin was dialyzed against a 0.35M NaCl buffer and then fractionated in a sucrose gradient containing 0.35M NaCI, most of the nuclease activity was solubilized into the above top region. On gel filtration of the mononucleosome fraction in the 0.35M NaCl buffer, the nuclease activity was eluted at the position of 36, 000 daltons. This nuclease cleaved heat-denatured DNA more rapidly than the native DNA in the presence of Mg2+, and had the ability to make both single-strand nicks and doublestrand cuts in pBR322 DNA; in other words, it had an endonucleolytic activity. Moreover, four different classes of mononucleosomes were fractionated by electrophoresis of the nucleosomes released by autodigestion of the nuclei. These mononucleosomes also showed nuclease activity with the heat-denatured DNA. Thus, the present studies suggest that an Mg2+-dependent endonuclease of about 36, 000 daltons is associated with the nucleosome particle (s) in rat-liver nuclei.View full abstractDownload PDF (1273K) -
Isao MATSUI-YUASA, Shuzo OTANI, Seiji MORISAWA1985 Volume 98 Issue 6 Pages 1591-1596
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhorbol 12-myristate-13-acetate (PMA) inhibited an increase in [3H] thymidine incorporation induced by phytohemagglutinin (PHA) in cultured bovine lymphocytes. Cellular levels of putrescine increased in the presence of PHA and PMA but the levels of spermidine and spermine had decreased to the control levels by 40 h. In cells treated with PHA and PMA, the activity of spermidine/spermine N1-acetyl-transferase, a rate-limiting enzyme in polyamine biodegradation, was stimulated synergistically. Phorbol esters with tumor-promoting ability also stimulated the enzyme activity and a reciprocal correlation between the enzyme activity and DNA synthesis was observed. Addition of spermine reversed the PHA- and PMA-induced inhibition of DNA synthesis but putrescine and spermidine failed to restore it. These results suggest that the enhancement of spermidine/spermine NI-acetyltrans-ferase activity results in the depletion of intracellular spermine and a concomitant decrease in DNA synthesis.View full abstractDownload PDF (460K) -
Hideaki TSUNEMATSU, Kumi ANDO, Yoshihiro HATANAKA, Koichi MIZUSAKI, Ry ...1985 Volume 98 Issue 6 Pages 1597-1602
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNα Benzyloxycarbonyl p-guanidine-L-phenylalanine β-naphthylamide (Z-GPA-βNA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of Nα benzyloxycarbonyl-L-arginine β-naphthylamide (Z-Arg-βNA). Both Z-GPA-βNA and Z-Arg-βNA were rapidly and almost completely hydrolyzed by trypsin and pronase. Z-Arg-βNA was hydrolyzed slowly by thrombin, while Z-GPA-βNA was not susceptible to this enzyme at all. The rate of hydrolysis of Z-GPA-βNA by papain was slower than that of Z-Arg-βNA. Neither β-naphthylamide substrate was hydrolyzed by α-chymotrypsin. The specificity constant (kcat/Km) for the hydrolysis of Z-GPA-βNA by trypsin was somewhat larger than that for the hydrolysis of Z-Arg-βNA. Contributions of the benzene ring in the side chain of Z-GPA-βNA to good binding of this substrate to the specificity site of this enzyme and to the poor fit of the scissile bond in the substrate molecule to the active serine residue are presumed from comparison of the individual kinetic parameters (Km and kcat) for the two β-naphthylamide substrates. Z-GPA-βNA was ascertained to be a useful substrate in the study of the binding and catalytic specificities of various trypsin-like enzymes.View full abstractDownload PDF (454K) -
Shun-ichiro KAWABATA, Takashi MORITA, Sadaaki IWANAGA, Hideo IGARASHI1985 Volume 98 Issue 6 Pages 1603-1614
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStaphylocoagulase with a molecular weight of 64, 000 and subspecies ranging in molecular weight from 36, 000 to 64, 000 were purified by affinity column chromatography on bovine prothrombin-Sepharose 4B from the culture filtrates of the Staphylococcus aureus strains, st-213 and 104. The samples containing all molecular species from both strains had the same NH2-terminal sequence, Ile-Val-Thr-Lys-Asp-Tyr-Ser-Lys-Glu-, implying that the molecular heterogeneity was due to proteolytic degradation to some extent of the COOH-terminal portion during cultivation or purification. Staphylocoagulase (Mr=64, 000) from strain st-213 formed an active complex, “staphylothrombin, ” with human prothrombin in a molar ratio of 1 to 1.1. Staphylothrombin was unstable at 37°C and some portions of staphylocoagulase in the complex were rapidly degraded into small fragments, together with the fragmentation of prothrombin into prethrombin I and prothrombin fragment 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent fluorography for the products of prothrombin activation by staphylocoagulase in the presence of [3H] diisopropylphosphofluoridate (DFP) demonstrated the formation of a DFP-sensitive active site in the prothrombin molecule, and no cleavage of the Arg-Ile bond linking the A and B chains of a-thrombin was found.
