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91.
Karasawa K Shirakura M Harada A Satoh N Yokoyama K Setaka M Inoue K 《Journal of biochemistry》2005,138(4):509-517
Although red blood cells account for about 30% of total PAF-AH activity found in the blood, the physiological function of this enzyme is unknown. To understand the role and regulatory mechanism of this enzyme, we purified it from easily obtainable pig red blood cells. PAF-AH activity was mainly found in the soluble fraction of the red blood cells. Two peaks of enzyme activity appeared with increasing concentration of imidazole on column chromatography on nickel-nitroacetic acid (Ni-NTA) resin. We called these peaks of small and large enzyme activities fractions X and Y, respectively, and then further purified the enzymes by sequential chromatofocusing on Mono P and gel filtration on TSK G-3000. In the final preparation from fraction Y, two proteins bands corresponding to 26 kDa and 28 kDa were related to enzyme activity. Determination of the partial amino acid sequences of the proteins of 26 kDa and 28 kDa revealed that these proteins were identical to alpha(1) and alpha(2), respectively, both of which are catalytic subunits of Type I intracellular PAF-AH. On Western analysis, the 26 kDa and 28 kDa protein bands cross-reacted with specific monoclonal antibodies to alpha(1) and alpha(2), respectively. Since the apparent molecular weight of the natural enzyme was estimated to be about 60 kDa, the enzyme activity in fraction Y was thought to be that of a heterodimer consisting of alpha(1) and alpha(2). On the other hand, the enzyme activity in fraction X was thought to be that of a homodimer consisting of alpha(2). Other blood cells such as polymorphonuclear leukocytes and platelets only contained the alpha(2)/alpha(2) homodimer. It has been reported that the alpha(1)/alpha(2) heterodimer is poorly expressed in adult animals except for in the spermatogonium. Taken altogether, these results suggest that high expression of the alpha(1)/alpha(2) heterodimer is important for the physiological function of mature red blood cells. 相似文献
92.
93.
Doi M Shigenaga A Emi T Kinoshita T Shimazaki K 《Journal of experimental botany》2004,55(396):517-523
Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses. 相似文献
94.
Tai A Takebayashi J Ueno A Gohda E Yamamoto I 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2006,840(1):38-43
2-O-alpha-D-glucopyranosyl-6-O-hexadecanoyl-L-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with alpha-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol-phosphate buffer (pH 2.1) (65:35, v/v) containing 20mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10-200 microM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives. 相似文献
95.
Yoshino H Sato H Shiraishi T Tachibana K Emura T Honma A Ishikura N Tsunenari T Watanabe M Nishimoto A Nakamura R Nakagawa T Ohta M Takata N Furumoto K Kimura K Kawata H 《Bioorganic & medicinal chemistry》2010,18(23):8150-8157
A series of 5,5-dimethylthiohydantoin derivatives were synthesized and evaluated for androgen receptor pure antagonistic activities for the treatment of castration-resistant prostate cancer. Since CH4933468, which we reported previously, had a problem with agonist metabolites, novel thiohydantoin derivatives were identified by applying two strategies. One was the replacement of the alkylsulfonamide moiety by a phenylsulfonamide to avoid the production of agonist metabolites. The other was the replacement of the phenyl ring with a pyridine ring to improve in vivo potency and reduce hERG affinity. Pharmacological assays indicated that CH5137291 (17b) was a potent AR pure antagonist which did not produce the agonist metabolite. Moreover, CH5137291 completely inhibited in vivo tumor growth of LNCaP-BC2, a castration-resistant prostate cancer model. 相似文献
96.
