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1.
Etsuko Kawashima Yukio Aoyama Mohamed F. Radwan Masayoshi Miyahara Takeshi Sekine Masatsune Kainosho 《Nucleosides, nucleotides & nucleic acids》2013,32(3-5):333-336
Abstract The four (2′S)-[2′-2H]-2′-deoxynucleosides (>90 atom % 2H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB2H4 in EtOH-H2O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu3SnH-Et3B reduction of the resulting bromide, and, finally, unmasking. 相似文献
2.
Vacuolar Localization of Endoproteinases EP(1) and EP(2) in Barley Mesophyll Cells 总被引:7,自引:5,他引:2
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The localization of two previously characterized endoproteinases (EP1 and EP2) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP1 and partially purified EP2. It is therefore concluded that EP1 and EP2 are localized in the vacuoles of mesophyll cells. 相似文献
3.
The uranium(IV) complexes [U(EDTA)(H2O)2], [U(HOEDTA)]+, and [U(DTPA)]? are well-formed in the pH fange 2–3 ([DTPA]5- = diethylenetriaminepentaacetate; [HOEDTA]3- N-(2-hydroxyethyl)ethylenediaminetriacetate). Of these, only [U(DTPA)]- is extracted from an aqueous phase at pH 2 by the perchlorate salt of the primary amine, Primene JM-T. As the aqueous phase pH was raised, extraction occurred in all three cases and hydrolysed species may be extracted from EDTA and HOEDTA solutions but [U(DTPA)]? resists hydrolysis. The addition of sulphate had a marked effect on the extraction of U(IV) from EDTA and HOEDTA through the formation of [U(EDTA)(SO4)(H2O)]2- and [U(HOEDTA)(SO4)(H2O)n]?. The equilibrium constant, log β1, for: [(U(EDTA)(H2O)2] 2 [SO4]2? ? [U(EDTA)(SO4)(H2O)]2- 2 H2O was found to be 2.43 ± 0.04 (I = 1 mol dm?3, NaClO4; pH 2.0; 20 °C) from spectrophotometric data.With tri-n-octylphosphine oxide (TOPO) electronic spectroscopy showed that the same U(IV) complex was extracted at pH 2 for Cs2UCl6, U(IV)/ HOEDTA, and U(IV)/DTPA and the aminepoly- carboxylates were aqueous phase masking agents but with [U(EDTA)(H2O)2] oxidation gave a uranyl(VI) organic phase species.Uranium(IV) is strongly extracted from aqueous solutions of HOEDTA at pH 2 or 3 by bis(2-ethyl- hexyl)phosphoric acid (HBEHP) but less so from EDTA and DTPA. Since U(IV) is completely extracted from Cs2UCl6 it could be that the amine- polycarboxylates were aqueous phase masking agents although spectral evidence did not support this. 相似文献
4.
Laboratory scale anoxic/aerobic sequencing batch reactor (A/O SBR) was operated around 15 °C to evaluate the effect of anoxic/aerobic phase fraction (PF) on N2O emission. The ammonia removal exhibited a decrease trend with the increase of PF, while the highest total nitrogen removal was achieved at PF = 0.5. Almost all the N2O was emitted during the aerobic phase, despite of the PF value. However, the net emission of N2O was affected by PF. Under the premise of completely aerobic nitrification, the lowest N2O emission was achieved at PF = 0.5, with a N2O-N conversion rate of 9.8%. At lower PF (PF = 0.2), N2O emission was stimulated by residual nitrite caused by uncompleted denitrification during the anoxic phase. On the other hand, the exhaustion of the easily degradable carbon was the major cause for the high N2O emission at higher PF (PF = 0.5). The N2O emission increased with the decreasing temperature. The time-weighted N2O emission quantity at 15 °C was 2.9 times higher than that at 25 °C. 相似文献
5.
G. Cecilia Toro Norbel Galanti Ulf Hellman Christer Wernstedt 《Journal of cellular biochemistry》1993,52(4):431-439
The existence of histone H1 has been questioned in Trypanosomatids. We report here the presence of a histone H1 in the chromatin of Trypanosoma cruzi. This protein was purified by narrow-bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H1 from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H1. 相似文献
6.