The enzymatic properties including the pH-dependency of the activity, substrate specificity and behavior towards thrombin inhibitors of staphylothrombin differed from those of α-thrombin, although the active site titration of staphylothrombin with p-nitrophenyl p'-guanidinobenzoate showed 0.95±0.2 mol of active site/mol of enzyme.View full abstractDownload PDF (3685K) -
Masayuki ISHIKAWA, Tetsuo MESHI, Yoshimi OKADA, Teruo SANO, Eishiro SH ...1985 Volume 98 Issue 6 Pages 1615-1620
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously we constructed infectious hop stunt viroid (HSV) cDNA clone, pHS-P2P, which carries two copies of full-length HSV cDNA tandemly and generates HSV RNA when it is inoculated into cucumber plants. The in vitro transcript of the cDNA clone was also infectious. To investigate the essential regions for infectivity of HSV, we introduced a short deletion or insertion into the HSV sequence of pHS-P2P using restriction sites (XhoI site for pHI-X1, PvuI site for pHI-P1, and BamHI site for pHI-B1) and assayed the infectivities of these mutagenized clones. None of these mutagenized clones and their transcripts were infectious under the conditions used. Simultaneous inoculation of two or three of non-infectious mutagenized clones or the transcripts from them did not restore the infectivity.View full abstractDownload PDF (419K) -
Michiko MORI, Isamu SHIIO1985 Volume 98 Issue 6 Pages 1621-1630
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPurification procedures for phosphoenolpyruvate carboxylase from B. flavum were improved by using hydrophobic chromatography. The carboxylase showed optimum pH values of 7.2 and 8.0 with Mn2+ and Mg2+ as metallic activators, respectively.