Yasushi Kanamori Ayako Saito Yuka Hagiwara-Komoda Daisuke Tanaka Kanako Mitsumasu Shingo Kikuta Masahiko Watanabe Richard Cornette Takahiro Kikawada Takashi Okuda 《Insect biochemistry and molecular biology》2010,40(1):30-37
We recently cloned a trehalose transporter gene (Tret1) that contributes to anhydrobiosis induction in the sleeping chironomid Polypedilum vanderplanki Hinton. Because trehalose is the main haemolymph sugar in most insects, they might possess Tret1 orthologs involved in maintaining haemolymph trehalose levels. We cloned Tret1 orthologs from four species in three insect orders. The similarities of the amino acid sequence to TRET1 in P. vanderplanki were 58.5–80.4%. Phylogenetic analysis suggested the Tret1 sequences were conserved in insects. The Xenopus oocyte expression system showed apparent differences in the Km and Vmax values for trehalose transport activity among the six proteins encoded by the corresponding orthologs. The TRET1 orthologs of Anopheles gambiae (Km: 45.74 ± 3.58 mM) and Bombyx mori (71.58 ± 6.45 mM) showed low trehalose affinity, whereas those of Apis mellifera (9.42 ± 2.37 mM) and Drosophila melanogaster (10.94 ± 7.70 mM) showed high affinity. This difference in kinetics might be reflected in the haemolymph trehalose:glucose ratio of each species. Tret1 was expressed not only in the fat body but also in muscle and testis. These findings suggest that insect TRET1 is responsible for the release of trehalose from the fat body and the incorporation of trehalose into other tissues that require a carbon source, thereby regulating trehalose levels in the haemolymph. 相似文献
97.
98.
Hiroshi Takashima Ayako Araki Keiko Takemoto Naokazu Yoshikawa Keiichi Tsukahara 《Journal of biological inorganic chemistry》2006,11(3):316-324
In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted
myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents,
such as N,N′-dimethylcinchoninium diiodide ([MCN]I2) and N,N′-dimethylcinchonidinium diiodide ([MCD]I2). The photoexcited triplet state of ZnMb, 3(ZnMb)*, was successfully quenched by [MCN]2+ and [MCD]2+ ions to form the radical pair of ZnMb cation (ZnMb·+) and reduced [MCN]·+ and [MCD]·+, followed by a thermal back ET reaction to the ground state. The rate constants (k
q) for the ET quenching at 25 °C were obtained as k
q(MCN)=(1.9±0.1)×106 M−1 s−1 and k
q(MCD)=(3.0±0.2)×106 M−1 s−1, respectively. The ratio of k
q(MCD)/k
q(MCN)=1.6 indicates that the [MCD]2+ preferentially quenches 3(ZnMb)*. The second-order rate constants (k
b) for the thermal back ET reaction from [MCN]·+ and [MCD]·+ to ZnMb·+ at 25 °C were k
b(MCN)=(0.79±0.04)×108 M−1 s−1 and k
b(MCD)=(1.0±0.1)×108 M−1 s−1, respectively, and the selectivity was k
q(MCD)/k
q(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy
differences of ΔΔH
≠(MCD–MCN) and ΔΔS
≠(MCD–MCN) at 25 °C were obtained as −1.1 and 0 kJ mol−1, respectively. On the other hand, ΔΔH
≠(MCD–MCN)=11±2 kJ mol−1 and TΔΔS
≠(MCD–MCN)=−10±2 kJ mol−1 were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD]·+ found at low temperature (10 °C) was due to the ΔΔS
≠ value obtained in the thermal back ET reaction.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
99.
Anabolic five subunit-type pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6
Ikeda T Ochiai T Morita S Nishiyama A Yamada E Arai H Ishii M Igarashi Y 《Biochemical and biophysical research communications》2006,340(1):76-82
The thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene cluster, porEDABG, encoding pyruvate:ferredoxin oxidoreductase (POR), which plays a key role in this cycle, was cloned and sequenced. The nucleotide sequence and the gene organization were similar to those of the five subunit-type 2-oxoglutarate:ferredoxin oxidoreductase from this strain, although the anabolic POR had been previously reported to consist of four subunits. A small protein (8 kDa) encoded by porE, which had not been detected in the previous work, was identified in the purified recombinant POR expressed in Escherichia coli, indicating that the enzyme is also a five-subunit type. Incorporation of PorE in the wild-type POR enzyme was confirmed by immunological analysis. PorA, PorB, PorG, and PorE were similar to the alpha, beta, gamma, and delta subunits of the four subunit-type 2-oxoacid oxidoreductases, respectively, and had conserved specific motifs. PorD had no specific motifs but was essential for the expression of the active enzyme. 相似文献
100.
Kakitani Y Nagae H Mizoguchi T Egawa A Akiba K Fujiwara T Akutsu H Koyama Y 《Biochemistry》2006,45(24):7574-7585
A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate. 相似文献