Cytochrome a1 was solubilized with Triton X-100 from a membrane-envelope preparation of Nitrosomonas and partially purified by repeated fractionation with (NH4)2SO4. The purified fraction of cytochrome a1 was enriched over the crude extract by a factor of 16 and 300 with respect to protein and c-type cytochrome, respectively. The cytochrome was characterized as cytochrome a1 on the basis of (a) reduced absorption maxima at 444 nm and 595 nm, (b) acid acetone extractibility and ether solubility of the heme and (c) absorption maximum of 587 nm of the ferro-hemochrome in alkaline pyridine. The α absorption band shifted from 600 nm to 595 nm upon solubilization of the cytochrome with Triton X-100. Spectral shifts were observed in the presence of cyanide and azide and the cytochrome changed with aging to a form with a reduced absorption band at 422 nm. Cytochrome a1 was reduced anaerobically in the presence of reduced mammalian cytochrome c and was rapidly reoxidized in the presence of O2. CO caused a shift in the soret peak of the reduced form but did not prevent reoxidation of cytochrome a1 in the presence of CO-O2 (95:5, v/v). 相似文献
7.
Makoto Ohkubo Kourin Miyagawa Kiyoshi Osatomi Kenji Hara Yukinori Nozaki Tadashi Ishihara 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2004,139(4):1143
A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α2-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α2-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α1-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α1-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α2-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed. 相似文献
8.
Stereochemistry of the decarboxylation of l-ornithine with ornithine decarboxylase from mouse kidney
Gary R. Orr Steven J. Gould Anthony E. Pegg James E. Seely James K. Coward 《Bioorganic chemistry》1984,12(3):252-258
Using highly purified ornithine decarboxylase isolated from androgen-treated mice, [1R-2H]putrescine was generated by the decarboxylation of l-ornithine in D2O, and [1S-2H]putrescine was generated from [2-2H]ornithine by carrying out the decarboxylation in H2O. Chirality of the putrescines was then determined from the 200-MHz 1H NMR spectra of their bis-camphanamides in the presence of Eu(fod)3. These results demonstrated that decarboxylation had taken place with retention of configuration. 相似文献
9.
Takahisa Miyamoto Keishi Yonemura Makoto Yoshimoto Shoji Hatano 《Bioscience, biotechnology, and biochemistry》2013,77(12):3045-3051
A novel ethanol dehydrogenase with high activity against dulcitol 1-phosphate (D1P-EDH) was purified from Salmonella typhimurium IFO 12529 grown in a medium containing dulcitol as a carbon source. D1P-EDH was purified from a crude extract of S. typhimurium cells by (NH4)2SO4 precipitation and column chromatographies on Blue-Cellulofine, Sephacryl S-300, and Zorbax GF-250. D1P-EDH was purified 277-fold with an activity yield of 21.3%. The purified preparation gave a single band on an electrophoregram. The activity staining of the electrophoregram of the (NH4)2SO4 precipitate indicated that there was no isozyme of D1P-EDH in the extract. The molecular weight of D1P-EDH was estimated to be 158,000 by gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis. D1P-EDH showed its maximal activity in a pH range from 9.0 to 9.5. D1P-EDH was stable in a pH range from 6.0 to 10.0 and was also stable at 30°C for 120 min. The purified preparation oxidized fructose 6-phosphate and galactose 6-phosphate to the same extent as D1P and oxidized much more ethanol than D1P. D1P-EDH activity was strongly inhibited by p-chloromercuribenzoic acid and NaN3 though it was activated by Al3 + , Ba2 + , Ca2 +, and Fe2 +. 相似文献
10.