Purified phosphoenolpyruvate carboxylase was found to be synergistically inhibited by aspartate and 2-oxoglutarate in the absence or presence of an activator, acetyl-CoA. Similarly to the aspartate inhibition, 2-oxoglutarate alone inhibited the enzyme competitively with respect to both substrates, with an inhibitor constant of 4.7mM. The dissociation constant for the combination of enzyme-2-oxoglutarate (-aspartate) complex with aspartate (2-oxoglutarate) was found to be one-third of that for the combination of the enzyme with aspartate (2-oxoglutarate). The Hill coefficient for phosphoenolpyruvate was increased from 1.0 to 2.3 by the simultaneous addition of the two inhibitors in a certain concentration range of phosphoenol-pyruvate where strong synergistic effects were observed. Outside this concentration range, the coefficient was not altered or was slightly increased by the addition of aspartate, 2-oxoglutarate, or both. The synergistic action seems to be caused by these effects, in addition to the decrease in dissociation constants of the inhibitors. Hill coefficients for aspartate and 2-oxoglutarate were both approximately 2.0. The coefficient for one inhibitor did not vary with the addition of the other inhibitor. Although many structural analogues of the two inhibitors, such as 2-oxoadipate and 3-hydroxyaspartate, were very weak inhibitors, their synergistic effects with aspartate or 2-oxoglutarate were comparable to the effects of the two natural inhibitors. On the other hand, malate and succinate, which markedly inhibited the enzyme, did not show synergistic action with aspartate or 2-oxoglutarate. Hill coefficients for the structural analogues showing synergistic effects were approximately 2.0 or above, whereas those for malate and succinate, which did not enhance the inhibitions, were about 1.0. Phosphoenolpyruvate carboxylase from an aspartateproducing mutant had the inhibitor constant of 5.8mM for 2-oxoglutarate, i.e., slightly higher than wild-type enzyme. The inhibitor constant for aspartate was three times higher than that of the wild-type enzyme as reported previously. The dissociation constant for aspartate of the enzyme-aspartate-2-oxoglutarate complex in the mutant enzyme was 8 times that in the wild-type enzyme, indicating that weaker synergistic inhibition was observed with the mutant enzyme.View full abstractDownload PDF (711K) -
Masayoshi TAKEUCHI, Naoki ASANO, Yukihiko KAMEDA, Katsuhiko MATSUI1985 Volume 98 Issue 6 Pages 1631-1638
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36, 000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(β-aminoethylether)-N, N´-tetraacetic acid and the inhibition was reversed by Ca2+ ion.The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-α-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-β-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24mM and 0.5mM, respectively.View full abstractDownload PDF (1397K) -
Shigeru TAKETANI, Hirao KOHNO, Rikio TOKUNAGA1985 Volume 98 Issue 6 Pages 1639-1646
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of transferrin receptors of cultured human lymphocytes has been investigated by using monoclonal antibody (5E9) specific for human transferrin receptors. When isolated lymphocytes were cultured in a medium containing fetal calf serum, the biosynthesis of transferrin receptor was barely detectable. The addition of concanavalin A or human serum to the medium caused a slight stimulation of the biosynthesis. The addition of concanavalin A and human serum in combination caused the highest biosynthetic activity. Appearance of the receptor on the cell surface increased in parallel with the degree of the synthesis. Treatment of concanavalin A- and human serum-treated cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a marked stimulation of the phosphorylation of the receptor. Enhancement of phosphorylation occurred within 20 min after the addition of TPA. The density of the receptor on the cell surface slightly increased upon TPA treatment of cells, and the treatment was without effect on iron incorporation from transferrin into the cells. The density of newly synthesized receptor in TPA-treated cells was similar to that in non-treated cells. These results indicated that TPA treatment of mitogen-activated human lymphocytes stimulated the phosphorylation of transferrin receptors, but TPA had no effect on the expression of the receptors thereafter.View full abstractDownload PDF (3743K) -
Yasumitsu TAKAGI, Tsuneo OMURA1985 Volume 98 Issue 6 Pages 1647-1652
