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971.
Yoshinobu Minami Etsushiro Doi Tadao Hata 《Bioscience, biotechnology, and biochemistry》2013,77(9):1419-1430
The heterogeneity of the proteolytic enzymes in the stem bromelain was investigated by the isoelectric focusing with carrier ampholytes. The isoelectric focusing of the stem bromelain demonstrated the presence of two types of proteolytic enzymes which were distinguishable from each other by their isoelectric points. One of these was a basic protein having an isoelectric point of 9.45. This basic enzyme comprised almost all of basic protein which are found in stem bromelain. The other was an acidic protein having an isoelectric point near pH 4.7. This was a minor compooent. The purification of the two enzymes was carried out by use of chromatographies on CM-Sephadex, DEAE-Sephadex and Sephadex at pH 7.0. 相似文献
972.
Kimihisa Ichikawa Koji Komiya Katsunori Suzuki Tadaatsu Nakahara Yoshifumi Jigami 《Bioscience, biotechnology, and biochemistry》2013,77(10):2687-2694
The effects of initial glucose concentrations on the cell growth, glucose usage, and human lysozyme (HLY) secretion under the ENOl promoter were examined in the Saccharomyces cerevisiae strain A2–1–1A harboring a multicopy plasmid. By increasing the initial glucose from 2 % to 10 %, the HLY secretion increased 7 ~ 8 fold although the cell growth was not affected. By adding a mixture of mineral salts to the basal medium, the HLY secretion was increased about twice due to the continuity of the HLY expression at the stationary phase of cell growth.The high HLY secretion (5.5 mg per liter, 47-fold higher than the original level) was achieved by the strain A2–1–1A grown in the synthetic basal medium containing 10% initial glucose, and supplemented with mineral salts containing ammonium sulfate, potassium phosphate, potassium chloride, magnesium sulfate, and iron sulfate. 相似文献
973.
Yoshinobu Kimura Hiroshi Suehisa Osamu Yamaguchi Shûhei Nakajima Shigeaki Takagi 《Bioscience, biotechnology, and biochemistry》2013,77(12):3259-3267
The structures of sugar chains from the water-soluble glycoproteins in developing castor beans have been identified. The structural analyses were done by a fluorescence method combined with exoglycosidase digestions and 1H-NMR spectroscopy. The identified structures fell into two categories; one was an oligomannose-type, the other xylomannose-type or xylose-containing type. Among these oligosaccharides, Man3Fuc1Xyl1GlcNAc2 (M3FX; 38%) and Man6GlcNAc2 (M6B; 22%) were the major structures. The higher mannose-content oligosaccharides (Man8 ? 7GlcNAc2) were only 4.1%, and the further-modified structures (GNM3FX, M2FX) than M3FX were 22% of the total. 相似文献
974.
Rikimaru Hayashi Yoshinobu Oka Etsushiro Doi Tadao Hata 《Bioscience, biotechnology, and biochemistry》2013,77(3):359-366
The existence of the inactive precursors of yeast proteinases B and C was confirmed in the autolysate of baker’s yeast and they were named as pro-proteinases B and C, respectively. The active and inactive forms of proteinase C were two distinct proteins, separable by chromatographical procedures. The two precursors were markedly activated by incubation at pH 5 or by treatment with denaturing agents, e.g. urea, dioxane, acetone and certain alcohols.These activations were also observed with extracts from acetone-dried cells and from mechanically destructed cells, but the activation of proteinase A was not demonstrated under any conditions tested. Therefore, it was assumed that most of proteinases B and C exist in vivo as inactive precursors, whereas proteinase A originally exists in an active form.Pro-proteinase C, the latent form of yeast proteinase C, was partially purified from the autolysate of baker’s yeast. It was strongly activated by incubation at pH 5 or by treatment with urea or dioxane. The former activation was prevented by treatment to inactivate yeast proteinase A, which co-existed with the pro-enzyme in the present preparation, but was promoted by addition of purified proteinase A. Thus, it was confirmed that A could activate pro-proteinase C. Furthermore, it was found that activation could be caused by extremes in pH or by heating to 55~60°C, accompanied by the simultaneous destruction of the enzyme produced. Pro-proteinase C was stable over a range of pH 5 to 8 after 60 min incubation at 50°C. 相似文献
975.