Vertucci CW 《Plant physiology》1989,90(4):1478-1485
The effect of cooling rate on seeds was studied by hydrating pea (Pisum sativum), soybean (Glycine max), and sunflower (Helianthus annuus) seeds to different levels and then cooling them to − 190°C at rates ranging from 1°C/minute to 700°C/minute. When seeds were moist enough to have freezable water (> 0.25 gram H2O/gram dry weight), rapid cooling rates were optimal for maintaining seed vigor. If the seeds were cooled while at intermediate moisture levels (0.12 to 0.20 gram H2O per gram dry weight), there appeared to be no effect of cooling rate on seedling vigor. When seeds were very dry (< 0.08 gram H2O per gram dry weight), cooling rate had no effect on pea, but rapid cooling rates had a marked detrimental effect on soybean and sunflower germination. Glass transitions, detected by differential scanning calorimetry, were observed at all moisture contents in sunflower and soybean cotyledons that were cooled rapidly. In pea, glasses were detectable when cotyledons with high moisture levels were cooled rapidly. The nature of the glasses changed with moisture content. It is suggested that, at high moisture contents, glasses were formed in the aqueous phase, as well as the lipid phase if tissues had high oil contents, and this had beneficial effects on the survival of seeds at low temperatures. At low moisture contents, glasses were observed to form in the lipid phase, and this was associated with detrimental effects on seed viability. 相似文献
11.
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13.
Rüdiger Lawaczeck 《The Journal of membrane biology》1979,51(3-4):229-261
Summary The permeation of water molccules across single-component lecithin or lecithin-cholesterol bilayers is studied by a new technique. The new technique makes use of the different fluorescence quantum yields of appropriate molecules in D2O and H2O. Water-soluble indole derivatives which by experimental manipulation reside almost entirely within the aqueous (H2O) intravesicular compartment thus can monitor D2O molecules permeating the bilayer by virtue of an increased quantum yield of the fluorescence. In a stopped-flow instrument, a vesicle solution containing the fluorescent chromophore in the intravesicular space is rapidly mixed with the deuterated solvent. The approach to the steady state, where the intra- and extravesicular D2O and H2O concentrations are equal, proceeds in a single-exponential manner. Consequently, the exchange relaxation time for the D2O molecules passing the bilayer can be deduced from the time-dependent increase of the fluorescence intensity. The method and results on lecithin and lecithin-cholesterol bilayer vesicles are discussed. The exchange relaxation times of temperature-dependent studies are interpreted within the framework of the solubility-diffusion theory. Below the crystalline to liquid-crystalline phase transition temperature and for cholesterol-free vesicles, the rate-limiting step for the D2O permeation is attributed to the intracore diffusion. Above the phase transition and for cholesterol-containing vesicles, the intracore diffusion seems not to be rate-limiting. Deviations from the linearity below the phase transition in the Arrhenius-type presentation of the data are related to changes of the partition coefficient of water between the solvent and the lipid phase at the premelting temperature. 相似文献
14.
The non-protein sulfhydryl (NPSH) content of cells moving into S from G1, plateau phase G1, and G0 was measured. Chinese hamster ovary (CHO) cells accumulated in G1 by growth into plateau phase contain only one-fourth the NPSH concentration of cycling C1 cells or G1 cells accumulated by brief growth in isoleucine-deficient medium. Upon dilution of plateau cultures with fresh medium, cellular NPSH content increases rapidly, reaching the same level as that in cycling cells within four hours. This increase is prevented by cycloheximide but not by actinomycin D or hydroxyurea. Neither CHO cells cycling in vitro nor salivary gland G0 cells stimulated with isoproterenol in vivo show significant changes in intracellular NPSH concentrations during S phase. This suggests that the concentration of intracellular NPSH (glutathione) remains constant during the cell cycle except when cells are grown to plateau phase in exhausted or deficient medium, in which case normal degradation exceeds synthesis and the gross level falls until fresh medium is provided and synthesis, apparently on preexisting RNA templates, accelerates. 相似文献
15.
Neville Marks Martin J. Berg Abba J. Kastin David H. Coy 《Neurochemistry international》1984,6(3):347-353
N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly·NH2), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly·NH2) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 μmol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I50, 5 × 10?5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn2+-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-1 but degraded MIF-1 with release of Leu-Gly·NH2 as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro·NH2), a μ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-1 and inactivating morphiceptin. 相似文献
16.
