共查询到20条相似文献,搜索用时 15 毫秒
1.
Lindsey J. Brown Matthias Baranowski Yun Wang Anna K. Schrey Thomas Lenz Sean D. Taverna Philip A. Cole Michael Sefkow 《Analytical biochemistry》2014
S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs. 相似文献
2.
The effects of β-glucan (BG) prepared from spent brewer’s yeast on gelatinization and retrogradation of rice starch (RS) were investigated as functions of mixing ratio and of storage time. Results of rapid visco-analysis (RVA) indicated that addition of BG increased the peak, breakdown, setback, and final viscosities, but decreased the pasting temperatures of the rice starch/β-glucan (RS/BG) mixtures. Differential scanning calorimetry (DSC) data demonstrated an increase in onset (To), peak (Tp), and conclusion (Tc) temperatures and a decrease in gelatinization enthalpy (ΔH1) with increasing BG concentration. Storage of the mixed gels at 4 °C resulted in a decrease in To, Tp, Tc, and melting enthalpy (ΔH2). The retrogradation ratio (ΔH2/ΔH1) and the phase transition temperature range (Tc − To) of the mixed gels increased with storage time, but this effect was reduced by the addition of BG. BG addition also slowed the syneresis of the mixed gels. Results of dynamic viscoelasticity measurement indicated that the addition of BG promoted RS retrogradation at the beginning and then retarded it during longer storage times. The added BG also retarded the development of gel hardness during refrigerated storage of the RS/BG mixed gels. 相似文献
3.
Hiroyuki Nozaki Shinji Kuroda Kunihiko Watanabe Kenzo Yokozeki 《Journal of Molecular Catalysis .B, Enzymatic》2009,59(4):237-242
The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The Vmax value was 1.40 μmol min−1 mg−1, and the Km value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid. 相似文献
4.
Robert M. Johnston Linda N. Chu Mark Liu Steven L. Goldberg Animesh Goswami Ramesh N. Patel 《Enzyme and microbial technology》2009,45(6-7):484-490
A synthetic gene encoding a Streptomyces l-proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l-proline to cis-3-hydroxy-l-proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy-l-proline. 相似文献
5.
Lucídio C. Fardelone J. Augusto R. Rodrigues Paulo J. S. Moran 《Journal of Molecular Catalysis .B, Enzymatic》2004,29(1-6):41
The enantioselective reduction of cinnamaldehyde derivatives is an attractive strategy to prepare various optically active multifunctional molecules that can be used as chiral building blocks for the synthesis of some HIV-protease inhibitors. The asymmetric reduction with pH adjusted to 5.5 of α-substituted-cinnamaldehydes (Br, N3) mediated by baker’s yeast (Saccharomyces cerevisiae) yielded α-substituted-3-phenyl-1-propanol in excellent enantiomeric excesses and yields. 相似文献
6.
Gao Min-tian Makoto Hirata Michiteru Koide Hirokazu Takanashi Tadashi Hano 《Process Biochemistry》2004,39(12):1903-1907
An efficient process for producing l-lactic acid using an, EDF method is described. The results showed that intermittent EDF with continuous medium feed was the best one among the experiment methods employed. Comparing with the conventional EDF, intermittent EDF (seven on–off) with continuous medium feed indicated that the maximum value of o.d.660 was not increased, but productivity was 1.5 times higher. The yield increased by above 30% and glucose transport decreased to 1/10 (from 0.46 to 0.05). 相似文献
7.
Paul Christakopoulos Mahalingeshwara K. Bhat Dimitris Kekos Basil J. Macris 《International journal of biological macromolecules》1994,16(6)
Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides. 相似文献
8.
α-
Jean-Paul Mornon Dyna Halaby Marc Malfois Patrick Durand Isabelle Callebaut Annette Tardieu 《International journal of biological macromolecules》1998,22(3-4)
New results obtained from a two-dimensional sequence analysis of the small heat shock protein (shsp) family are described. It is confirmed that the conserved C-terminal α-crystallin domain is essentially made of β-strands, most probably two groups of β-strands separated by a large loop. A direct correspondence between the putative β-strands that have been identified in shsps and the seven β-strands of a classical immunoglobulin-like fold is proposed. The hypothesis that the shsp family could belong to the immunoglobulin superfamily (IgSF) is consistent with the ubiquitous distribution and the multifunctional properties of the crystallins that are now emerging. 相似文献
9.