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe biogenesis of a microsomal NAD (P)+-dependent aldehyde dehydrogenase (AIDH) in rat liver was studied using specific antibody raised against the purified enzyme prepared by the method of Nakayasu et al. (Nakayasu, H., Mihara, K., & Sato, R. (1978) Biochem. Biophys. Res. Commum. 83, 697-703). The antibody raised against purified AlDH inhibited the activity of microsome-bound AlDH as effectively as that of the detergent-solubilized enzyme, suggesting that AlDH molecules are present on the outer surface of microsomal vesicles and that a major portion of each molecule, including the active site, is exposed. The cell-free translation of RNA preparations extracted from free and membrane-bound polysomes showed that AlDH was exclusively synthesized on free polysomes. The in vitro synthesized AlDH had the same molecular weight as the authentic enzyme. When a single intravenous injection of [3H] leucine was given to rats, there was no detectable difference in the specific radioactivities of AIDH in rough and smooth microsomes even at the shortest time point, suggesting random incorporation of newly synthesized AIDH molecules into rough and smooth endoplasmic reticulum.View full abstractDownload PDF (1186K) -
Yasuo SUZUKI, Yoshio HIRABAYASHI, Takashi SUZUKI, Makoto MATSUMOTO1985 Volume 98 Issue 6 Pages 1653-1659
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUnique high molecular weight (M. W. 4, 000-9, 000) sugar chains termed erythroglycan II have been obtained from alkali/sodium borohydride digests of I-active asialoglycoprotein derived from sialoglycoprotein GP-2, which was isolated recently from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y. et al. (1983) J. Biochem. 93, 1621-1633; (1984) J. Biochem, 95, 1193-1200). It was found that these sugar chains comprise about 40% of total alkali-labile oligosaccharides of asialo GP-2 and contain endo-β-galactosidase (Flavobacterium keratolyticus)-resistant highly branched and heterogeneous oligosaccharides of poly-N-acetyllactosamine type which are linked O-glycosidically to the peptide backbone through N-acetylgalactosamine. Erythroglycan II also contains endo-β-galactosidase-susceptible straight terminal polylactosaminyl side chains. A major oligosaccharide released by the enzyme cochromatographed with Galβ-4 GlcNAcβ-3Gal.
Inhibitory activity of Sendai virus-mediated hemagglutination and the receptor activity for the virus were reduced significantly but not completely by the endo-β-galactosidase. These results indicate that both linear and branched sialosylpolylactosamine sequences in erythroglycan II are important for the reception of the virus into the target cells.View full abstractDownload PDF (1941K) -
Taneaki HIGASHI, Masumi FURUKAWA, Kunihiko HIKITA, Akiko NARUSE, Norik ...1985 Volume 98 Issue 6 Pages 1661-1667
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe extent of intracellular glutathione binding to proteins through a disulfide linkage in rat liver was examined quantitatively. The content of glutathione associated with the acid-precipitable fraction and releasable on borohydride treatment was 0.024±0.016 μmol/g liver, which accounted for less than one per cent of the total glutathione (6-7 μmol/g liver) in the liver of fed rats.
Most of the thiol (2-4 μmol/g liver) liberated from liver proteins into the acidsoluble fraction on borohydride reduction in the presence of guanidine hydrochloride was not glutathione but was proteinous in nature. The amounts of thiols liberated per g of liver were similar in fed, fasted, and dibutyryl-3', 5'-cyclic AMP-treated rats.View full abstractDownload PDF (601K) -
Yutaka YOSHIDA, Kiyoshi ARIMOTO, Mitsuru SATO, Norio SAKURAGAWA, Masat ...1985 Volume 98 Issue 6 Pages 1669-1679
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAY-9944 (trans-1, 4-bis (2-chlorobenzylaminoethyl) cyclohexane dihydrochloride), a cationic amphiphilic drug, caused a rapid, irreversible and dose-dependent reduction of acid sphingomyelinase activity in normal human fibroblasts without changing the activities of other lysosomal hydrolases tested. Examinations of activities against synthetic substrates and of the pH-dependency of sphingomyelinase in the drugtreated cells also suggested that the reduction of activity was specific to acid sphingomyelinase. Such a specific reduction was also found with 12 other cationic amphiphilic drugs, most of which have been shown to