Takeshi Uozumi Gakuzo Tamura Kei Arima 《Bioscience, biotechnology, and biochemistry》2013,77(5):636-652
An intracellular nuclease inhibitor was 1270 times purified from a heat treated cell free extract of fresh mycelia of Aspergillus oryzae, by ammonium sulfate fractionation and chromatographies using DEAE-cellulose and Sephadex G-75. The purified sample of the inhibitor showed a UV absorption curve typical for protein, and it was inactivated by proteases such as chymotrypsin. The inhibitor stoichiometrically inactivated nuclease O (an intracellular nuclease of Asp. oryzae), forming an enzyme-inhibitor complex. But, it did not affect nuclease S1, RNase T1, RNase T2 or pancreatic RNase. The inhibitor was insensitive to 10?5m p-chloromercuribenzoate or 10?4m Pb2+. Molecular weights estimated by the method of Andrews were 23,000 for the inhibitor, 47,000 for nuclease O, and 82,000 for the enzyme-inhibitor complex. The nuclease activity was recovered from the inactive complex by the action of chymotrypsin.Nuclease O of Asp. oryzae was purified and crystallized from 113.5 kg of wet mycelia and 2 kl of culture filtrate, by salting out with ammonium sulfate and by chromatographies on CM-Sephadex C-50 and Sephadex G-100. The purified nuclease showed a single peak with apparent sedimentation constant 2.9S in an ultracentrifuge. The molecular weight measured by short column method was 64,000. The nuclease was completely inhibited by the specific nuclease inhibitor obtained from Asp. oryzae. The nuclease was activated by 0.1 mm Mg2+ and Mn2+, and completely inhibited by 1 mm EDTA. Optimum pH for activity was 7.6 for RNA and 7.4 for DNA. The nuclease degraded polyadenylic acid, polyuridylic acid and polycytidylic acid without forming detectable amount of mononucleotides. And, the main product from RNA was oligonucleotides. The enzyme showed no nonspecific phosphodiesterase activity. 相似文献
17.
Identifying sources of nitrous oxide emission in anoxic/aerobic sequencing batch reactors (A/O SBRs) acclimated in different aeration rates 总被引:1,自引:0,他引:1
Both long term and batch experiments were carried out to identify the sources of the N2O emission in anoxic/aerobic sequencing batch reactors (A/O SBRs) under different aeration rates. The obtained results showed that aeration rate has an important effect on the N2O emission of A/O SBR and most of the N2O was emitted during the aerobic phase. During the anoxic phase, nitrate ammonification was the major source of N2O emission while denitrification performed as a sink of N2O, in all three bioreactors. The N2O emission mechanisms during the aerobic phase differed with the aeration rate. At low and high aeration rates (Run 1 and Run 3), both coupled-denitrification and nitrifier denitrification were ascribed to be the source of N2O emission. At mild aeration rate (Run 2), nitrifier denitrification by Nitrosomonas-like ammonia oxidizing-bacterial (AOB) was responsible for N2O emission while coupled-denitrification turned out to be a sink of N2O because of the presence of inner anaerobic region in sludge flocs. 相似文献
18.
Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation
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Christian Voitenleitner Christoph Rehfuess Melissa Hilmes Lynda ORear Pao-Chi Liao Douglas A. Gage Robert Ott Heinz-Peter Nasheuer Ellen Fanning 《Molecular and cellular biology》1999,19(1):646-656
DNA polymerase α-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase α-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase α-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase α-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner. 相似文献
19.
Mariano J. Levin Dominique Daegelen Marie-Claire Meienhofer Jean-Claude Dreyfus Axel Kahn 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,699(2):77-83
A study was performed to determine whether M1 and M2 pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M1 and M2 pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M1 or M2. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M1 and M2 with V8 protease from Staphylococcus aureus confirmed that these polypeptides were clearly different. 相似文献
20.
Efficient Expression and Rapid Purification of Human T-Cell Leukemia Virus Type 1 Protease
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Y. Shirley Ding Sherry M. Owen Renu B. Lal Richard A. Ikeda 《Journal of virology》1998,72(4):3383-3386
Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus that is clinically associated with adult T-cell leukemia. We report here the construction of a pET19-based expression clone containing HTLV-1 protease fused to a decahistidine-containing leader peptide. The recombinant protein is efficiently expressed in Escherichia coli, and the fusion protein can be easily purified by affinity chromatography. Active mature protease in yields in excess of 3 mg/liter of culture can then be obtained by a novel two-step refolding and autoprocessing procedure. The purified enzyme exhibited Km and Kcat values of 0.3 mM and 0.143 sec−1 at pH 5.3 and was inhibited by pepstatin A. 相似文献