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31.
Kinetics of proton transfer between lysozyme and a pH indicator p-nitrophenol (p-Np) were measured by the temperature-jump method in a pH range of 6.0–7.0. Two well-defined relaxation processes were observed. The fast process (τ ? 15 μsec) was also observed for a lysozyme derivative succinylated at the terminal α-amino group of Lys 1. Therefore, the fast process was found to be attributable to the proton transfer reaction of His 15 with p-Np. The slow process (τ ? 50 μsec) was found to be characteristic of the proton transfer reaction of Glu 35, because it disappeared completely in solution containing a lysozyme derivative having an ester crosslink between the carboxyl group of Glu 35 and indol C-2 of Trp 108. The rate constants for proton transfer from Glu 35 and His 15 to p-Np were found to be 9 × 106/sec/M (±65%, 23°C) and 3 × 108/sec/M (±20%, 25°C), respectively. These data indicate that the proton of the carboxyl group of Glu 35 is kinetically stabilized in lysozyme. 相似文献
32.
Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction 总被引:1,自引:0,他引:1 下载免费PDF全文
Masaru Wada Ayumi Yoshizumi Yumiko Noda Michihiko Kataoka Sakayu Shimizu Hiroshi Takagi Shigeru Nakamori 《Applied microbiology》2003,69(2):933-937
A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD+, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound. 相似文献
33.
Uchino S Wada H Honda S Nakamura Y Ondo Y Uchiyama T Tsutsumi M Suzuki E Hirasawa T Kohsaka S 《Journal of neurochemistry》2006,97(4):1203-1214
A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons. 相似文献
34.
Acidification inside the vacuo-lysosome systems is ubiquitous in eukaryotic organisms and essential for organelle functions. The acidification of these organelles is accomplished by proton-translocating ATPase belonging to the V-type H+-ATPase superfamily. However, in terms of chemiosmotic energy transduction, electrogenic proton pumping alone is not sufficient to establish and maintain those compartments inside acidic. Current studies have shown that thein situ acidification depends upon the activity of V-ATPase and vacuolar anion conductance; the latter is required for shunting a membrane potential (interior positive) generated by the positively charged proton translocation. Yeast vacuoles possess two distinct Cl– transport systems both participating in the acidification inside the vacuole, a large acidic compartment with digestive and storage functions. These two transport systems have distinct characteristics for their kinetics of Cl– uptake or sensitivity to a stilbene derivative. One shows linear dependence on a Cl– concentration and is inhibited by 4,4-diisothiocyano-2,2-stilbenedisulfonic acid (DIDS). The other shows saturable kinetics with an apparentK
m
for Cl– of approximately 20 mM. Molecular mechanisms of the chemiosmotic coupling in the vacuolar ion transport and acidification inside are discussed in detail. 相似文献
35.
36.
We prepared highly crystalline samples of a cellulose I-ethylenediamine (EDA) complex by immersing oriented films of algal (Cladophora) cellulose microcrystals in EDA at room temperature for a few days. The unit-cell parameters were determined to be a = 0.455, b = 1.133, and c = 1.037 nm (fiber repeat) and gamma = 94.02 degrees. The space group was P2(1). On the basis of unit cell, density, and thermogravimetry analyses, the asymmetric unit is composed of one anhydrous glucose residue and one EDA molecule. The chemical and thermal stabilities of the cellulose I-EDA complex were also investigated by the use of X-ray diffraction. When the cellulose I-EDA complex was immersed in methanol or water at room temperature, cellulose III I or I beta was obtained, respectively. However, immersion in a nonpolar solvent such as toluene did not affect the crystal structure of the complex. The cellulose I-EDA complex was stable up to a temperature of approximately 130 degrees C, whereas the boiling point of EDA is 117 degrees C. This thermal stability of the complex is probably caused by intermolecular hydrogen bonds between EDA molecules and cellulose. When heated above 150 degrees C, the cellulose I-EDA complex decomposed into cellulose I beta. 相似文献
37.
Organization of microtubules (MTs) in relation to the behavior of nuclei was examined in dividing binucleate cells ofAdiantum capillus-veneris L. To induce binucleate cells, caffeine, an inhibitor of formation of the cell plate, was applied at 4 mM to synchronously
dividing protonemal cells during cytokinesis (Murata and Wada 1993). Formation of the preprophase band (PPB) during the next
cell cycle was examined in non-centrifuged and centrifuged cells. The two nuclei were separated or associated with one another
in both non-centrifuged and centrifuged cells, although the location of the nuclei in the cylindrical protonemal cells was
different (Murata and Wada 1993). Irrespective of centrifugation, a single PPB was formed around the nuclei in cells with
associated nuclei. Two PPBs were formed in cells with separated nuclei in centrifuged cells. Patterns of mitosis and cytokinesis
varied, depending on the location of the PPB and the distribution of the nuclei. The role of the nucleus in formation of the
PPB is discussed. 相似文献
38.
T Ohnishi A Wada Y Nonaka T Sugiyama T Yamano M Okamoto 《Journal of biochemistry》1986,100(4):1065-1076
Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta. 相似文献
39.
40.
We examined the expression and functions of Pax1 and Pax9 in a teleost fish, the medaka Oryzias latipes. While Pax1 and Pax9 show distinct expression in the sclerotome in amniotes, we could not detect the differential expression of Pax1 and Pax9 in the developing sclerotome of the medaka. Furthermore, unlike the mouse, in which Pax1 is essential for development of the vertebral body, and where the neural arch is formed independent of either Pax1 or Pax9, our morpholino knockdown experiments revealed that both Pax1 and Pax9 are indispensable for the development of the vertebral body and neural arch. Therefore, we conclude that after gene duplication, Pax1 and Pax9 subfunctionalize their roles in the sclerotome independently in teleosts and amniotes. In Stage-30 embryo, Pax9 was strongly expressed in the posterior mesoderm, as was also observed for mouse Pax9. Since this expression was not detected for Pax1 in the mouse or fish, this new expression in the posterior mesoderm likely evolved in Pax9 of ancestral vertebrates after gene duplication. Two-month-old fish injected with Pax9 morpholino oligonucleotide showed abnormal morphology in the tail hypural skeletal element, which may have been related to this expression. 相似文献