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91.
Toshiko Kido Katsuyuki Tanizawa Kenji Inagaki Tohru Yoshimura Masaaki Ishida Katsumi Hashizume 《Bioscience, biotechnology, and biochemistry》2013,77(10):2549-2554
2-Nitropropane dioxygenase, purified to homogeneity by an improved method from a yeast, Hansenula mrakii, has a molecular weight of 42,000, and consists of a single polypeptide. The enzyme contains 1 mol of FAD per mol of enzyme. The iron protein associated with previous preparations was removed by the present purification procedures. The enzyme catalyzes the oxygenative denitrification of anionic nitroalkanes much more effectively than that of the neutral ones with the optimum pH of 6.5. The Michaelis constants for the anionic substrates are as follows: 2-nitropropane, 1.61 mM; 1-nitropropane, 3.23 mM; nitroethane, 3.13 mM, and 3-nitro-2-butanol, 0.59 mM. 相似文献
92.
Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same. 相似文献
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94.
Osaki Y Shirabe T Nakanishi H Wakagi T Yoshimura E 《Metallomics : integrated biometal science》2009,1(4):353-358
Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides. 相似文献
95.
96.
97.
Tomono S Miyoshi N Shiokawa H Iwabuchi T Aratani Y Higashi T Nukaya H Ohshima H 《Journal of lipid research》2011,52(1):87-97
3β-Hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentified, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3β-hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ~30% was converted to its aldehyde-oxidation product, 3β-hydroxy-5β-hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophil-like differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, significantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-deficient mice. Our results demonstrate that secoterol-A is formed by an ozone-like oxidant generated with PMA-activated neutrophils through the MPO-dependent mechanism. 相似文献
98.
H Sakurai H Uchikubo T Yoshimura M Maeda K Kawasaki 《Biochemical and biophysical research communications》1985,130(3):1254-1260
Thiol-containing penta (Leu-Ala-Cys-Ser-Leu) and nona (Leu-Ala-Cys-Ser-Leu-Ile-Glu-Ser-Leu) peptides corresponding to residues 132-136 and 132-140, respectively, of apo-P450 from Psuedomonas putida were synthesized to examine heme-binding by the enzymes in the oxidized (ferric) form. The peptide-hemin complexes prepared in solution were characterized by their optical and ESR spectra. In these complexes without nitrogenous ligands, no ferric complexes in the low-spin state were observed, suggesting that simultaneous axial coordination of Cys-134 (thiolate) and Ser-139 (hydroxyl) of apo-P450cam to the heme is unlikely to occur. In the presence of nitrogenous ligands, such as py, Im and MeIm, the resulting complexes were good models of apo-P450cam-nitrogenous ligand adducts in the low-spin ferric state retaining a thiolate-Fe(III)-nitrogen axial coordination mode, as judged by their spectral pattern as well as by crystal field analysis of ESR g-values. 相似文献
99.
Linford A Yoshimura S Nunes Bastos R Langemeyer L Gerondopoulos A Rigden DJ Barr FA 《Developmental cell》2012,22(5):952-966
Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. 相似文献
100.