be inducers of experimental phospholipidosis in animals and/or cultured cells. These results strongly suggest that acid sphingomyelinase is involved in the process of drug-induced lipidosis. The reduction of acid sphingomyelinase seemed not to be due to direct inhibition by these drugs, a specific loss of the enzyme into the culture medium, the presence of inhibitor in the drug-treated cells, or impaired synthesis of the enzyme. There was no indication that changes in the catalytic properties of the enzyme, or changes in the requirement of detergents for its activity occurred in the cell. These results suggest that AY-9944 and other cationic amphiphilic drugs may cause the reduction of acid sphingomyelinase activity by inducing an increased rate of degradation of the enzyme or by causing an irreversible inactivation via some undetected factor.View full abstractDownload PDF (911K) -
Kyoichi KOBASHI, Sachiko TAKEBE, Akiko NUMATA1985 Volume 98 Issue 6 Pages 1681-1688
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBenzoyl- and isopentenoyl phosphoric triamides (BPA and IPA) strongly inhibited urease activities from jack bean, soybean, watermelon seed, Proteus mirabilis, P. rettgeri, P. vulgaris, Mycobacterium smegmatis, and Ureaplasma urealyticum. Their I50 values (the final concentration causing 50% inhibition), independent of enzyme source, were 2-21 nM, which are about 1, 000-fold lower than that of caprylohydroxamic acid, one of the most potent urease inhibitors. ATP-urea amidolyase activity was inhibited 50% by BPA at a higher concentration of 0.28mM, but was not affected by IPA even at 1.3mM. Thirteen kinds of hydrolases (trypsin, chymotrypsin, thermolysin, leucine aminopeptidase, papain, lipase, α-amylase, glucuronidase, asparaginase, arylsulfatase, alkaline phosphatase, acid phosphatase, and true cholinesterase), two oxidoreductases (catalase and alcohol dehydrogenase), three transferases (glutamic-oxaloacetic aminotransferase, γ-glutamyl transpeptidase, and arylsulfotransferase) and two kinases (pyruvate kinase and creatine kinase) were not affected at all even at 1mM BPA and IPA. Exceptionally, pseudo-cholinesterase from human serum was inhibited by BPA and IPA, whose I50 values were 70 nM and 10 μM, respectively, using acetylthiocholine as a substrate. These values increased to 0.55 μM and 54 μM, respectively, when acetylcholine was used as a substrate. These results show that N-acylphosphoric triamides potently and specifically inhibit urease activity at concentrations of nM order.View full abstractDownload PDF (679K) -
Makoto SHIMOSAKA, Yasuki FUKUDA, Kousaku MURATA, Akira KIMUIRA1985 Volume 98 Issue 6 Pages 1689-1697
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOrotate phosphoribosyltransferase (OPT) was purified from both Escherichia coli K-12 strain and its derivative, a purine-sensitive mutant. The wild-type OPT had a molecular weight (M. W.) of 47, 000 and was composed of two identical subunits (M. W. 23, 500). The wild-type OPT showed maximum activity at pH 9.5, and no activity was seen in the absence of Mg2+ or Mn2+ ion. It also catalyzed a reverse reaction, namely orotidine-5'-monophosphate (OMP) pyrophosphorolysis. In this reverse reaction, tripolyphosphate, tetrapolyphosphate, and trimetaphosphate were also effective as pyrophosphate donors. The apparent Km values of the wild-type OPT were 30 μM for orotate and 40 μM for 5-phosphoribosyl 1-pyrophosphate (PRibPP), and also 3.6 μM for OMP and 13 μM for PP, . On the other hand, the mutant OPT showed increased apparent Km values for all four substrates, 440 μM for orotate, 360 μM for PRib-PP, 33 μM for OMP, and 250 μM for PPi. The mutant OPT required a higher concentration of Mg2+ ion for maximum activity than the wild-type OPT. The nature of the purine-sensitive phenotype of the mutant is discussed from the standpoint of the reactivity of the mutant OPT, which has an increased Km value for PRib-PP (about 9-fold).View full abstractDownload PDF (1243K) -
Dongchon KANG, Hiroko TSUDA, Koichiro TAKESHIGE, Yosaburo SHIBATA, Shi ...1985 Volume 98 Issue 6 Pages 1699-1706
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A 23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A 23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of β-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A 23187 at a concentration higher than 0.1 μM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A 23187 less markedly. The release of lysozyme reached a plateau at about 0.1 μm A 23187 and increased again at higher concentrations of A 23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A 23187 at low concentrations may be due to the activation of the C-kinase; the effects of A 23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.View full abstractDownload PDF (1480K) -
