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91.
Nishio T Kanai C Hakamata W Ogawa M Nakajima K Hoshino S Matsuishi A Kawachi R Oku T 《Bioscience, biotechnology, and biochemistry》2003,67(5):1024-1029
Enzymatic transglycosylation using four possible monodeoxy analogs of p-nitrophenyl alpha-D-glucopyranoside (Glc alpha-O-pNP), modified at the C-2, C-3, C-4, and C-6 positions (2D-, 3D-, 4D-, and 6D-Glc alpha-O-pNP, respectively), as glycosyl donors and six equivalents of ethyl beta-D-thioglucopyranoside (Glc beta-S-Et) as a glycosyl acceptor, to yield the monodeoxy derivatives of glucooligosaccharides were done. The reaction was catalyzed using purified Aspergillus niger alpha-glucosidase in a mixture of 50 mM sodium acetate buffer (pH 4.0)/CH3CN (1:1 v/v) at 37 degrees C. High activity of the enzyme was observed in the reaction between 2D-Glc alpha-O-pNP and Glc beta-S-Et to afford the monodeoxy analogs of ethyl beta-thiomaltoside and ethyl beta-thioisomaltoside that contain a 2-deoxy alpha-D-glucopyranose moiety at their glycon portions, namely ethyl 2-deoxy-alpha-D-arabino-hexopyranosyl-(1,4)-beta-D-thioglucopyranoside and ethyl 2-deoxy-alpha-D-arabino-hexopyranosyl-(1,6)-beta-D-thioglucopyranoside, in 6.72% and 46.6% isolated yields (based on 2D-Glc alpha-O-pNP), respectively. Moreover, from 3D-Glc alpha-O-pNP and Glc beta-S-Et, the enzyme also catalyzed the synthesis of the 3-deoxy analog of ethyl beta-thioisomaltoside that was modified at the glycon alpha-D-glucopyranose moiety, namely ethyl 3-deoxy-alpha-D-ribo-hexopyranosyl-(1,6)-beta-D-thioglucopyranoside, in 23.0% isolated yield (based on 3D-Glc alpha-O-pNP). Products were not obtained from the enzymatic reactions between 4D- or 6D-Glc alpha-O-pNP and Glc beta-S-Et. 相似文献
92.
Characterization of factors involved in the production of 2(E)-nonenal during mashing 总被引:1,自引:0,他引:1
Kuroda H Furusho S Maeba H Takashio M 《Bioscience, biotechnology, and biochemistry》2003,67(4):691-697
To characterize the factors involved in the production of volatile aldehydes during mashing, a model mashing experiment was done. After we inactivated the endogenous lipoxygenase (LOX) activity in the mash by mashing at 70 degrees C for 30 min, further incubation with recombinant barley LOX-1 stimulated the accumulation of 2(E)-nonenal; however, this effect was significantly reduced by boiling the mash sample. The result suggests that both LOX-1 and a heat-stable enzymatic factor are involved in the production of 2(E)-nonenal during mashing. Malt contained fatty acid hydroperoxide lyase-like activity (HPL-like activity) that transformed 9-hydroperoxy-10(E), 12(Z)-octadecadienoic and 13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid into 2(E)-nonenal and hexanal, respectively. Proteinase K sensitivity tests showed that they are distinct factors. 9-HPL-like activity survived through the mashing at 70 degrees C for 30 min but was inactivated by boiling, suggesting it will be the heat-stable enzymatic factor found in the model mashing experiment. 相似文献
93.
94.
Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol 总被引:9,自引:0,他引:9
Niemann-Pick type C (NPC) is a disease that affects intracellular cholesterol-trafficking pathways. By cloning the hamster ortholog of NPC1, we identified the molecular lesions in two independently isolated Chinese hamster ovary cell mutants, CT60 and CT43. Both mutants lead to premature translational terminations of the NPC1 protein. Transfecting hamster NPC1 cDNA complemented the defects of the mutants. Investigation of the CT mutants, their parental cells, and an NPC1-stable transfectant allow us to present evidence that NPC1 is involved in a post-plasma membrane cholesterol-trafficking pathway. We found that the initial movement of low density lipoprotein (LDL)-derived cholesterol to the plasma membrane (PM) did not require NPC1. After reaching the PM and subsequent internalization, however, cholesterol trafficking back to the PM did involve NPC1. Both LDL-derived cholesterol and cholesterol originating from the PM accumulated in a dense, intracellular compartment in the CT mutants. Cholesterol movement from this compartment to the PM or endoplasmic reticulum was defective in the CT mutants. Our results functionally distinguish the dense, intracellular compartment from the early endocytic hydrolytic organelle and imply that NPC1 is involved in sorting cholesterol from the intracellular compartment back to the PM or to the endoplasmic reticulum. 相似文献
95.
Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis
by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted
with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in
which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal
amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated
that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In
an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although
this effect was weaker than that with adenosine diphosphate, a well-known activator.
Received October 15, 1999; accepted January 4, 2000. 相似文献
96.
Isometric skeletal muscle fatigue is usually assumed to be a linear process based upon the monotonic decrease in spectral
frequency of the EMG. Since spectral analysis by fast Fourier transform (FFT) constitutes a linear transformation of the data,
the present study was designed to reevaluate the time-course of muscle fatigue with a nonlinear tool, recurrence quantification
analysis (RQA). Surface EMG recordings were obtained from the multifidus muscle of 17 human subjects during isometric posture-holding
of the upper torso. The process of muscle fatigue was found to be linear for 59% of the subjects by FFT criteria, but nonlinear
for 76% by RQA criteria. As a demonstrative control, both slow and fast transients occurring within a nonlinear mathematical
process could be accurately depicted by RQA, but not by FFT. It is concluded that assessment of EMG patterns by nonlinear
techniques can give insight into the time-course of fatiguing muscles attributed to the summation of several nonlinear and
competing processes.
Received: 12 November 1998 / Accepted in revised form: 29 November 1999 相似文献
97.
Hajime Shibuya Hiroaki Nagasaki Satoshi Kaneko Shigeki Yoshida Gwi Gun Park Isao Kusakabe Hideyuki Kobayashi 《Applied and environmental microbiology》1998,64(11):4489-4494
The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.α-Galactosidase (αGal) (EC 3.2.1.22) is of particular interest in view of its biotechnological applications. αGal from coffee beans demonstrates a relatively broad substrate specificity, cleaving a variety of terminal α-galactosyl residues, including blood group B antigens on the erythrocyte surface. Treatment of type B erythrocytes with coffee bean αGal results in specific removal of the terminal α-galactosyl residues, thus generating serological type O erythrocytes (8). Cyamopsis tetragonoloba (guar) αGal effectively liberates the α-galactosyl residue of galactomannan. Removal of a quantitative proportion of galactose moieties from guar gum by αGal improves the gelling properties of the polysaccharide and makes them comparable to those of locust bean gum (18). In the sugar beet industry, αGal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (28). Raffinose and stachyose in beans are known to cause flatulence. αGal has the potential to alleviate these symptoms, for instance, in the treatment of soybean milk (16).αGals are also known to occur widely in microorganisms, plants, and animals, and some of them have been purified and characterized (5). Dey et al. showed that αGals are classified into two groups based on their substrate specificity. One group is specific for low-Mr α-galactosides such as pNPGal (p-nitrophenyl-α-d-galactopyranoside), melibiose, and the raffinose family of oligosaccharides. The other group of αGals acts on galactomannans and also hydrolyzes low-Mr substrates to various extents (6).We have studied the substrate specificity of αGals by using galactomanno-oligosaccharides such as Gal3Man3 (63-mono-α-d-galactopyranosyl-β-1,4-mannotriose) and Gal3Man4 (63-mono-α-d-galactopyranosyl-β-1,4-mannotetraose). The structures of these galactomanno-oligosaccharides are shown in Fig. Fig.1.1. Mortierella vinacea αGal I (11) and yeast αGals (29) are specific for the Gal3Man3 having an α-galactosyl residue (designated the terminal α-galactosyl residue) attached to the O-6 position of the nonreducing end mannose of β-1,4-mannotriose. On the other hand, Aspergillus niger 5-16 αGal (12) and Penicillium purpurogenum αGal (25) show a preference for the Gal3Man4 having an α-galactosyl residue (designated the stubbed α-galactosyl residue) attached to the O-6 position of the third mannose from the reducing end of β-1,4-mannotetraose. The M. vinacea αGal II (26) acts on both substrates to almost equal extents. The difference in specificity may be ascribed to the tertiary structures of these enzymes. Open in a separate windowFIG. 1Structures of galactomanno-oligosaccharides.Genes encoding αGals have been cloned from various sources, including humans (3), plants (20, 32), yeasts (27), filamentous fungi (4, 17, 24, 26), and bacteria (1, 2, 15). αGals from eukaryotes show a considerable degree of similarity and are grouped into family 27 (10).Here we describe the cloning of P. purpurogenum αGal cDNA, its expression in Saccharomyces cerevisiae, and the purification and characterization of the recombinant enzyme. 相似文献
98.
99.
A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition 总被引:7,自引:6,他引:1
Shigeki Moriya Eitoku Tsujikawa Anwarul K. M. Hassan Kei Asai Takeko Kodama & Naotake Ogasawara 《Molecular microbiology》1998,29(1):179-187
We have analysed the function of a gene of Bacillus subtilis , the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed. 相似文献
100.
Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb 总被引:8,自引:3,他引:5
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