Shinichi Kitamura Takashi Hirano Kenichi Takeo Mitsuru Mimura Kanji Kajiwara Bjrn T. Stokke Tsutomu Harada 《International journal of biological macromolecules》1994,16(6)
The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (Mw) from 1.49 × 104 to 5.29 × 106 were obtained from a native sample by ultrasonic degradation and fractional precipitation. For Mw < 4 × 104, the intrinsic viscosity [η] varies with Mw0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For Mw > 105, [η] exhibits an unusually weak dependence on Mw and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed. 相似文献
10.
Deok Kun Oh Hyo Jung Oh Hye Jung Kim Jina Cheon Pil Kim 《Journal of Molecular Catalysis .B, Enzymatic》2006,43(1-4):108
l-Arabinose isomerase from Geobacillus stearothermophilus (GSAI; EC 5.3.1.4) has been genetically evolved to increase the reaction rate toward d-galactose, which is not a natural substrate. To change the optimal pH of GSAI for d-galactose isomerization (pH optimum at 8.5), we investigated the single point mutations influencing the activity based on the sequences of the previously evolved enzymes. Among the seven point mutations found in the evolved enzymes, mutations at Val408 and Asn475 were determined to be highly influential mutation points for d-galactose isomerization activity. A random mutation was introduced into sites Val408 and Asn475 (X408V and X475N), and candidates were screened based on non-optimal pH conditions. Among the mutations of X408V and X475N, mutations of Q408V and R408V were selected. The optimal pH of the both mutations Q408V and R408V was shifted to pH 7.5. At the shifted optimal pH, the d-galactose isomerization activities of Q408V and R408V were 60 and 30% higher than that of the wild type at pH 8.5, respectively. 相似文献
11.
Danuta Gillner Nicola Armoush Richard C. Holz Daniel P. Becker 《Bioorganic & medicinal chemistry letters》2009,19(22):6350-6352
The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC50 = 3.3 μM, Ki = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli. 相似文献
12.
An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH4)2SO4 precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, Km 0.29 mM, Vmax 2.56 μkat mg−1) and pNP-β-d-GalNAc (optimum pH 4.4, Km 0.27 mM, Vmax 2.50 μkat mg−1). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The Kis for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)2 and pNP-β-d-(GlcNAc)3 than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)2 (optimum pH 5.0) and (GlcNAc)3−6 (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)n were predominantly (GlcNAc)n−1 and GlcNAc. 相似文献
13.
Free l-amino acids and d-aspartate content in the nervous system of Cephalopoda. A comparative study
Antimo D'Aniello Giovanna Nardi Amedeo De Santis Amedeo Vetere Anna di Cosmo Rosangela Marchelli Arnaldo Dossena George Fisher 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,112(4)
We have determined the content of free l-amino acids and d-aspartate in the nervous tissue of three representative cephalopods: Sepia officinalis, Octopus vulgaris, and Loligo vulgaris, and the optic lobes of adult and embryo Sepia officinalis. Taurine is the most abundant amino acid in the cephalopod nervous tissue. Its content amounts to more than 50% of the total free amino acids. The other most concentrated amino acids are Glu, Ala, Asp, and GABA. High concentrations of d-aspartate were found in the nervous tissue of all cephalopods examined (7–12 μmol/g wet tissue) which represents 50–80% of the total aspartate (d + l), depending on the animal. Among the various regions of the brain of Octopus vulgaris, d-aspartate was found to be evenly distributed in the various regions of the brain. In nerve tissue of Sepia officinalis, there is no significant difference in the pattern of free l-amino acids, in particular of the d-aspartate concentration, between adults and embryos, except for GABA, Gly, His and Thr. This suggests that d-aspartate in nerve tissue of the Cephalopoda is of endogenous origin and not a product of accumulation from exogenous sources. From a comparative study of the content of d-aspartate in the nervous tissue of different animals, we found that protostomia contain a significantly higher amount than deuterostomia. Thus, d-aspartate could be a criterion to distinguish the protostomia phyla from the deuterostomia phyla. 相似文献
14.
Hisato Takei Yoichiro Nakai Naoko Hattori Masahiro Yamamoto Shuichi Takeda Masako Yamamoto Kazuyoshi Arishima 《Phytomedicine》2007,14(9):591-600
The chronic inhibition of nitric oxide (NO) synthesis with N(omega)-Nitro-L-arginine methyl ester (L-NAME) induces a pre-eclampsia-like syndrome including hypertension in pregnant rats. We tested the traditional herbal medicines Saireito (SR) and Boiogito (BO), which have been used clinically for the treatment of pre-eclampsia, in this model. L-NAME was infused subcutaneously into pregnant rats from gestational day 14 (G14). SR and BO (both at 1, 2g/kg) were administered by gavage from G14 to G20. Systolic blood pressure was measured on G19. SR and BO (both at 1, 2g/kg) inhibited L-NAME-induced hypertension. SR was effective in both pregnant and non-pregnant rats while BO was effective only in pregnant rats. BO increased blood levels of CGRP and decreased levels of endothelin-1; both are known to play important roles in regulation of blood pressure in pre-eclampsia. SR and BO may be beneficial for the treatment and prevention of hypertension in pre-eclampsia. 相似文献
15.