Shigeaki Ichikawa Yuzo Shibuya Kunio Matsumoto Tadashiro Fujii Kenichi Komatsu Ryoji Kodaira 《Bioscience, biotechnology, and biochemistry》2013,77(10):2231-2236
7β-(4-Carboxybutanamido)cephalosporanic acid acylase (penicillin amidohydrolase, EC 3.5.1.11) was crystallized from cell-free extracts of a mutant derived from Pseudomonas SY-77-1. Purification of the enzyme was performed by a procedure involving ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex, TEAE-cellulose and Sephadex G-200. The crystalline enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the acylase was estimated to be 1.3 × 105 by gel filtration. The enzyme was fully active at pH above 6.5 and was highly stable at a pH range of 6.0 to 8.0 and below 38°C. The Michaelis-Menten constant (Km) and Vmax for 7β-(4-carboxybutanamido)cephalosporanic acid were 0.16mM and 4.91 μmol/min/mg-protein, respectively. It was also indicated that this enzyme-protein occupied 2.3% of the dry-cell weight. 相似文献
976.
Kazutoshi Fujii Hiroki Takata Michiyo Yanase Yoshinobu Terada Kohji Ohdan Takeshi Takaha 《Biocatalysis and Biotransformation》2013,31(4-5):167-172
AbstractGlucan phosphorylase, branching enzyme, and 4-α-glucanotransferase were employed to produce glucose polymers with controlled molecular size and structures. Linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with bracnhing enzyme, where the molecular weight of linear glucan was strictly controlled by the glucose-1-phosphate/primer molar ratio, and the branching pattern by the relative branching enzyme/glucan phosphorylase activity ratio. Cyclic glucans were produced by the cyclization reaction of 5-αglucanotransferases and branching enzyme on amylose and amylopectin. Molecular size and structure of cyclic glucan was controlled by the type of enyzyme and substrate chosen and by the reaction conditions. This in vitro approach can be used to manufacture novel glucose polymers with applicable value. 相似文献
977.
978.
Sharma V Ichikawa M He P Scott DA Bravo Y Dahl R Ng BG Cosford ND Freeze HH 《The Journal of biological chemistry》2011,286(45):39431-39438
Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos. 相似文献
979.
Fukaya M Kamata A Hara Y Tamaki H Katsumata O Ito N Takeda S Hata Y Suzuki T Watanabe M Harvey RJ Sakagami H 《Journal of neurochemistry》2011,116(6):1122-1137
SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both β-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM. 相似文献
980.
Kabir SR Zubair MA Nurujjaman M Haque MA Hasan I Islam MF Hossain MT Hossain MA Rakib MA Alam MT Shaha RK Hossain MT Kimura Y Absar N 《Bioscience reports》2011,31(6):465-475
A lectin (termed NNTL) was purified from the extracts of Nymphaea nouchali tuber followed by anion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on HiTrap Phenyl HP and by repeated anion-exchange chromatography on HiTrap Q FF column. The molecular mass of the purified lectin was 27.0 ± 1.0 kDa, as estimated by SDS/PAGE both in the presence and in the absence of 2-mercaptoethanol. NNTL was an o-nitrophenyl β-D-galactopyranoside sugar-specific lectin that agglutinated rat, chicken and different groups of human blood cells and exhibited high agglutination activity over the pH range 5-9 and temperatures of 30-60 °C. The N-terminal sequence of NNTL did not show sequence similarity with any other lectin and the amino acid analysis revealed that NNTL was rich in leucine, methionine and glycine residues. NNTL was a glycoprotein containing 8% neutral sugar and showed toxicity against brine shrimp nauplii with an LC(50) value of 120 ± 29 μg/ml and exerted strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). In addition, antiproliferative activity of this lectin against EAC (Ehrlich ascites carcinoma) cells showed 56% and 76% inhibition in vivo in mice at 1.5 and 3 mg·kg(-1)·day(-1) respectively. NNTL was a divalent ion-dependent glycoprotein, which lost its activity markedly in the presence of denaturants. Furthermore, measurement of fluorescence spectra in the presence and absence of urea and CaCl(2) indicated the requirement of Ca(2+) for the stability of NNTL. 相似文献