Shoji WAKASUGI, Shuichiro MAEDA, Kazunori SHIMADA, Hiroshi NAKASHIMA, ...1985 Volume 98 Issue 6 Pages 1707-1714
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an attempt to construct model systems for familial amyloidotic polyneuropathy, prealbumin cDNA was cloned from a mouse liver cDNA library, using previously cloned human prealbumin cDNA as a hybridization probe. The primary structure of mouse prealbumin deduced from the cDNA sequence shows that it consists of 147 amino acids, including a whole prealbumin sequence (127 amino acids) and a putative signal sequence (20 amino acids). These numbers are in complete agreement with those determined for the human prealbumin. Among the 127 amino acid residues of the mature human prealbumin, 25 are replaced by different amino acids in the mouse prealbumin. Interestingly, 24 out of the 25 substituted amino acids are located at the outer surface of the protein, and the regions corresponding to the core and central channel of the protein are almost completely conserved. The cloned cDNA provided essential information for manipulating amyloidosis in mice.View full abstractDownload PDF (611K) -
Nobuyuki NUKINA, Yasuo IHARA1985 Volume 98 Issue 6 Pages 1715-1718
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe found that at least a part of Alzheimer's paired helical filaments (PHF) was cleaved by proteases to release three major polypeptides, the apparent molecular weights of which were 10K, 26K, and 36K daltons. These proteolytic fragments were strongly labeled with the antibodies specific to PHF. Absorption with highly purified PHF eliminated this labeling. The monospecific antibodies bound to the 10K daltons protein, the most intensely immunolabeled one, stained isolated neurofibrillary tangles composed of PHF. From these observations, we conclude that these polypeptides released by proteases, especially the 10 K daltons protein, are derived from PHF themselves.View full abstractDownload PDF (1920K) -
Yoshihiro FUKUMORI, Koji NAKAYAMA, Tateo YAMANAKA1985 Volume 98 Issue 6 Pages 1719-1722
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom Pseudomonas AM 1 grown in a medium deficient in Cu, aa3-type cytochrome c oxidase was purified which contained 2 molecules of haem α and one atom of Cu per molecule. The enzyme showed absorption peaks at 428 ad 595 nm in the oxidized form and at 442 and 604 nm in the reduced form, and its CO complex showed peaks at 432 and 602 nm. The enzyme in the oxidized state showed an obscure absorption peak around 800 nm instead of a peak at 820 nm.
One mol of the enzyme oxidized maximally 76, 75, and 98 mol of the ferrocytochromes c of Candida krusei, horse and Pseudomonas AM 1 per sec, respectively. These reactions were 50% inhibited by 7 μM KCN. The product of reduction of O2 catalyzed by the enzyme was concluded to be H2O on the basis of the ratio of ferrocytochrome c oxidized to O2 consumed.View full abstractDownload PDF (292K) -
Shyh-Horng CHIOU, Wen-Chang CHANG, Yuh-Shan JOU, Hui-Min M. CHUNG, Tun ...1985 Volume 98 Issue 6 Pages 1723-1726
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe DNA-cleavage specificity of ascorbate in the presence of copper ion is analyzed with end-labeled pBR 322 DNA fragments. The nonenzymatic reaction of Cu (II)/ascorbate and DNA shows certain degrees of cleavage preference toward purinecontaining short segments in the labeled DNA under mild conditions (at 0&C and 10min). The segments of pyrimidine clusters are least susceptible to cleavage. The DNA scission cannot be detected in the absence of metal ions, and is greatly diminished in the presence of EDTA and metal-chelating peptide. It is more specific than the nuclease-like scission activity induced by cuprous-phenanthroline complex. This scission activity in relation to the antiviral and antitumor activities of vitamin C reported in the literature deserves a crucial consideration.View full abstractDownload PDF (1187K) -
1985 Volume 98 Issue 6 Pages 1727a
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (23K) -
1985 Volume 98 Issue 6 Pages 1727b
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (23K)
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