A general and flexible synthetic approach to biologically important 5,6-unsaturated C18-phytosphingosines was developed via olefin cross-metathesis employing truncated C6-phytosphingosines as the key intermediates. These were efficiently prepared in high yields by zinc-mediated reductive opening of methyl 2-amino-2-deoxy-β-hexopyranosides.
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16.
Marciane Magnani Caroline M. Calliari Fernando C. de Macedo Jr. Mateus P. Mori Ilce M. de Syllos Clus Raul J.H. Castro-Gomez 《Carbohydrate polymers》2009,78(4):159
The cell wall of Saccharomyces cerevisiae is an important source of β-d-glucan, a glucose homopolymer with immunostimulant properties. The standard methodologies described for its extraction involve acid and alkaline washings, which degrade part of its glucose chains and reduce the final yield. In the present study, an optimized methodology for extraction of β-d-glucan from S. cerevisiae cells, involving sonication and enzyme treatment, with a yield of 11.08 ± 0.19%, was developed. The high-purity (1 → 3)(1 → 6)-β-d-glucan was derivatized to carboxymethyl-glucan (CM-G). In vitro tests with CM-G in Chinese hamster epithelial cells (CHO-k1) did not reveal any cytotoxic or genotoxic effects or influences of this molecule on cell viability. The method described here is a convenient alternative for the extraction of (1 → 3)(1 → 6)-β-d-glucan under mild conditions without the generation of wastes that could be potentially harmful to the environment. 相似文献
17.
Kazuki Oka Hiroshi Maeda Shoichi Ikeda 《International journal of biological macromolecules》1983,5(6)
The effects of eight divalent metal ions on fully neutralized poly(S-carboxyethyl-l-cysteine) have been studied by means of circular dichroism. Four ionic species (Cd2+, Cu2+, Zn2+ and Ni2+) effectively induce the β-form, while the other four species (Co2+, Ba2+, Ca2+ and Mg2+) are not effective. Specifically, Mg(ClO4)2 is ineffective, even at 1.86 m. The effect of Cu2+ ions on the polypeptide conformation is significant at pH values other than in the neural range. Comparison of the present results with previous ones from the lower side chain homologue, poly(S-carboxymethyl-l-cysteine), shows that Cd2+ and Zn2+ ions are more effetive but Co2+ ions are much less effective in the polypeptide studied here. Random coils of poly(S-carboxyethyl-l-cysteine) are more soluble while the β-form is less soluble compared with the respective conformations of the lower side-chain homologue. 相似文献
18.
A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation–reduction sequence at C-2′. The β-manno configurations of the final deprotected congeners 2–7 were confirmed by measurement of 1JC1,H1 heteronuclear and 3J1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the protective epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site. 相似文献
19.
Pavla Simersk Marek Kuzma Daniela Monti Sergio Riva Martina Mackov Vladimír Ken 《Journal of Molecular Catalysis .B, Enzymatic》2006,39(1-4):128
The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail. 相似文献
20.
Cytogenetic evaluation of occupational exposure to external γ-rays and internal
M. Bauchinger E. Schmid H. Braselmann 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》1997,395(2-3)
Chromosome analyses were performed on peripheral lymphocytes from seven radiation workers exposed to external γ-radiation within maximum annual permissible dose limits during 11 to 22 years of employment. Five years prior to blood sampling, six workers had additionally incorporated the α-emitting radionuclide
which contributed between five to 25% of the total accumulated whole body dose in five workers and about 70% in one worker. For the radiation workers as a group, both the mean frequencies of symmetrical translocations measured by fluorescence in situ hybridisation (FISH)/chromosome painting and dicentrics+ring chromosomes scored in first division metaphases of conventional preparations were significantly elevated compared with respective controls. The mean biodosimetry estimate for the group was 270 mSv when based on stable translocations which compares well with the mean dose of 247 mSv based on official dosimetry records. The lower mean dose estimate of 160 mSv based on unstable dicentrics is compatible with the well-known loss of dicentrics from the circulating blood with time after exposure. 相似文献
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